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A collection of articles from the J. Craig Venter Institute’s

Global Ocean Sampling expedition
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PLoS Biology | www.plosbiology.org i March 2007 | Oceanic Metagenomics Collection

 PUBLIC LIBRARY of SCIENCE www.plos.org Oceanic Metagenomics Collection | March 2007

Editorial ____________________________________________________________________________
Global Ocean Sampling Collection S1
Hemai Parthasarathy, Emma Hill, e83
Catriona MacCallum

Synopses of Research Articles _________________________________________

About the Cover Untapped Bounty: Sampling the Seas S3
Aboard the Sorcerer II, the Global Ocean Sampling to Survey Microbial Biodiversity e85
expedition made its way from Canada, through the Liza Gross
Panama Canal, into the South Pacific, collecting
genomic sequences from marine microorganisms. Feature _____________________________________________________________________________
The resulting data are explored in three papers in this Sorcerer II: The Search for Microbial Diversity S9
special collection from the March 2007 issue of PLoS Roils the Waters e74
Biology (see Rusch et al., e77; Yooseph et al., e16; and Henry Nicholls
Kannan et al., e17).
Essay _________________________________________________________________________________
Cover credit: Image provided by the J. Craig Venter Institute
doi:10.1371/journal.pbio.0050088.g001 Environmental Shotgun Sequencing: S13
Its Potential and Challenges for Studying e82
the Hidden World of Microbes
Jonathan A. Eisen

Community Page _____________________________________________________________

CAMERA: A Community Resource S18
for Metagenomics e75
Rekha Seshadri, Saul A. Kravitz, Larry Smarr,
Paul Gilna, Marvin Frazier

Research Articles ______________________________________________________________

The Sorcerer II Global Ocean Sampling S22
Expedition: Northwest Atlantic through e77
Eastern Tropical Pacific
Douglas B. Rusch, Aaron L. Halpern,
Granger Sutton, et al.

The Sorcerer II Global Ocean Sampling S56

Expedition: Expanding the Universe e16
of Protein Families
Shibu Yooseph, Granger Sutton,
Douglas B. Rusch, et al.

Structural and Functional Diversity S91

of the Microbial Kinome e17
Every paper we publish is freely available Natarajan Kannan, Susan S. Taylor,
Yufeng Zhai, et al.
online for you to read, download, copy,
distribute and use—no permissions required.
All articles are archived in PubMed Central.

PLoS Biology | www.plosbiology.org iii March 2007 | Oceanic Metagenomics Collection

Editorial
Global Ocean Sampling Collection
Hemai Parthasarathy*, Emma Hill, Catriona MacCallum
T
oday, PLoS Biology publishes
landmark metagenomics
papers from the J. Craig
Venter Institute’s Global Ocean
Sampling expedition [1–3]. These
papers describe the initial analyses
of several gigabasepairs’ worth of
sequence data from oceanic microbes
collected during the Sorcerer II
expedition, as the ship made her
way down from Canada, through the
Panama Canal, and finally out beyond
the Galapagos Islands well into the
tropical Pacific and the South Pacific
Gyre. Results from the first foray of
this research mission into the Sargasso
Sea were published three years ago
[4]. As described in the accompanying
Synopsis [5], the new voyage has
added information from multiple
biomes and several-fold more data.
Analysis of these data poses not
only scientific challenges [6], but also
significant legal hurdles. Craig Venter
is no stranger to issues of intellectual
property—his previous incarnation
as the president of Celera saw him
embroiled in controversy over the
decision to “privatize” aspects of his
company’s work in sequencing the
human genome. Now, at the head of
the Global Ocean Sampling project,
Venter finds himself on the side of
greater accessibility, negotiating the
claims of individual governments
on the genomic wealth within their
waters. In particular, as of this writing,
there is an active negotiation with
the Ecuadorian government (which
has seen more than one change of
power since the expedition began)
over restricting commercial reuse of
these data. Henry Nicholls describes
this tangled legal landscape in an
accompanying Feature [7].
PLoS Biology | www.plosbiology.org | S1
Although extensive in scope, the
papers presented here only touch the
surface of the wealth of information
to be gleaned from these data, which
are freely available for all to explore
from their desktops: the trace reads
and processed data have been
deposited in the National Center for
Biotechnology Information’s Trace
Archive (http://www.ncbi.nlm.nih.
gov/Traces) (with the exception
of that fraction of the trace data
acquired from Ecuadorian coastal
waters), annotated with extensive
geographical and physicochemical
metadata. The assemblies and
associated annotated peptides will be
delivered to GenBank (http:⁄⁄www.
ncbi.nlm.nih.gov/Genbank) around
the time of publication, and will
become available after GenBank has
processed them. More immediately,
and potentially more usefully, these
data are also freely available through
a specially built database, CAMERA—
Cyberinfrastructure for Advanced
Marine Microbial Ecology Research and
Analysis (http:⁄⁄camera.calit2.net)—
which provides greater annotation and
analysis capabilities [8]. (CAMERA was
funded by the Gordon and Betty Moore
Foundation, which also supports PLoS.)
The proponents of open-access
publishing, ourselves included, often
cite as an inspiration the power that
open access to DNA sequence databases
has had in transforming scientific
discovery. As our founders noted in
the inaugural issue of PLoS Biology,
“With great foresight, it was decided
in the early 1980s that published
DNA sequences should be deposited
in a central repository, in a common
format, where they could be freely
accessed and used by anyone. Simply
giving scientists free and unrestricted
access to the raw sequences led them
to develop the powerful methods,
tools, and resources that have made
the whole much greater than the sum
of the individual sequences....Now
imagine the possibilities if the same
creative explosion that was fueled by
open access to DNA sequences were
to occur for the much larger body of
published scientific results.” [9]
0369 Special Section from March 2007 | Volume 5 | Issue 3 | e83
But the publishing reality in
genomics research has been less
inspiring. Although sequence data are
publicly available and free to be reused
by the community, the same creative
license has not yet been awarded to
the key papers resulting from the
major genome projects, which are
commonly published in subscription-
based journals. Many of these genomics
papers are “freely” available from
publisher Web sites, but their use
remains restricted, and to claim that
freedom to read an article is the main
benefit of open access is to miss the
promise inspired by DNA sequence
databases.
While we and other open-access
journals have both enjoyed and been
grateful for strong support from the
genomics community, we are also
disappointed that authors of landmark
genomics papers, who adamantly
support open access to sequence
data, have not taken the opportunity
to provide further leadership for
their community by promoting open
access to the scientific literature. We
encourage all researchers to apply the
same standards to their papers as they
would to their data, regardless of the
publisher. As Jensen et al. stated in a
recent review about the benefits of text
mining for the scientific community, “It
is the restricted access to the full text of
papers…that is currently the greatest
limitation…” [10].
Citation: Parthasarathy H, Hill E, MacCallum C (2007)
Global Ocean Sampling collection. PLoS Biol 5(3): e83.
doi:10.1371/journal.pbio.0050083
Copyright: © 2007 Parthasarathy et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Hemai Parthasarathy is Managing Editor, Emma Hill
is Associate Editor, and Catriona MacCallum is Senior
Editor at PLoS Biology.
* To whom correspondence should be addressed.
E-mail: hemai@plos.org
This article is part of the Oceanic Metagenomics
collection in PLoS Biology. The full collection is
available online at http://collections.plos.org/
plosbiology/gos-2007.php.
Acknowledgments
PLoS Biology relies on the support of our
academic editors and reviewers in selecting
and improving manuscripts for publication.
We would like to extend particular thanks
to our editorial board members Sean Eddy,
Jonathan Eisen, and Nancy Moran, our
guest editors Simon Levin and Tony Pawson,
and our anonymous peer reviewers for their
contributions to this collection of articles.
References
1. Rusch DB, Halpern AL, Sutton G,
Heidelberg KB, Williamson S, et al. (2007)
The Sorcerer II Gobal Ocean Sampling
expedition: Northwest Atlantic through
PLoS Biology | www.plosbiology.org | S2
eastern tropical Pacific. PLoS Biol 5: e77.
doi:10.1371/journal.pbio.0050077
2. Yooseph S, Sutton G, Rusch DB, Halpern
AL, Williamson SJ, et al. (2007) The Sorcerer
II Global Ocean Sampling expedition:
Expanding the universe of protein families.
PLoS Biol 5: e16. doi:10.1371/journal.
pbio.0050016
3. Kannan N, Taylor SS, Zhai Y, Venter JC,
Manning G (2006) Structural and functional
diversity of the microbial kinome. PLoS Biol 5:
e17. doi:10.1371/journal.pbio.0050017
4. Venter JC, Remington K, Heidelberg
JF, Halpern AL, Rusch D, et al (2004)
Environmental genome shotgun sequencing of
the Sargasso Sea. Science 304: 58–60.
5. Gross L (2007) Untapped bounty: Sampling
the seas to survey microbial biodiversity. PLoS
Biol 5: e85. doi:10.1371/journal.pbio.0050085
0370 Special Section from March 2007 | Volume 5 | Issue 3 | e83
6. Eisen JA (2007) Environmental shotgun
sequencing: The potential and challenges
of random and fragmented sampling of the
hidden world of microbes. PLoS Biol 5: e82.
doi:10.1371/journal.pbio.0050082
7. Seshadri R, Kravitz SA, Smarr L, Gilna P,
Frazier M (2007) CAMERA: A community
resource for metagenomics. PLoS Biol 5: e75.
doi:10.1371/journal.pbio.0050075
8. Nicholls H (2007) Sorcerer II: The search for
microbial diversity roils the waters. PLoS Biol 5:
e74. doi:10.1371/journal.pbio.0050074
9. Brown PO, Eisen MB, Varmus HE (2003) Why
PLoS became a publisher. PLoS Biol 1: e36.
doi:10.1371/journal.pbio.0000036
10. Jensen LJ, Saric J, Bork P (2006) Literature
mining for the biologist: From information
retrieval to biological discovery. Nat Rev Genet
7: 119–129.
Untapped Bounty: Sampling the Seas to Survey Microbial Biodiversity
Liza Gross | doi:10.1371/journal.pbio.0050085
Being invisible to the naked eye, microbes managed to
escape scientific scrutiny until the mid-17th century, when
Leeuwenhoek invented the microscope. These cryptic
organisms continued to thwart scientists’ efforts to probe,
describe, and classify them until about 40 years ago, owing
largely to a limited morphology that defies traditional
taxonomic methods and an enigmatic physiology that makes
them notoriously difficult to cultivate.
Most of what we know about the biochemical diversity of
microbes comes from the tiny fraction that submit to lab
investigations. Not until scientists determined that they could
use molecular sequences to identify species and determine
their evolutionary heritage, or phylogeny, did it begin to
become apparent just how diverse microbes are. We now
know that microbes are the most widely distributed organisms
on earth, having adapted to environments as diverse as
boiling sulfur pits and the human gut. Accounting for half
of the world’s biomass, microbes provide essential ecosystem
services by cycling the mineral nutrients that support life on
earth. And marine microbes remove so much carbon dioxide
from the atmosphere that some scientists see them as a
potential solution to global warming.
Yet even as scientists describe seemingly endless variations
on the cosmopolitan microbial lifestyle, the concept of a
bacterial species remains elusive. Some bacterial species
(such as anthrax) appear to have little genetic variation
while in others (such as Escherichia coli) individuals can have
completely different sets of genes, challenging scientists to
explain the observed diversity.
The emerging field of environmental genomics (or
metagenomics) aims to capture the full measure of microbial
diversity by trading the lens of the microscope (and
biochemistry) for the lens of genomics (and bioinformatics).
By recovering communities of microbial genes where they
live, environmental genomics avoids the need to culture
uncooperative organisms. And by linking these data to details
relating to sequence collection sites, such as pH, salinity, and
water temperature, it sheds light on the biological processes
encoded in the genes.
The largest metagenomic dataset collected so far comes
from the Sorcerer II expedition, named after the yacht J. Craig
Venter transformed into a marine research vessel. In a pilot
study of the Sargasso Sea, Venter’s team identified 1.2 million
genes and inferred the presence of at least 1,800 bacterial
species. But the genetic and taxonomic diversity of the
PLoS Biology | www.plosbiology.org | S3
data imposed new challenges on existing genome assembly
methods and other analysis techniques. The researchers
designed the Sorcerer II Global Ocean Sampling (GOS)
expedition to see if collecting more samples would improve
their assembly and lead to a better estimate of the number
and diversity of microbial genes in the oceans.
And now, in three new studies, Venter’s team has
combined the expedition’s latest bounty—6.5 million
sequencing “reads”—with the Sargasso Sea data. The result
is a geographically diverse environmental genomic dataset of
6.3 billion base pairs—twice the size of the human genome.
(To learn about the voyage and sampling methods, see
Box 1.) In the first paper, Douglas Rusch, Aaron Halpern,
and colleagues attempt to describe the immense amount
of microbial diversity in the seas, and determine how—or
if—that diversity is structured and what might be shaping that
structure. In the second paper, Shibu Yooseph et al. study the
millions of proteins in the GOS sequences to see if we’re close
to discovering all the proteins in nature. And in the third
study, Natarajan Kannan, Gerard Manning, and colleagues
classify thousands of kinases into 20 distinct families,
revealing their structural and functional diversity and an
unexpected importance in prokaryotic regulation.
Extracting Meaning from Metagenomic Datasets
The GOS samples used in the Rusch et al. study were
collected over the course of a year from a wide range of
aquatic environments—including estuaries, lakes, and open
oceans—then pumped through serial filters. After extracting
the genetic material from the microbe-encrusted filters,
Rusch et al. used shotgun sequencing to study the genes
present in the samples. DNA is forced through a tiny nozzle
that smashes it into bits; the fragments are cloned and the
letters of the genetic code are scanned from both ends to
create “reads.” Reads are then assembled, much like a jigsaw
puzzle, starting with contiguous fragments (“contigs”) that are
then mapped onto “scaffolds,” which order and orient sets of
contigs on a chromosome. (For more on shotgun sequencing,
see Box 2.) Using a conservative sequence similarity
requirement, most reads failed to assemble, suggesting that
the samples contained great microbial diversity.
With only a bare bones assembly to guide their
investigation, Rusch et al. tried a different approach. They
used the 584 completed and draft microbial genomes already
available in public databases as points of reference and
relaxed search parameters to detect even remote similarity
to GOS sequences. Although the majority of GOS reads
matched up with one or more of the reference genomes,
the loose criteria prevented the researchers from drawing
meaningful inferences about kinship.
To boost their inference power, they required that
similarity to a reference genome extend nearly the full
length of a read (producing “recruited reads”). A substantial
majority of reads failed this criterion, with only 30% of the
GOS data being recruited. The bulk of these aligned to three
genera of widely distributed marine microbes—Pelagibacter,
Synechococcus, and Prochlorococcus—which accounted for about
15% of the recruited reads. The remaining recruited reads
appear to signal conserved genes rather than closely related
0371 Special Section from March 2007 | Volume 5 | Issue 3 | e85
Box 1. Following the Sorcerer II’s Hunt for Microbes
The Sorcerer II expedition was inspired by the British
Challenger expedition (1872–1876), a pioneering oceanography
research project that discovered hundreds of new genera and
nearly 5,000 new marine species. Its gun stations replaced
with research stations, the Challenger circumnavigated the
oceans, stopping every 320 kilometers to recover specimens
from bottom, intermediate, and surface depths to explore the
diversity of macroscopic marine life. At each stop, the crew
recorded the location, what they used to extract the sample, the
such environments. Continuing down the Atlantic seaboard, the
expedition stopped near Cape Hatteras, North Carolina, and the
Florida Keys before passing through the Caribbean and ending
near Panama, where the crew collaborated with scientists at the
Smithsonian Tropical Research Institute.
The fourth leg of the voyage sampled sites in the Eastern
Pacific, including Cocos Island, about 500 kilometers southwest
of Costa Rica. A highly productive ecosystem inhabits the waters
off the island, a result of ocean currents buffeting the coast and
causing nutrient upwellings that mix with warm surface waters.
The crew made one last stop in the open ocean, then headed for
the Galapagos Islands.
Owing in part to its position near major ocean currents and
atmospheric transition zones, the Galapagos Archipelago
sits within a hydrographically complex region. Unique
oceanographic features there support a diverse set of habitats
and endemic species, found within several discrete zones
distinguished by temperature. This microbial mother lode held
the crew’s attention for two months, while they extensively
sampled the region.
By early March 2004, the crew had collected the last three
samples used in these studies, from two open ocean sites
and a lagoon in a coral reef in the South Pacific Gyre. Follow
these links to learn more about the Sorcerer II (http://www.
sorcerer2expedition.org/version1/HTML/main.htm) and the
Challenger (http://hercules.kgs.ku.edu/hexacoral/expedition/
challenger_1872-1876/challenger.html) expeditions.

doi:10.1371/journal.pbio.0050085.g001

H.M.S. Challenger (Image: NOAA, Steve Nicklas)

depth of the sample, and several observations related to water

and atmospheric conditions. The Sorcerer II followed a similar
sampling schedule, traveling nearly 9,000 kilometers to collect
samples of microbial marine life and record the water’s location,
depth, pH, salinity, and temperature.
The GOS crew collected samples from surface waters of
diverse, mostly marine aquatic environments. The samples were
collected between August 2003 and May 2004 during a six-leg
journey that followed a path from northeastern Canada to the
South Pacific Gyre. Venter’s crew collected microbial samples
by pumping 200 liters of surface seawater through a series of
increasingly fine filters, which they labeled, froze, and sent back
to the lab of the J. Craig Venter Institute in Maryland.
After a stop in the Gulf of Maine, the expedition sampled three
sites along Nova Scotia, including a “highly eutrophic” coastal
embayment in Halifax. The crew set sail again in November,
starting in Newport Harbor, Rhode Island, and ending in the
Delaware Bay, one of several estuaries targeted on the journey.
The next leg began in Chesapeake Bay. The largest US estuary,
Chesapeake Bay contains a rich mix of freshwater and marine
organisms. Estuaries are complex hydrodynamic environments
that are highly sensitive to runoff from agricultural and urban
development (which can dump massive amounts of nitrogen
and phosphorous into watersheds). Microbial communities
doi:10.1371/journal.pbio.0050085.g002
collected from estuaries promise to provide valuable insights
into the metabolic and physiological adaptations required by The Sorcerer II (Image: J. Craig Venter Institute)

PLoS Biology | www.plosbiology.org | S4 0372 Special Section from March 2007 | Volume 5 | Issue 3 | e85
organisms. Most of the GOS sequences failed to be identified,
in part because so few surface water microbes have been
sequenced.
A novel comparative genomic method. Focusing on
the reads that recruited to these most abundant genera,
Rusch et al. generated “fragment recruitment plots.” These
graphics represent relatedness and diversity of environmental
sequences to a reference genome by showing where a read
aligns with the reference genome (indicated by a horizontal
bar) and its degree of similarity to the reference sequence
(indicated by its vertical position). Recruited reads were
color-coded based on sample origin to indirectly depict their
associated metadata (for example, salinity and pH). These
plots provided a visual tool to explore genetic diversity at the
sequence and gene level, genome structure and evolution,
and taxonomic and evolutionary relationships. (For more on
fragment recruitment plots, see the accompanying poster,
doi:10.1371/journal.pbio.0050077.sd001.)
Distinct recruitment patterns, easily detected by bands of
color, emerged for each organism. In some cases, a single
reference genome had multiple color bands, distinguished
by their similarity and sample provenance. Because
bands appeared to represent unique, closely related, and
geographically distinct populations—and showed a novel level
of diversity across the entire genome—the researchers termed
each band a subtype. A tremendous amount of sequence
diversity appeared in the subtypes, which also harbored
substantial sequence variation at the protein level, some likely
reflecting adaptations to local environments. This finding
reveals a potential locus of microbial diversity—at the level of
subtype rather than at the level of species, or ribotype (based
on a segment of a ribosomal RNA gene called 16S rRNA)—
and offers clues to why it emerged (perhaps in response to
local pressures) and how it evolved.
A novel sequence assembly method. Because such high
levels of sequence diversity among organisms confound
standard whole genome assembly software, and most of
the GOS data correspond to organisms for which there is
no appropriate reference genome, Rusch et al. used an
“extreme assembly” approach to investigate the genomes of
other abundant GOS populations. They used greatly reduced
requirements for sequence similarity in the assemblers to
generate longer contigs and capture more of the GOS data
in an assembly. While some of the resulting larger assemblies
corresponded to known reference genomes, others did not,
allowing the researchers to study microbes without cultivated
or sequenced counterparts. And because these larger
assemblies could potentially provide functional insights into
uncharacterized organisms, they might identify conditions
that would allow scientists to grow them in the lab.
Many of the large contigs failed to align in any significant
way with known genomes, so the researchers tried to match
them with “seed fragments” from known taxonomic groups. By
starting assembly from reads mated to the 16S rRNA gene—
one of the most common marker genes used for classifying
microbes—they could generate large contigs associated with
many of the abundant GOS ribotypes. Fragment recruitment
plots of these assemblies again revealed multiple subtypes,
providing further support for the presence of multiple
evolutionarily distinct subtypes within a given ribotype.
Evidence for environmental adaptations. A computational
approach designed to identify groups of samples with similar
PLoS Biology | www.plosbiology.org | S5
genomic content revealed that tropical and temperate
samples shared the least amount of genomic material. Some
samples, however, were very similar.
While untangling all the factors that may affect genetic
makeup of a sample is beyond current datasets and methods,
the researchers demonstrated that specific genetic differences
can be related to environmental factors. Several genes
occurred up to seven times more frequently in a pair of
samples from the Caribbean than they did in a pair from
the eastern Pacific, even though both pairs had similar
ribotype and genetic profiles. Many of these genes govern
the metabolism and transport of phosphate (required for
microbial growth), likely reflecting functional adaptations in
the microbial communities to the measured differences in
phosphate availability in the Caribbean and Pacific samples.
The researchers also explored diversity at the gene level
by looking for evidence of functional differences in one
gene family, proteorhodopsins, light-activated proton pumps
with a slightly murky biological role. Proteorhodopsins
were abundant in all the GOS and Sargasso Sea samples. In
keeping with the diverse light environments sampled during
the expedition, the researchers found a strong correlation
between sequence variation and sample provenance. They
hypothesize that the distribution of given variants reflects
adaptation to the most abundant light spectra in their
habitats.
Altogether, these results reveal the power of metagenomic
approaches to capture the true measure of microbial diversity
by uncovering genomic differences that would not have
been apparent using traditional marker-based approaches.
The breadth of this newly revealed diversity may come as a
surprise to even inveterate microbe hunters.
The Expanding Protein Universe
Along with insights into microbial diversity, metagenomics
promises to help us understand the vast number of proteins
in nature. By randomly sampling DNA sequences from
communities of organisms, metagenomic studies overcome
selection and culturing biases that arise from focusing on
a particular organism or a set of proteins, to provide an
expansive view of protein diversity and evolution.
Proteins are typically grouped into families based on
their evolutionary relationship, which can then be used
to guide investigations of their biological roles. Proteins
in the same family share similar amino acid sequences
and three-dimensional conformations. Using amino acid
sequence similarity as a measure to identify and group
protein sequences from the GOS data with sequences from
a comprehensive set of known proteins, Shibu Yooseph et al.
evaluated the impact of the GOS data on our understanding
of known proteins and studied the rate of discovery of protein
families with new sequences. To group related sequences and
predict proteins, they developed a novel sequence clustering
technique based on full-length sequence similarity.
Identifying proteins in metagenomics data. Hypothetical
proteins can be predicted by searching for open reading
frames (ORFs), sequences flanked by nucleotide triplets
(called codons) that signal the beginning and end of
translation but don’t necessarily encode a protein. Because
the GOS data contain many fragmentary sequences,
Yooseph et al. allowed ORFs to be terminated at the end of a
sequence, resulting in a partial or truncated ORF. They used
0373 Special Section from March 2007 | Volume 5 | Issue 3 | e85
Box 2. Bioinformatic Methods at a Glance
Bioinformatics relies on statistics and computer power
to synthesize and interpret huge datasets. Here’s a brief
introduction to some of the environmental genomics methods
used in the GOS studies.
Shotgun sequencing decodes genetic material by randomly
shredding it into millions of fragments. The DNA sequence of
each end of a fragment is determined; the two ends of a given
fragment (or insert) can be associated, and constitute a “mate
pair.” These random sequencing “reads” are then reassembled
with a computer. Based on sequence similarity, overlapping
reads are identified and merged into longer sequences called
“contigs.” Contigs are organized into larger (but not necessarily
continuous) pieces of a genome, called “scaffolds,” based
on mate pairs. The resulting assemblies can link genes to
their regulatory elements, guide investigations of biological
pathways, and connect unknown sequences with taxonomic
markers to suggest evolutionary relationships.
Sequence similarity detection allows functional and taxonomic
characterization of genomic sequences. Once the shotgunned
sequences have been organized into a library of sequence
“scaffolds” and translated into hypothetical proteins, the next
step uses sequence similarity to figure out what the proteins
are and to identify families. Similarity can also associate a new
sequence with an approximate location on the tree of life.
Sequence–sequence (pairwise) methods, the first step for
identifying closely related sequences, compare all sequences
to all other sequences in a pairwise manner. These methods
(such as BLAST) allow all collected sequences to be compared
with one another (and with all sequences already available in
public databases) and reliably clustered into families of related
sequences with high sequence similarity, or homology.
Profile methods are used to identify more remote
relationships. Profile methods use multiple sequence
the ORFs to generate a set of predicted proteins based on the
results of a series of clustering steps and statistical analyses.
After performing pairwise comparisons (of every sequence
against every other sequence) of the more than 28 million
sequences in the combined dataset, the researchers identified
conserved groups of sequences after accounting for
redundancy due to identical and near-identical sequences.
They then used profile methods to merge and expand these
groups of sequences. While pairwise comparisons capture
the most closely related sequences (or homologs), profile
methods (the researchers used both PSI-BLAST and hidden
Markov models) detect more distantly related sequences by
combining homologs into multiple sequence alignments to
generate “profiles.” (For more on these methods, see Box 2.)
From the clusters obtained by the above procedure, clusters
of spurious sequences (that overlap true protein regions
on the genome) were identified in addition to clusters of
noncoding conserved sequences (based on tests showing
no selection on their codons). Sequences in these clusters
were removed; those remaining were labeled as predicted
proteins. The researchers identified nearly 6 million proteins
in the GOS dataset—1.8 times the number already in public
databases. Comparing the predicted protein clusters to
known prokaryotic and nonprokaryotic protein databases
PLoS Biology | www.plosbiology.org | S6
alignments of previously identified families to compute
“position-specific scoring matrixes” (PSSMs). Each position in
the alignment is associated with a set of scores that reward or
penalize the alignment of a given amino acid to the position.
Profile methods can be more sensitive than simple sequence
similarity methods because they give more weight to signals at
sites that are conserved within a protein family and less weight
to more variable positions.
Initially, the advantages of profile methods for detecting
remote homology were limited to well-characterized families,
as construction of a profile required some expertise. However,
this changed with the fully automated integration of this step
into PSI-BLAST. PSI-BLAST begins with a pairwise (sequence–
sequence) similarity search, but then iteratively runs alternating
steps of building a profile from the current set of similar
sequences and using the profile to re-search the database for
additional matching sequences.
Hidden Markov models (HMMs) employ statistical methods
to model the likelihood of different amino acids at any given
position of the sequence in an underlying alignment. Like
some profile methods, HMMs use a probability-based method
to determine the score of aligning an observed amino acid to
a given position in a protein family, but HMMs improve upon
profiles by more sophisticated modeling of variation in protein
length, storing the probabilities of insertions or deletions at
each position of the model. HMMs have a good track record for
identifying more distantly related protein sequences.
Profile–profile methods are the most recent enhancement to
sequence homology detection methods. As the name suggests,
profile–profile methods compare one profile to another. Because
each profile implicitly encodes more information than a single
sequence, these methods identify relationships that cannot be
detected by comparing individual sequences.
revealed GOS counterparts in nearly all known prokaryotic
protein families; nearly 2,000 clusters appeared unique to the
GOS dataset.
Since they couldn’t use sequence similarity to infer
function for the unique GOS sequences, the researchers
relied on the assumption that proteins with similar roles are
more likely to reside in the same genomic neighborhood.
This analysis implicated several GOS-only clusters in
photosynthesis or electron transport. Such clusters may come
from viruses, as many viral parasites of photosynthetic bacteria
express the photosynthetic genes of their hosts. Interestingly,
though most of the sequences in GOS-only clusters appeared
to be bacterial, a higher than expected proportion of them
were flagged as viral. If such novel GOS protein families pan
out as viral, the researchers argue, “we are far from exploring
the molecular diversity of viruses.”
Insights into evolutionary and functional diversity. To
compare ocean versus terrestrial life at the biochemical level,
Yooseph et al. compared GOS sequences to those of land-
dwelling prokaryotes. Nearly 70% of protein domains varied
between the two classes of microbes, mostly reflecting the
distinct biochemical requirements of the two environments,
as well as the different taxonomic groupings in the two
datasets. The researchers were surprised to find little evidence
0374 Special Section from March 2007 | Volume 5 | Issue 3 | e85
of domains specific to gram-positive bacteria (defined by their
unique cell wall), even though this bacterial group makes up
nearly 12% of the GOS dataset. They also found a relative
dearth of components related to flagella (the whip-like tail of
microbial motility), possibly reflecting the reduced need for
self-propulsion in the ocean.
Using a comprehensive protein family database (called
Pfam), the researchers compared the kingdom distribution
of known protein domains in the GOS data to that of
proteins in public databases. In this process, some families
that were previously thought to be single-kingdom turned
out to have members in multiple kingdoms. For example,
indoleamine 2,3-dioxygenase (IDO), an enzyme linked to
the immune system in mammals, was considered unique to
eukaryotes. But the IDO Pfam search turned up matches to
ten GOS sequences identified as bacterial—suggesting that
the proteins may have arisen much earlier than previously
thought, or perhaps arose through lateral gene transfer (from
an unrelated organism).
The sheer size of the GOS dataset—which nearly doubles
the number of proteins—greatly expands the functional
diversity of known protein families, providing valuable
insights into their evolution. For example, the researchers
found a 10-fold increase in the number and type of proteins
involved in repairing ultraviolet radiation damage, likely
reflecting the hazards of living in surface waters. A similar
boost in phosphatases—which function in such fundamental
biological processes as cell signaling, development, and
cell division—highlighted important differences in the way
one phosphatase (protein phosphatase 2C) functions in
prokaryotes and eukaryotes.
And the unexpected abundance of a nitrogen metabolism
catalyst typically associated with eukaryotes (type II glutamine
synthetase) suggested two possible evolutionary mechanisms:
either lateral gene transfer from eukaryotes, or gene
duplication prior to the divergence of prokaryotes and
eukaryotes. (The researchers suspect gene duplication.) The
diversity of the GOS sequences also promises to characterize
sequences with no similarity to known sequences (known as
ORFans): over 6,000 ORFans pair up with GOS sequences
representing some 600 organisms, paving the way for further
study of their identity and function.
As GOS protein predictions are tested, some of these
proteins will expand existing protein families while others
will carve out GOS-specific families. Both results will help
researchers determine priority targets for structural studies—
an essential strategy for dealing with the flood of protein
discoveries. And given that the GOS sequences represent
mostly microbes from the ocean’s surface—yet point to
substantial viral diversity as well—the rate of protein discovery
indicates that a comprehensive catalog of proteins in nature is
far from complete.
Variations on a Theme: A Single Fold Spawns a Diverse
Kinase Superfamily
Cellular life chugs along under the power of enzymes,
proteins that catalyze the scores of chemical reactions
required for life. One of the largest protein families
in eukaryotes, the eukaryotic protein kinases (ePKs),
regulates the activity of a large fraction of all proteins
and almost all biological pathways by phosphorylating
proteins. Phosphorylation activates its target by transferring
PLoS Biology | www.plosbiology.org | S7
a phosphate group from adenosine triphosphate (ATP)
to a specific amino acid on the protein, releasing energy
and inducing structural changes that alter the protein’s
activity. (Dephosphorylation removes the phosphate group,
restoring the protein to its original conformation and
inactive state.) One cell can contain hundreds of different
protein kinases, each charged with phosphorylating one or
many different proteins.
Bacteria and other prokaryotes, conventional wisdom held,
rely mostly on structurally distinct kinases (histidine kinases)
to mediate protein phosphorylation and cell signaling. But
it now emerges that ePK-like kinases (ELKs), once thought
to be minor players, are more prevalent and widespread
than the histidine kinases. Although ePKs and ELKs typically
exhibit very low sequence similarity, they share similar
phosphorylation mechanisms and the same structural fold
(the protein kinase–like, or PKL, fold).
Since PKL kinases conserve both fold and mechanism
of action, they provide a robust model for determining
how sequence variation corresponds to functional
diversity. Unfortunately, comprehensive comparisons had
been frustrated by a lack of sequence information for
the prokaryotic ELK families relative to the well-studied
eukaryotic domains. But now, thanks to the Sorcerer
II expedition, sequence databases are brimming with
microbial sequences, including a 3-fold increase in ELK
sequences. Taking advantage of the bounty, Natarajan
Kannan, Gerard Manning, and colleagues surveyed the
global PKL landscape, and identified over 45,000 PKLs,
which they classified into 20 families. Surprisingly, PKLs
appear to usurp the histidine kinases as the core regulator
of prokaryotic signaling and cell behavior.
Cataloging the number and diversity of PKL families. To
detect kinase sequences, Kannan et al. searched over 17
million predicted proteins in the GOS dataset and 5 million-
plus predicted and known protein sequences in public
databases. Kinase sequences were detected using hidden
Markov model (HMM) profiles of known PKLs along with
a model that predicts kinases on the basis of a few ultra-
conserved motifs. The sensitivity of the HMMs allowed the
researchers to discover very remote new members of these
families and to classify and organize the tens of thousands of
sequences. Both approaches iterate through multiple runs of
the clustered results to refine the family alignments and to
classify clusters with little similarity to known PKL families as
potentially novel. (For more on these methods, see Box 2.)
The public databases, it turned out, harbored nearly 25,000
ePKs and over 5,000 ELKs. Over 16,000 GOS sequences fell
into 20 PKL families—doubling the size of most families.
Three main superfamily clusters emerged, distinguished by
the most abundant members: choline and aminoglycoside
kinases (CAKs), a “particularly diverse” family harboring
kinases that facilitate colonization by beneficial and
pathogenic bacteria; ePKs, almost exclusively eukaryotic
except for a similar bacterial kinase (pknB); and a cluster of
kinases, including Rio and Bud32, that are conserved between
archaea and eukaryotes. Three families bore no sequence
similarity to any other families save for a group of key motifs.
Overall, the 20 families exhibit significant functional and
sequence diversity. Most of the families have not yet been
fully investigated, though they do include some characterized
members. Those with known kinase activity target small
0375 Special Section from March 2007 | Volume 5 | Issue 3 | e85
molecules (such as lipids and amino acids) and seem to
play regulatory roles, in contrast to many other structurally
unrelated small molecule kinases, which affect metabolism.
Functional diversity springs from a set of core residues.
Because sequence similarity ranged from “very low to
almost undetectable,” the researchers used sequence
profiles—models built from entire families to highlight their
core characteristics—to both discover and classify kinase
sequences. They found several novel families, and greatly
extended the breadth of previously defined families. With
these methods to refine the relationships within and between
PKL families, the researchers explored the traits that unite or
distinguish them.
Ten key amino acid residues of the catalytic domain
consistently turned up in each family. This “core pattern of
conservation,” the researchers explain, represents an ancient
evolutionary innovation, spanning not just the three divisions
of life—which diverged 1–2 billion years ago—but also the
diverse families. The conservation of these residues across
and within the families suggests that they play an essential
role. And, indeed, six of those already characterized mediate
ATP binding and catalysis.
Yet despite the seemingly universal presence of the ten
residues, their occurrence in individual subfamilies showed
a surprising pattern: all but one of these “core” residues had
either disappeared or changed in individual families—though
the proteins retained their fold and function—suggesting an
unexpected flexibility for catalytic cores. To test this possibility,
the researchers focused on one of the ten residues—the
catalytic lysine K72, which repositions ATP’s phosphates.
Present in ePKs, K72 is replaced by a different conserved
amino acid in three CAK subfamilies. These subfamilies
had corresponding substitutions near other key motifs,
and structural modeling showed how these coordinated
replacements could still result in an active enzyme.
A number of features (including amino acid motifs and
secondary structure) emerged as family-specific, being highly
conserved within but not between families. And as was seen
in the CAK analysis, many family-specific residues occur near
one of the ten key residues, suggesting that they may help
PLoS Biology | www.plosbiology.org | S8
direct substrates to the catalytic core or influence the nature
of the reaction.
Evolutionary insights and beyond. Altogether these results
reveal the vast functional and phylogenetic diversity that can
occur in even just a subset of proteins, even though they retain
a common catalytic fold and function. The massive sequence
comparisons in this study not only identified the core of the
PKL kinase, but also revealed the specific motifs underlying
each family, including the ePKs. And the flexibility of several
key regions within ePKs may underlie the huge expansion of
these enzymes in eukaryotes. This structural flexibility may
give kinases the ability to integrate multiple regulatory signals,
and account for their almost universal involvement in the
regulation of eukaryotic pathways.
These results set the stage for more in-depth structural and
biochemical studies to elucidate the diverse functions carried
out by these critical regulators of cell behavior. This study
also demonstrates how metagenomic datasets, by covering
an unbiased diversity of life, can refine our understanding
of well-studied protein families, such as the ePKs, and shed
light on their evolution. Kannan et al. hope that others take
advantage of the environmental metagenomic largesse to
pursue “similar insights into virtually every gene family with
prokaryotic relatives.”
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al.
(2007) The Sorcerer II Gobal Ocean Sampling expedition: Northwest
Atlantic through eastern tropical Pacific. doi:10.1371/journal.
pbio.0050077
Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, et al. (2007)
The Sorcerer II Global Ocean Sampling expedition: Expanding the
universe of protein families. doi:10.1371/journal.pbio.0050016
Kannan N, Taylor SS, Zhai Y, Venter JC, Manning G (2006) Structural
and functional diversity of the microbial kinome. doi:10.1371/journal.
pbio.0050017
This article is part of the Oceanic Metagenomics collection
in PLoS Biology. The full collection is available online at
http:⁄⁄collections.plos.org/plosbiology/gos-2007.php.
0376 Special Section from March 2007 | Volume 5 | Issue 3 | e85
Feature
Sorcerer II: The Search for Microbial Diversity
Roils the Waters
Henry Nicholls
C
raig Venter is not short of ambition. With the human
genome fresh off the sequencing machines, he
set his sights on a project of even grander scale: to
describe the immense wealth of genetic information living in
the world’s oceans. This voyage into biologically uncharted
waters was, according to the Web site of the expedition vessel
Sorcerer II, inspired in part by the voyage of H. M. S. Beagle [1].
Venter, it seems, would like to be remembered as the Charles
Darwin of the 21st century (Figure 1).
This is the largest effort to describe the genetic diversity
in the world’s oceans. The voyage around national and
international waters, collecting from around 150 sites and
interrogating samples at the level of the gene rather than at
the level of the organism, has already turned up between 5
and 6 million genes. Most of these genes have never been
seen before, says Venter. Analysing this immense collection
of data, the researchers discovered that many of the genes
encode proteins that fall outside standard classification
schemes. Proteins grouped within their own unique
kingdoms are turning up in other kingdoms as well—forcing
the team to reconsider the evolutionary relationships of
established kingdoms. “This project is revealing some of the
biggest discoveries about the environment,” says Venter. (For
more on these discoveries see the synopsis of the research
articles [2].)
“If Darwin were alive today trying to
do his experiments, he would not have
been allowed to.”
Untapped Diversity
The Sorcerer II probably captured only a tiny fraction of the
genetic diversity out there, says Mitchell Sogin, Director of the
Josephine Bay Paul Center in Comparative Molecular Biology
and Evolution at the Marine Biological Laboratory in Woods
Hole, Massachusetts. In August 2006, Sogin and his colleagues
published a detailed analysis of variable stretches of ribosomal
PLoS Biology | www.plosbiology.org | S9
doi:10.1371/journal.pbio.0050074.g001
Figure 1. Two of a Kind?
The young Charles Darwin (left) and Craig Venter (right). (Photo: J. Craig
Venter Institute)
RNA collected from the marine microbial world (Figure 2)
[3]. “We estimate there are at least 25,000 different kinds of
microbes per litre of seawater,” says Sogin. “But I wouldn’t
be surprised if it turns out there are 100,000 or more.” A few
of these microbes are common, and Venter will probably use
them to recover complete gene sequences, he says. “The vast
majority of low-abundance organisms are going undetected.”
Venter is more than aware that there’s a lot more to be
discovered, but for the moment the goal is to sequence as many
genes, in their entirety, as possible from these ecologically rich
environments. These data raise a host of intriguing questions:
in particular, what is the structure and function of the novel
proteins these genes encode, and what role do they play in the
metabolism of these undescribed microbes? Just as Darwin’s
work drove a change in the way we see the world, so Venter is
hoping these marine data will do the same in years to come.
Legal Framework
But times have changed. In the 21st century, there are plenty
of hurdles to clear before the collecting and describing of
biodiversity—even microscopic biodiversity—can go ahead.
Citation: Nicholls H (2007) Sorcerer II: The search for microbial diversity roils the
waters. PLoS Biol 5(3): e74. doi:10.1371/journal.pbio.0050074
Copyright: © 2007 Henry Nicholls. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: UNCLOS, United Nations Convention on the Law of the Sea
Henry Nicholls is a freelance science journalist based in London, United Kingdom.
His book Lonesome George was nominated for the 2006 Guardian First Book Award.
E-mail: henry.nicholls@tiscali.co.uk
This article is part of the Oceanic Metagenomics collection in PLoS Biology. The full
collection is available online at http://collections.plos.org/plosbiology/gos-2007.php.
0380 Special Section from March 2007 | Volume 5 | Issue 3 | e74
The 1982 United Nations Convention on the Law of the Sea
(UNCLOS) endowed coastal nations with the sovereign right
to explore and exploit all resources within their “exclusive
economic zone”—usually a body of water stretching 200
nautical miles out to sea [4]. Most coastal states exercise
this right, granting permits to outsiders wanting to conduct
research in their waters.
The 1992 Convention on Biological Diversity went on to
set out some basic principles that might encourage sharing
of benefits arising from genetic resources [5]. Where parties
to the convention have got round to incorporating these
principles into their own legislation, the result has been that
anyone wishing to conduct research on these resources must
agree to terms set by the host government.
Beyond national waters (with a few exceptions) are the
“high seas”. Here, there is little regulation. According
to UNCLOS, mineral resources on the deep seabed are
considered the “common heritage of mankind”; this means
that any benefits deriving from them should be shared with
the international community. But when it comes to biological
resources, just about anything goes.
The Rise of Bioprospecting
In areas beyond national jurisdiction, there has been an
increase in so-called bioprospecting, the search for and
exploitation of commercially valuable compounds from
genetic resources. In 2005, researchers at the United Nations
University scoured patent office databases for inventions
based on the genomic features of deep seabed organisms
[6].They found that private companies such as Roche,
Diversa, and New England Biolabs are after patents on DNA
polymerases developed from deep-sea thermophilic bacteria
that promise to enhance the molecular biologist’s expanding
toolbox. Others like Sederma (based in France) and
California Tan (based in the US) have used enzymes from
similar microorganisms to develop skin products boasting UV-
and heat-resistant properties.
There are plenty of not-for-profit organisations interested
in the applications of discoveries from the deep. For example,
doi:10.1371/journal.pbio.0050074.g002
Figure 2. A Remotely Operated Platform Samples Vent Fluids from
the Northeast Pacific Ocean
(Photo: NOAA, http://oceanexplorer.noaa.gov)
PLoS Biology | www.plosbiology.org | S10
Harbor Branch Oceanographic Institution, an oceanographic
research and education institution based in Florida, is
after compounds from marine organisms that might have
biomedical potential. The institution has patents on, among
others, potential anti-cancer agents derived from the marine
sponges Discodermia dissoluta and Forcepia triabilis (Figure 3).
Deep-sea exploration, and the lengthy research and
development that follows, is an expensive business. This
means it’s a realistic option for only the world’s wealthiest
nations. At least that’s the concern being expressed by some
developing countries that would like to see a piece of this
action, says David Leary of the Centre for Environmental Law
at Macquarie University in Sydney, Australia.
“No effort ever attempted to
incorporate data from such vastly
divergent sources to meet the needs
of such a wide range of scientific
interests.”
These countries are seeking a change to UNCLOS that
requires biological resources to be treated in the same
way as mineral resources and any benefits deriving from
them to be shared with the wider community. But others
fear tighter regulation of such activities will only stifle pure
marine scientific research. The Philippines was one of the
first countries to regulate access to its genetic resources, says
Sam Johnston, an expert on international environmental law
based in Melbourne, Australia, and a senior research fellow at
the United Nations University Institute of Advanced Studies
in Yokohama, Japan. “It basically closed down all research,”
he says. “A lot of researchers around the world have found
the red tape prohibitive.”
Finding a balance between the unregulated status quo and
cumbersome controls over research on marine biodiversity
is now the concern of a United Nations working group [7].
“Some countries see this as the early stage of negotiating a
new UNCLOS,” says Leary. But, he warns, “this could take 10
or 15 years before we see a result.”
One compromise might be for coastal states to allow all
research on their genetic resources with the proviso that
exploitation of any commercial application is subject to
further negotiation. Another possibility is for the patent
system to take responsibility for seeing that benefits are
shared fairly, only granting patents based on biological
resources if a royalty is paid into a global commons trust
fund.
Ecological Impact
Whilst the UN goes in search of this kind of middle ground,
both pure and applied research in the high seas continues
apace—and this is cause for another concern. “There’s a
number of sites that are so popular that there’s concern
about the intensity of research,” says Leary. Repeated visits to
the same deep-sea spot could not only result in unsustainable
collection of some species and influence local hydrological
and environmental conditions, but increase the likelihood
that one person’s experiment will influence that of another.
So far, little thought has been devoted to this consequence of
unregulated access, says Leary. “I haven’t yet seen any clear
0381 Special Section from March 2007 | Volume 5 | Issue 3 | e74

doi:10.1371/journal.pbio.0050074.g003
Figure 3. Marine Sponges That Have Generated Products with Anti-
Cancer Promise
(A) Discodermia dissoluta. (Photo: NOAA)
(B) Forcepia triabilis. (Photo: T. Piper, NOAA)
scientific data on the extent of the environmental impact of
bioprospecting or marine scientific research,” he says.
Clearly, the environmental impact of carrying off 150-odd
barrels of seawater for analysis isn’t something that Venter
and his colleagues had to worry about. But navigating the
complex legal territory was. “If Darwin were alive today trying
to do his experiments, he would not have been allowed to,”
says Venter.
At least, that is, without help from a lawyer. Sorcerer II
collected samples in the waters of 17 coastal states and
obtained all necessary permits, says Bob Friedman, Vice
President for Environmental and Energy Policy at the J.
Craig Venter Institute. Some countries required detailed
agreements thrashing out how benefits deriving from these
data would be shared. All of these are posted on the Sorcerer
II Web site, says Friedman [8]. Most countries, however, have
not decided how they might regulate access to their genetic
resources, he says.
In addition to getting the paperwork in order, Venter
encouraged collaboration with local scientists. What’s more,
the entire metagenomic database will be put in the public
domain. The gene sequences should be of tremendous value
to each of the countries involved, says Venter. In particular,
it will help them monitor and manage the health of their
marine ecosystems more effectively, he predicts. To ensure
that this vast dataset will be available to all, the Gordon and
Betty Moore Foundation has stumped up $24.5 million
dollars for a seven-year project to design a new database to
host it and new tools to interrogate it (Box 1).
Yet, it seems, all these undertakings and assurances
have not been enough to steer this expedition clear of
controversy. In 2004, when the Sorcerer II dropped anchor
PLoS Biology | www.plosbiology.org | S11
Box 1. Zooming in on CAMERA
CAMERA is the convenient acronym for the cumbersomely
named Community Cyberinfrastructure for Advanced Marine
Microbial Ecology Research and Analysis. “This resource
will focus on providing easy-to-use tools for uploading,
downloading, searching, and analysis of genomic datasets,” says
Paul Gilna, CAMERA’s executive director, based at the California
Institute for Telecom and Information Technology in La Jolla,
California.
Researchers will also be able to clothe the bare genetic
sequences in a wealth of other data, such as GPS coordinates
and depth of collection, the water temperature, its oxygen
content, salinity and pH. The site could well draw upon other
resources that enrich these metadata, says Gilna. For example,
satellite imagery associated with the sampling sites, and other
data types, such as microscopy stills and high-definition video,
could become important metadata that help researchers
characterise the environments from which samples were taken.
Crucially, CAMERA will allow researchers to record the source
of each genetic sequence. Many coastal countries now want a
share of commercial applications that derive from their marine
resources. Countries may be happy to see genetic sequences
placed in CAMERA provided they are acknowledged and
commercial exploitation of their sequence is not permitted
without their consent.
But handling such immense datasets poses considerable
technological challenges. The GOS database alone contains
around 6 billion bases—the equivalent of two entire human
genomes. And the number and size of this kind of database
will only mushroom in coming years, making it necessary to
develop high-speed optical networks, grid-based computing,
and new visualisation technologies. “We are quickly approaching
a ‘tipping point’,” says Gilna. “These datasets will start to follow
exponential, rather than linear trends, much as was the case for
DNA sequencing.”
Finally, there’s the tricky task of satisfying all researchers who
could benefit from this resource. “The scientific communities—
from studies on biodiversity and biogeochemistry to evolution
and genomes—have different interests, different data
expectations, different vocabularies, and different levels of
experience with using computational tools and databases,” says
John Wooley, a pharmacologist at the University of California,
San Diego, who is working on CAMERA. “Before metagenomics,
no effort ever attempted to incorporate data from such vastly
divergent sources to meet the needs of such a wide range of
scientific interests.” For more on CAMERA, see the Community
Page article by Seshadri et al. [13].
just off Hiva Oa, an island in the Marquesas archipelago in
the Pacific Ocean, tensions escalated. Although the plan to
sample seawater around the islands had the backing of local
French Polynesian authorities and scientists, the French
government in Paris had other ideas, says Venter. “We
were placed under house arrest.” Eventually, after a further
round of intense negotiations, the Sorcerer II was allowed out
of the harbour to collect its seawater samples and continue
on its way.
Last year, a Canadian-based non-governmental
organisation—the Action Group on Erosion, Technology and
Concentration—dedicated to “the advancement of cultural
0382 Special Section from March 2007 | Volume 5 | Issue 3 | e74
and ecological diversity and human rights” labelled Venter a
“biopirate”, accusing him of “flagrant disregard for national
sovereignty over biodiversity” [9]. In several countries, there’s
real concern about how he managed his collecting, claims
Pat Mooney, Executive Director of the group. Although the
data are going into the public domain, it is laboratories like
Venter’s that are best placed to exploit it, he argues. “There’s
a handful of folk around the planet that can understand such
stuff,” says Mooney.
Venter is adamant that this whole project is just pure, clean
marine scientific research. Indeed, the Sorcerer II Web site
explicitly states that “no intellectual property rights will be
sought by the Venter Institute on these genomic sequence
data” [10]. Venter sums up the goal of the project: “We were
just trying to answer some basic questions about the diversity
of microbes on the planet,” he says.
But, says environmental lawyer Johnston, the distinction
between pure and applied research is becoming increasingly
blurred. To illustrate this, he cites a strain of thermophilic
Bacillus collected from Antarctica in the early 1980s as part
of a study into the worldwide distribution and characteristics
of such extremophiles. Years later, the same sample, taken
out of storage and subjected to further study, turned out
to contain a talented enzyme that has the promise to
revolutionise DNA extraction for forensic analysis [11]. “The
collector undertook the act in the purest form but ultimately
the use of it has changed in the course of two decades,” says
Johnston. “So much depends on the perspective at which you
look at the issue.”
This means that there are likely to be several different
takes on the same research. What for one person is
pure marine scientific research can be another person’s
bioprospecting and yet another’s biopiracy. There are very
few cases where everyone agrees there has been outright
theft of a biological resource and very few cases where
everyone is happy there’s been proper benefit sharing,
says Johnston. “Even the best-designed programmes where
there’s enormous consultation with the local people have
found it’s difficult to get the right kind of consensus and
buy-in,” he says [12].
PLoS Biology | www.plosbiology.org | S12
So, keen as Venter might be to put the controversy of his
human-genome-sequencing days behind him, this kind of
research strays into unknown biological, legal, and ethical
territory. And in this environment, allegations of biopiracy
are almost inevitable. This, however, is unlikely to deter a
man like Venter. “If it’s in the Darwin school of biopiracy,
then fine,” he says. 
References
1. Sorcerer II Expedition (2005) Expedition info—Environmental genomics.
Available: http:⁄⁄www.sorcerer2expedition.org. Accessed 19 January 2007.
2. Gross L (2007) Untapped bounty: Sampling the seas to survey microbial
biodiversity. PLoS Biol 5: e85. doi:10.1371/journal.pbio.0050085
3. Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, et al. (2006)
Microbial diversity in the deep sea and the underexplored “rare biosphere”.
Proc Natl Acad Sci U S A 103: 12115–12120.
4. United Nations (1982) United Nations convention on the law of the sea of
10 December 1982. New York: United Nations. Available: http:⁄⁄www.un.
org/Depts/los/convention_agreements/convention_overview_convention.
htm. Accessed 18 January 2007.
5. Secretariat of the Convention on Biological Diversity (2002) Bonn
guidelines on access to genetic resources and fair and equitable sharing
of the benefits arising out of their utilization. Montreal: Secretariat of the
Convention on Biological Diversity. Available: https:⁄⁄www.biodiv.org/doc/
publications/cbd-bonn-gdls-en.pdf. Accessed 16 January 2007.
6. Arico S, Salpin C (2005) UNU-IAS report—Bioprospecting of genetic
resources in the deep seabed: Scientific, legal and policy aspects. Yokohama
(Japan): United Nations University Institute of Advanced Studies. Available:
http:⁄⁄www.ias.unu.edu/binaries2/DeepSeabed.pdf. Accessed 16 January
2007.
7. International Institute for Sustainable Development (2006) Ad Hoc Open-
ended Informal Working Group to study issues relating to the conservation
and sustainable use of marine biological diversity beyond areas of national
jurisdiction. Winnipeg (Canada): International Institute for Sustainable
Development. Available: http:⁄⁄www.iisd.ca/oceans/marinebiodiv. Accessed
16 January 2007.
8. Sorcerer II Expedition (2005) Collaborative agreements. Available:
http:⁄⁄www.sorcerer2expedition.org/permits. Accessed 22 January 2007.
9. Coalition Against Biopiracy (2006) Captain Hook awards for biopiracy
2006. Available: http:⁄⁄www.captainhookawards.org/winners/2006_pirates.
Accessed 18 January 2007.
10. Sorcerer II Expedition (2005) Agreements. Available: http:⁄⁄www.
sorcerer2expedition.org. Accessed 19 January 2007.
11. Moss D, Harbison AS, Saul DJ (2003) An easily automated, closed-tube
forensic DNA extraction procedure using a thermostable proteinase. Int J
Legal Med 117: 340–349.
12. Laird SA, Wynberg R, Johnston S (2006) Recent trends in the biological
prospecting. 29th Antarctic Treaty Consultative Meeting. Available:
http:⁄⁄www.ias.unu.edu/binaries2/ATCM29_May2006.doc. Accessed 16
January 2007.
13. Seshadri R, Kravitz SA, Smarr L, Gilna P, Frazier M (2007) CAMERA: A
community resource for metagenomics. PLoS Biol 5: e75. doi:10.1371/
journal.pbio.0050075
0383 Special Section from March 2007 | Volume 5 | Issue 3 | e74
Essay
Environmental Shotgun Sequencing:
Its Potential and Challenges for Studying
the Hidden World of Microbes
Jonathan A. Eisen
S
ince their discovery in the 1670s
by Anton van Leeuwenhoek,
an incredible amount has been
learned about microorganisms and
their importance to human health,
agriculture, industry, ecosystem
functioning, global biogeochemical
cycles, and the origin and evolution
of life. Nevertheless, it is what is not
known that is most astonishing. For
example, though there are certainly
at least 10 million species of bacteria,
only a few thousand have been formally
described [1]. This contrasts with the
more than 350,000 described species
of beetles [2]. This is one of many
examples indicative of the general
difficulties encountered in studying
organisms that we cannot readily see
or collect in large samples for future
analyses. It is thus not surprising that
most major advances in microbiology
can be traced to methodological
advances rather than scientific
discoveries per se.
Examples of these key revolutionary
methods (Table 1) include the use of
microscopes to view microbial cells,
the growth of single types of organisms
in the lab in isolation from other
types (culturing), the comparison
of ribosomal RNA (rRNA) genes to
construct the first tree of life that
included microbes [3], the use of the
polymerase chain reaction (PCR) [4]
to clone rRNA genes from organisms
Essays articulate a specific perspective on a topic of
broad interest to scientists.
PLoS Biology | www.plosbiology.org | S13
without culturing them [5–7], and
the use of high-throughput “shotgun”
methods to sequence the genomes of
cultured species [8]. We are now in the
midst of another such revolution—this
one driven by the use of genome
sequencing methods to study microbes
directly in their natural habitats, an
approach known as metagenomics,
environmental genomics, or
community genomics [9].
In this essay I focus on one
particularly promising area of
metagenomics—the use of shotgun
genome methods to sequence random
fragments of DNA from microbes
in an environmental sample. The
randomness and breadth of this
environmental shotgun sequencing
(ESS)—first used only a few years ago
[10,11] and now being used to assay
every microbial system imaginable
from the human gut [12] to waste
water sludge [13]—has the potential to
reveal novel and fundamental insights
into the hidden world of microbes and
their impact on our world. However,
the complexity of analysis required
to realize this potential poses unique
interdisciplinary challenges, challenges
that make the approach both
fascinating and frustrating in equal
measure.
Who Is Out There? Typing
and Counting Microbes in
the Environment
One of the most important and
conceptually straightforward steps
in studying any ecosystem involves
cataloging the types of organisms and
the numbers of each type. For a long
time, such typing and counting was
an almost insurmountable problem in
microbiology. This is largely because
physical appearance does not provide
a valid taxonomic picture in microbes.
Appearance evolves so rapidly that two
closely related taxa may look wildly
different and two distantly related
0384 Special Section from March 2007 | Volume 5 | Issue 3 | e82
taxa may look the same. This vexing
problem was partially overcome in
the 1980s through the use of rRNA-
PCR (Table 1). This method allows
microorganisms in a sample to be
phylogenetically typed and counted
based on the sequence of their rRNA
genes, genes that are present in all
cell-based organisms. In essence, a
database of rRNA sequences [14,15]
from known organisms functions
like a bird field guide, and finding a
rRNA-PCR product is akin to seeing a
bird through binoculars. Rather than
counting species, this approach focuses
on “phylotypes,” which are defined as
organisms whose rRNA sequences are
very similar to each other (a cutoff of
>97% or >99% identical is frequently
used). The ability to use phylotyping
to determine who was out there in any
microbial sample has revolutionized
environmental microbiology [16],
led to many discoveries [e.g.,17],
and convinced many people (myself
included) to become microbiologists.
Citation: Eisen JA (2007) Environmental shotgun
sequencing: Its potential and challenges for studying
the hidden world of microbes. PLoS Biol 5(3): e82.
doi:10.1371/journal.pbio.0050082
Series Editor: Simon Levin, Princeton University,
United States of America
Copyright: © 2007 Jonathan A. Eisen. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Abbreviations: ESS, environmental shotgun
sequencing; PCR, polymerase chain reaction; rRNA,
ribosomal RNA
Jonathan A. Eisen is at the University of California
Davis Genome Center, with joint appointments
in the Section of Evolution and Ecology and
the Department of Medical Microbiology and
Immunology, Davis, California, United States of
America. Web site: http://phylogenomics.blogspot.
com. E-mail: jaeisen@ucdavis.edu
This article is part of the Oceanic Metagenomics
collection in PLoS Biology. The full collection is
available online at http://collections.plos.org/
plosbiology/gos-2007.php.
Table 1. Some Major Methods for Studying Individual Microbes Found in the Environment
Method Summary Comments

Microscopy Microbial phenotypes can be studied by making them more visible. In conjunction
with other methods, such as staining, microscopy can also be used to count taxa
and make inferences about biological processes.
Culturing Single cells of a particular microbial type are grown in isolation from other
organisms. This can be done in liquid or solid growth media.
rRNA-PCR The key aspects of this method are the following: (a) all cell-based organisms
possess the same rRNA genes (albeit with different underlying sequences); (b) PCR
is used to make billions of copies of basically each and every rRNA gene present in
a sample; this amplifies the rRNA signal relative to the noise of thousands of other
genes present in each organism’s DNA; (c) sequencing and phylogenetic analysis
places rRNA genes on the rRNA tree of life; the position on the tree is used to infer
what type of organism (a.k.a. phylotype) the gene came from; and (d) the numbers
of each microbe type are estimated from the number of times the same rRNA gene
is seen.
Shotgun genome The DNA from an organism is isolated and broken into small fragments, and then
sequencing of cultured portions of these fragments are sequenced, usually with the aid of sequencing
species machines. The fragments are then assembled into larger pieces by looking
for overlaps in the sequence each possesses. The complete genome can be
determined by filling in gaps between the larger pieces.
Metagenomics DNA is directly isolated from an environmental sample and then sequenced.
One approach to doing this is to select particular pieces of interest (e.g., those
containing interesting rRNA genes) and sequence them. An alternative is ESS,
which is shotgun genome sequencing as described above, but applied to an
environmental sample with multiple organisms, rather than to a single cultured
organism.
The appearance of microbes is not a reliable indicator of
what type of microbe one is looking at.
This is the best way to learn about the biology of a
particular organism. However, many microbes are
uncultured (i.e., have never been grown in the lab in
isolation from other organisms) and may be unculturable
(i.e., may not be able to grow without other organisms).
This method revolutionized microbiology in the 1980s by
allowing the types and numbers of microbes present in
a sample to be rapidly characterized. However, there are
some biases in the process that make it not perfect for all
aspects of typing and counting.
This has now been applied to over 1,000 microbes, as well
as some multicellular species, and has provided a much
deeper understanding of the biology and evolution of life.
One limitation is that each genome sequence is usually a
snapshot of one or a few individuals.
This method allows one to sample the genomes of
microbes without culturing them. It can be used both for
typing and counting taxa and for making predictions of
their biological functions.

doi:10.1371/journal.pbio.0050082.t001

The selective targeting of a single
gene makes rRNA-PCR an efficient
method for deep community sampling
[18]. However, this efficiency comes
with limitations, most of which are
complemented or circumvented by the
randomness and breadth of ESS. For
example, examination of the random
samples of rRNA sequences obtained
through ESS has already led to the
discovery of new taxa—taxa that were
completely missed by PCR because of
its inability to sample all taxa equally
well (e.g., [19]). In addition, ESS
provides the first robust sampling of
genes other than rRNA, and many of
these genes can be more useful for
some aspects of typing and counting.
Some universal protein coding
genes are better than rRNA both for
distinguishing closely related strains
(because of third position variation in
codons) and for estimating numbers
of individuals (because they vary less
in copy number between species
than do rRNA genes) [10]. Perhaps
most significantly, ESS is providing
groundbreaking insights into the
diversity of viruses [20,21], which lack
rRNA genes and thus were left out of
the previous revolution.
Certainly, many challenges remain
before we can fully realize the potential
of ESS for the typing and counting of
species, including making automated
yet accurate phylogenetic trees of every
gene, determining which genes are
most useful for which taxa, combining
data from different genes even when
we do not know if they come from
the same organisms, building up
databases of genes other than rRNA,
and making up for the lack of depth of
sampling. If these challenges are met,
ESS has the potential to rewrite much
of what we thought we knew about the
phylogenetic diversity of microbial life.
What Are They Doing? Top Down
and Bottom Up Approaches to
Understanding Functions in
Communities
A community is, of course, more
than a list of types of organisms.
One approach to understanding
the properties and functioning of
a microbial community is to start
with studies of the different types of
organisms and build up from these
individuals to the community. Ideally,
to do this one would culture each of
the phylotypes and study its properties
in the lab. Unfortunately, many, if
not most, key microbes have not yet
been cultured [22]. Thus, for many
years, the only alternative was to
make predictions about the biology of
particular phylotypes based on what
was known about related organisms.
Unfortunately, this too does not work
well for microbes since very closely
related organisms frequently have
major biological differences. For
example, Escherichia coli K12 and E.
coli O157:H7 are strains of the same
species (and considered to be the same
phylotype), with genomes containing
only about 4,000 genes, yet each
possesses hundreds of functionally
important genes not seen in the
other strain [23]. Such differences
are routine in microbes, and thus one
cannot make any useful inferences
about what particular phylotypes are
doing (e.g., type of metabolism, growth
properties, role in nutrient cycling, or
pathogenicity) based on the activities of
their relatives.
These difficulties—the inability
to culture most microbes and the
functional disparities between close
relatives—led to one of the first kinds

PLoS Biology | www.plosbiology.org | S14 0385 Special Section from March 2007 | Volume 5 | Issue 3 | e82
Table 2. Methods of Binning
Method Description Comments

Genome assembly
Reference genome alignment
Phylogenetic analysis
Word frequency and nucleotide
composition analysis
Population genetics
Identify regions of overlap between different fragments
from the same organism to build larger contiguous pieces
(contigs).
Identify ESS fragments or contigs that are very similar
to already assembled sections of the genome of single
microbial types.
Build evolutionary trees of genes encoded by ESS fragments (a) Very powerful, but level of resolution depends on whether
or contigs. Assign fragments or contigs to taxonomic
groups based on nearest neighbor(s) in trees.
Measure word frequency and composition of each
fragment. Group by clustering algorithms or principal
component analysis.
Build alignments of fragments or contigs with similarity
to each other (but not as much as needed for assembly).
Examine haplotype structure, predicted effective
population size, and synonymous and non synonymous
substitution patterns.
Getting deep enough sampling for this to work is very expensive
except for low diversity systems or for very abundant taxa.
(a) One of the most effective ways to sort through ESS data, if the
reference genome is very closely related to an organism in the sample;
(b) the reason why more reference genomes are needed; (c) does
not handle regions present in uncultured organisms but not in the
reference.
fragments encode useful phylogenetic markers and on how well
sampled the database is for the neighbor analysis; (b) would work
much better if more genomes were available from across the tree of
life.
(a) Has the potential to work because organisms sometimes have
“signatures” of word frequencies that are found throughout the
genome and are different between species; (b) very challenging for
small fragments.
May be most useful as a way of subdividing bins created by other
methods.

Note that some methods can be applied to ESS fragments or to bins identified by other methods.
doi:10.1371/journal.pbio.0050082.t002

of metagenomic analyses, wherein
predictions of function were made
from analysis of the sequence of large
DNA fragments from representatives
of known phylotypes. This approach
has provided some stunning insights,
such as the discovery of a novel form
of phototrophy in the oceans [24].
However, this large insert approach
has the same limitation as predicting
properties from characterized
relatives—a single cell cannot possibly
represent the biological functions of all
members of a phylotype.
ESS provides an alternative, more
global way of assessing biological
functions in microbial communities. As
when using the large insert approach,
functions can be predicted from
sequences. However, in this case the
predicted functions represent a random
sampling of those encoded in the
genomes of all the organisms present.
This approach has unquestionably
been wildly successful in terms of gene
discovery. For example, analysis of
ESS data has revealed novel forms of
every type of gene family examined, as
well as a great number of completely
novel families (e.g., [25]). However,
there is a major caveat when using
ESS data to make community-level
inferences. Ecosystems are more than
just a bag of genes—they are made up of
compartments (e.g., cells, chromosomes,
and species), and these compartments
matter. The key challenge in analyzing
ESS data is to sort the DNA fragments
(which are usually less than 1,000 base
pairs long relative to genome sizes of
millions or billions of bases) into bins
that correspond to compartments in the
system being studied.
A recent study by myself and
colleagues illustrates the importance
of compartments when interpreting
ESS data. When we analyzed ESS data
from symbionts living inside the gut
of the glassy-winged sharpshooter (an
insect that has a nutrient-limited diet),
we were able to bin the data to two
distinct symbionts [26]. We then could
infer from those data that one of the
symbionts synthesizes amino acids for
the host while the other synthesizes
the needed vitamins and cofactors.
Modeling and understanding of this
ecosystem are greatly enhanced by the
demonstration of this complementary
division of labor, in comparison to
simply knowing that amino acids,
vitamins, and cofactors are made by
“symbionts.”
How does one go about binning
ESS data? A variety of approaches have
been developed, some of which are
described in Table 2. In considering
the different binning methods and
their limitations, the first question
one needs to ask is, what are we
trying to bin? Is it fragments from the
same chromosome from a single cell,
which would be useful for studying
chromosome structure? If so, then
perhaps genome assembly methods
are the best. What if instead, as in the
sharpshooter example, we are trying to
have each bin include every fragment
that came from a particular species,
knowledge which may be useful for
predicting community metabolic
potential? If the level of genetic
polymorphism among individual
cells from the same species is high,
then genome assembly methods may
not work well (the polymorphisms
will break up assemblies). A better
approach might be to look for species-
specific “word” frequencies in the
DNA, such as ones created by patterns
in codon usage. The challenge is, how
do we tune the methods to find the
right target level of resolution? If we
are too stringent, most bins will include
only a few fragments. But if we are
too relaxed, we will create artificial
constructs that may prove biologically
misleading, such as grouping together
sequences from different species. To
make matters more complex, most
likely the stringency needed will vary
for different taxa present in the sample.
Another critical issue is the diversity
of the system under study. Generally,
binning works better when there are

PLoS Biology | www.plosbiology.org | S15 0386 Special Section from March 2007 | Volume 5 | Issue 3 | e82
few different phylotypes present, all
of which are distantly related and
form discrete populations. This is why
binning works well for the sharpshooter
system and other relatively isolated,
low diversity environments. Binning
increases in difficulty exponentially
as the number of species increases:
the populations and species start to
merge together, and the populations
get more and more polymorphic and
variable in relative abundance (such as
in the paper about the Global Ocean
Sampling expedition in this issue [27]).
Further complicating binning is the
phenomenon of lateral gene transfer,
where genes are exchanged between
distantly related lineages at rates that
are high enough that random sampling
of a genome will frequently include
genes with multiple histories.
Despite these challenges, I believe we
can develop effective binning methods
for complex communities. First, we
can combine different approaches
together, such as using one method
to sort in a relaxed manner and then
using another to subdivide the bins
provided by the first method. Second,
we can incorporate new approaches
such as population genetics into
the analysis [28]. In addition, the
lessons learned here can be applied to
other aspects of metagenomics (e.g.,
the counting and typing discussed
above) and provide insights into the
nature of microbial genomes and the
structure of microbial populations and
communities.
Comparative Metagenomics
So far, I have discussed issues relating
mostly to intrasample analysis of
ESS data. However, the area with
perhaps the most promise involves
the comparative analysis of different
samples. This work parallels the
comparative analysis of genomes of
cultured species. Initial studies of
that type compared distantly related
taxa with enormous biological
differences. What has been learned
from these studies pertains mostly to
core housekeeping functions, such
as translation and DNA metabolism,
and to other very ancient processes
[29,30]. It was not until comparisons
were made between closely related
organisms that we began to understand
events that occurred on shorter
time scales, such as selection, gene
transfer, and mutation processes [31].
PLoS Biology | www.plosbiology.org | S16
Similarly, the initial comparisons of
ESS data involved comparisons of wildly
different environments [32], yielding
insights into the general structure of
communities. But as more comparisons
are made between similar communities
[33,34], such as those sampled during
vertical and horizontal ocean transects
[27,35–37], we will begin to learn
about shorter time scale processes such
as migration, speciation, extinction,
responses to disturbance, and
succession. It is from a combination
of both approaches—comparing
both similar and very divergent
communities—that we will be able to
understand the fundamental rules of
microbial ecology and how they relate
to ecological principles seen in macro-
organisms.
Conclusions
In promoting some of the exciting
opportunities with ESS, I do not
want to give the impression that it is
flawless. It is helpful in this respect to
compare ESS to the Internet. As with
the Internet, ESS is a global portal for
looking at what occurs in a previously
hidden world. Making sense of it
requires one to sort through massive,
random, fragmented collections of bits
of information. Such searches need
to be done with caution because any
time you analyze such a large amount
of data patterns can be found. In
addition, as with the Internet, there
is certainly some hype associated with
ESS that gives relatively trivial findings
more attention than they deserve.
Overall, though, I believe the hype
is deserved. As long as we treat ESS
as a strong complement to existing
methods, and we build the tools and
databases necessary for people to use
the information, it will live up to its
revolutionary potential. 
Acknowledgments
I thank Simon Levin, Joshua Weitz,
Jonathan Dushoff, Maria-Inés Benito,
Doug Rusch, Aaron Halpern, and Shibu
Yooseph for helpful discussions, and
Melinda Simmons, Merry Youle, and three
anonymous reviewers for helpful comments
on the manuscript. The writing of this
paper was supported by National Science
Foundation Assembling the Tree of Life
Grant 0228651 to Jonathan A. Eisen and by
the Defense Advanced Research Projects
Agency under grants HR0011-05-1-0057
and FA9550-06-1-0478.
0387 Special Section from March 2007 | Volume 5 | Issue 3 | e82
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Community Page
CAMERA: A Community Resource
for Metagenomics
Rekha Seshadri*, Saul A. Kravitz, Larry Smarr, Paul Gilna, Marvin Frazier
M
icrobes are responsible
for most of the chemical
transformations that are
crucial to sustaining life on Earth.
Their ability to inhabit almost any
environmental niche suggests that
they possess an incredible diversity of
physiological capabilities. However,
we have little to no information on a
majority of the millions of microbial
species that are predicted to exist,
mainly because of our inability to
culture them in the laboratory.
A growing discipline called
metagenomics allows us to study these
uncultured organisms by deciphering
their genetic information from
DNA that is extracted directly from
their environment, thus effectively
bypassing the laboratory culture step.
Metagenomics allows us to address the
questions “who’s there?”, “what are they
doing?”, and “how are they doing it?”,
offering insights into the evolutionary
history as well as previously
unrecognized physiological abilities of
uncultured communities.
Studies such as the J. Craig Venter
Institute’s Global Ocean Sampling
(GOS) expedition (in this issue) reveal
a remarkable breadth and depth of
microbial diversity in the oceans. To
date, researchers have made significant
but largely preliminary inroads into
understanding the biogeography
of microbial populations across
ecosystems. We know even less about
the dynamic physiological processes
The Community Page is a forum for organizations
and societies to highlight their efforts to enhance the
dissemination and value of scientific knowledge.
PLoS Biology | www.plosbiology.org | S18
and complex interactions that impact
global carbon cycles and ocean
productivity. Marine microbes are
thought to act as part of the biological
conduit that transports carbon dioxide
from the surface to the deep oceanic
realms. By removing carbon from the
atmosphere and sequestering it (in
the form of organic matter), marine
microorganisms may significantly
affect global climate. Although we
now have numerous global and real-
time methods to measure physical
We invite the research
community to submit its
metagenomics data to
CAMERA.
and chemical parameters within the
ocean, few methods or concepts have
been developed to measure important
microbial processes on a global scale.
Even if the technology to make such
measurements existed, we would
presently not know what to measure or
how to interpret those measurements.
We need a systematic way to explore
the structure and function of ocean
ecosystems, and their impact on
global carbon processing and climate.
Metagenomics has the potential to
shed light on the genetic controls
of these processes by investigating
the key players, their roles, and
community compositions that may
change as a function of time, climate,
nutrients, carbon dioxide, and
anthropogenic factors. These studies
include a substantial informatics
component, requiring researchers to
take on complex computational and
mathematical challenges. Nonetheless,
microbiologists have been quick to seize
upon this modern technique, resulting
in a deluge of sequence data, and an
ever-widening gap between the rates of
collecting data and interpreting it.
The Community Cyberinfrastructure
for Advanced Marine Microbial
Ecology Research and Analysis
0394 Special Section from March 2007 | Volume 5 | Issue 3 | e75
(CAMERA) project [1] is an important
first step in attempting to bridge
these gaps and in developing global
methods for monitoring microbial
communities in the ocean and their
response to environmental changes.
The aim is to create a rich, distinctive
data repository and bioinformatics
tools resource that will address
many of the unique challenges of
metagenomics and enable researchers
to unravel the biology of environmental
microorganisms (Figure 1). CAMERA’s
database includes environmental
metagenomic and genomic sequence
data, associated environmental
parameters (“metadata”), pre-
computed search results, and software
tools to support powerful cross-analysis
of environmental samples.
Citation: Seshadri R, Kravitz SA, Smarr L, Gilna P,
Frazier M (2007) CAMERA: A community resource
for metagenomics. PLoS Biol 5(3): e75. doi:10.1371/
journal.pbio.0050075
Copyright: © 2007 Seshadri et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Abbreviations: CAMERA, Community
Cyberinfrastructure for Advanced Marine Microbial
Ecology Research and Analysis; GOS, Global Ocean
Sampling
Rekha Seshadri, Saul A. Kravitz, and Marvin Frazier
are at the J. Craig Venter Institute (JCVI) in Rockville,
Maryland, United States of America. Larry Smarr
and Paul Gilna are at the California Institute for
Telecommunications and Information Technology
(Calit2), a University of California San Diego
(UCSD)/University of California Irvine partnership,
La Jolla, California, United States of America. Larry
Smarr is also the Harry E. Gruber Professor of
Computer Science and Engineering at UCSD, La Jolla,
California, United States of America. CAMERA is being
developed by Calit2 at UCSD in collaboration with
the JCVI, UCSD’s Center for Earth Observations and
Applications (anchored by the Scripps Institution
of Oceanography), the San Diego Supercomputer
Center, and the University of California Davis.
* To whom correspondence should be addressed.
E-mail: rseshadri@venterinstitute.org
This article is part of the Oceanic Metagenomics
collection in PLoS Biology. The full collection is
available online at http://collections.plos.org/
plosbiology/gos-2007.php.
 The initial release will include
data and tools associated with the
companion set of GOS expedition
publications [2–4]; metagenome data
from the Hawaii Ocean Time Series
Station ALOHA [5] and marine
viromes from four different oceanic
regions[6]; standard nonredundant
sequence databases (e.g., nrnt for
nucleotides and nraa for amino
acids[7]); and collections of microbial
genome sequences, including a set
of 155 marine microbial genomes
funded by the Gordon and Betty
Moore Foundation. The focal point
for the CAMERA project is its Web
site: http://camera.calit2.net. We
invite the research community to
submit its metagenomics data to
CAMERA, and are establishing
mechanisms to streamline this
process. Here we describe some of
the key challenges and features of the
CAMERA project.
Accessibility of Metadata
Existing data repositories provide
limited support for metadata and
metadata-based queries—including
any supplemental information
for the sequence data, such as pH
and temperature of water at the
collection site—and therefore these
metadata go underutilized by the
research community. CAMERA
will integrate sequence data with
all available, relevant metadata,
including physical information (e.g.,
temperature and sample method),
chemical information (e.g., salinity
and pH), temporal information,
geospatial information, methodology
and instrumentation used for data
collection, and satellite images of
the collection site. These contextual
data allow researchers to derive
correlations between deciphered
ecology and the environmental
conditions that may favor one
community structure over another.
One can envision a future where
metadata from satellites and weather
stations, and other physicochemical
data, can be used to help interpret and
inform scientists on how these factors
affect microbial processes as well as
community composition. CAMERA
is working with other groups (e.g.,
Genome Standards Consortium) to
establish standards for the information
content and format of metagenomic
data and metadata submissions.
PLoS Biology | www.plosbiology.org | S19
doi:10.1371/journal.pbio.0050075.g001
Figure 1. Schematic of Intended Core
Functions of the CAMERA Project
CBD, Convention on Biological Diversity.
New-Generation Bioinformatics
Tools
Analysis and comparison of complex
metagenomic data is driving the
development of a new class of
bioinformatics and visualization
software. CAMERA will integrate these
tools with its database, couple them
with large-scale compute resources,
and make them widely available to
the research community. Initially,
CAMERA will support analytical
tools used for analyses in the GOS
publications [2–6]. An example
is shown in Figure 2: a subset of
metagenome sequence reads from
GOS environmental samples is
compared to a reference genome
sequence (Synechococcus spp.) using
BLASTN. The results and underlying
metadata are displayed through an
interactive graphical viewer, which
helps users quickly identify sequence
reads that are similar to the reference
genome sequence, and potentially
identify metabolic similarities between
microbes in environmental samples
and a reference microbe. A detailed
description of this tool and its
applications are provided in the GOS
companion paper by Rusch et al. [2].
CAMERA will work closely with the
community to identify and incorporate
additional tools and workflows.
Large-Scale, Robust, and
Expandable Cyberinfrastructure
The enormousness of metagenomics
datasets requires terascale
computation and storage facilities.
CAMERA is building a state-of-the-art
0395
computational infrastructure to provide
high-performance networking access
and grid-based computing (applying
the resources of many computers in
a network to a single problem at the
same time), and to support new ways
of visualizing and interacting with the
data. The distributed architecture of
the CAMERA computational engine
will be based on the National Science
Foundation–funded OptIPuter
project [8,9], which allows for use of
dedicated 1- or 10-Gbps optical fiber
links between remote user laboratory
clusters and the CAMERA compute
complex. The data server complex
itself will contain a large amount of
rotating storage (ultimately several
tens of terabytes replicated) and a
large computational cluster (upwards
of a thousand processors). It will be
augmented on demand by a scalable
back end provided by the recently
upgraded National Science Foundation
TeraGrid.
Recognition of the Sources
of Samples
The Convention on Biological Diversity
grants countries certain rights over
their genetic resources, including,
for example, metagenomic sequence
data of marine microbes taken from
a country’s territorial waters. Many
countries require, at minimum, that
databases explicitly identify the country
of origin of the DNA. Rules vary by
country, and it is not a simple task
to find out what might be required.
International harmonization of these
rules is currently being debated by the
over 150 countries that are party to the
Convention on Biological Diversity.
Agreements about the use of genetic
resources are negotiated on a case-by-
case basis with each researcher who
wishes to sample within a country’s
“exclusive economic zone,” typically
200 miles from its shoreline. Some of
these “memoranda of understanding”
impose additional requirements on
the researchers. For example, the J.
Craig Venter Institute’s agreement
with Australia requires us to “use
reasonable effort to notify Australia as
soon as possible of any inquiries for
commercial purposes.”
Current databases do not allow the
original investigators to inform others
about the details of an agreement,
thus creating a significant roadblock to
both the collection and public release
Special Section from March 2007 | Volume 5 | Issue 3 | e75
doi:10.1371/journal.pbio.0050075.g002

Figure 2. CAMERA Fragment Recruitment Viewer

This tool graphically displays the results of a BLASTN sequence comparison of an available microbial genome against selected sequence read datasets. The
example shown displays the abundance and distribution of Synechococcus spp. genome sequence in the selected sampling sites. The Synechococcus spp.
genome coordinates are shown on the x-axis, while the y-axis shows the percent identity scores of the alignment to the selected Sargasso Sea and GOS
sequence reads. The viewer incorporates metadata associated with the reads, allowing a user to quickly identify data of interest for further examination.
The utility of the plot is to examine the biogeography and genomic variation of abundant microbes when a close reference genome exists.

of metagenomics data. To address and who acknowledge the potential Convention on Biological Diversity, all
this issue, CAMERA data will only be restriction on commercial use by data objects served by CAMERA will
made available to users who register countries from which the data were possess a mapping to the country of
by supplying a suitable E-mail address collected. To further comply with the origin of the underlying DNA sample.

PLoS Biology | www.plosbiology.org | S20 0396 Special Section from March 2007 | Volume 5 | Issue 3 | e75
Outreach and Training
Since the ultimate success of CAMERA
will depend on the broader research
community’s ability to make use of the
novel cyberinfrastructure, a series of on-
site and Web-based training programs
will be provided to keep users apprised
of CAMERA’s functionalities and to
support integration of CAMERA’s
service-oriented architecture into
their computational fabrics. Finally,
we envision interacting with the
community on several fronts, including
standardization of ontology, metadata,
nomenclature, and tools, and
incorporation or federation of existing
tools and resources with CAMERA.
We believe that the data and
community cyberinfrastructure
provided by CAMERA will help
researchers to advance understanding
of the codependence or feedback
between microbial communities
and biogeochemical processes
in oceans over time, and of how
perturbations in the environment
cause compositional changes
(including extinction). Eventually,
the expanded global environmental
metagenomics datasets will enable
better monitoring of environmental
PLoS Biology | www.plosbiology.org | S21
change and the processes that control
climate. Systematic and routine
monitoring of genomic signatures
of global microbial populations and
processes overlaid with meteorological
information and other metadata may
help researchers explain past shifts in
global climate as well as predict future
changes. This knowledge may someday
guide decisions about acceptable
atmospheric levels of greenhouse
gases, or guide strategies to increase
sequestration of atmospheric carbon
dioxide by changing ocean microbial
compositions, in order to reverse the
effects of global warming. 
Acknowledgments
The authors benefited from many
discussions with members of the CAMERA
team. We wish to thank Robert Friedman,
Michael Press, Jasmine Pollard, and
Matthew LaPointe at the J. Craig Venter
Institute for their assistance in preparing
the manuscript.
Funding. The authors acknowledge
funding from the Gordon and Betty Moore
Foundation to the California Institute for
Telecommunications and Information
Technology at the University of California,
San Diego, and from National Science
Foundation OptIPuter grant SCI-0225642.
0397 Special Section from March 2007 | Volume 5 | Issue 3 | e75
References
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KB, Williamson S, et al. (2007) The Sorcerer II
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Atlantic through eastern tropical Pacific.
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pbio.0050077
3. Yooseph S, Sutton G, Rusch DB, Halpern AL,
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6. Angly FE, Felts B, Breitbart M, Salamon P,
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Papadopoulos PM (2003) The OptIPuter.
Commun ACM 46: 58–67.
9. Taesombut N, Uyeda F, Chien AA, Smarr L,
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for emerging e-science applications. IEEE
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 PLoS BIOLOGY

The Sorcerer II Global Ocean Sampling

Expedition: Northwest Atlantic through
Eastern Tropical Pacific
Douglas B. Rusch1*, Aaron L. Halpern1, Granger Sutton1, Karla B. Heidelberg1,2, Shannon Williamson1, Shibu Yooseph1,
Dongying Wu1,3, Jonathan A. Eisen1,3, Jeff M. Hoffman1, Karin Remington1,4, Karen Beeson1, Bao Tran1,
Hamilton Smith1, Holly Baden-Tillson1, Clare Stewart1, Joyce Thorpe1, Jason Freeman1, Cynthia Andrews-Pfannkoch1,
Joseph E. Venter1, Kelvin Li1, Saul Kravitz1, John F. Heidelberg1,2, Terry Utterback1, Yu-Hui Rogers1, Luisa I. Falcón5,
Valeria Souza5, Germán Bonilla-Rosso5, Luis E. Eguiarte5, David M. Karl6, Shubha Sathyendranath7, Trevor Platt7,
Eldredge Bermingham8, Victor Gallardo9, Giselle Tamayo-Castillo10, Michael R. Ferrari11, Robert L. Strausberg1,
Kenneth Nealson1,12, Robert Friedman1, Marvin Frazier1, J. Craig Venter1
1 J. Craig Venter Institute, Rockville, Maryland, United States of America, 2 Department of Biological Sciences, University of Southern California, Avalon, California, United
States of America, 3 Genome Center, University of California Davis, Davis, California, United States of America, 4 Your Genome, Your World, Rockville, Maryland, United States
of America, 5 Departmento de Ecologı́a Evolutiva, Instituto de Ecologı́a, Universidad Nacional Autónoma de México, Mexico City, Mexico, 6 Department of Oceanography,
University of Hawaii, Honolulu, Hawaii, United States of America, 7 Bedford Institute of Oceanography, Dartmouth, Nova Scotia, Canada, 8 Smithsonian Tropical Research
Institute, Balboa, Ancon, Republic of Panama, 9 Departamento de Oceanografı́a, Universidad de Concepción, Concepción, Chile, 10 Escuela de Quı́mica, Universidad de Costa
Rica, San Pedro, Costa Rica, 11 Department of Environmental Sciences, Rutgers University, New Brunswick, New Jersey, United States of America, 12 Department of Earth
Sciences, University of Southern California, Los Angles, California, United States of America

The world’s oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both
genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which
surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition.
These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and
ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp).
Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with
85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff.
Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and
assembly methods. One comparative genomic method, termed ‘‘fragment recruitment,’’ addressed questions of
genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes
and gene families. A second method, termed ‘‘extreme assembly,’’ made possible the assembly and reconstruction of
large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found
extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions
throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual
sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3)
hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into
genetically isolated populations that have overlapping but independent distributions, implying distinct environmental
preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show
how they may be grouped into several community types. Specific functional adaptations can be identified both within
individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or
absence of the phosphate-binding gene PstS.
Citation: Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al. (2007) The Sorcerer II Global Ocean Sampling expedition: Northwest Atlantic through eastern
tropical Pacific. PLoS Biol 5(3): e77. doi:10.1371/journal.pbio.0050077
Academic Editor: Nancy A. Moran, University of Arizona, United States of America
Received July 14, 2006; Accepted January 16, 2007; Published March 13, 2007
Copyright: Ó 2007 Rusch et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: CAMERA, Cyberinfrastructure for Advanced Marine Microbial
Ecology Research and Analysis; GOS, Global Ocean Sampling; NCBI, National
Center for Biotechnology Information
* To whom correspondence should be addressed. E-mail: DRusch@venterinstitute.
org
This article is part of Global Ocean Sampling collection in PLoS Biology. The full
collection is available online at http://collections.plos.org/plosbiology/gos-2007.
php.

PLoS Biology | www.plosbiology.org | S22 0398 Special Section from March 2007 | Volume 5 | Issue 3 | e77

Author Summary
Marine microbes remain elusive and mysterious, even though they
are the most abundant life form in the ocean, form the base of the
marine food web, and drive energy and nutrient cycling. We know
so little about the vast majority of microbes because only a small
percentage can be cultivated and studied in the lab. Here we report
on the Global Ocean Sampling expedition, an environmental
metagenomics project that aims to shed light on the role of marine
microbes by sequencing their DNA without first needing to isolate
individual organisms. A total of 41 different samples were taken
from a wide variety of aquatic habitats collected over 8,000 km. The
resulting 7.7 million sequencing reads provide an unprecedented
look at the incredible diversity and heterogeneity in naturally
occurring microbial populations. We have developed new bioinfor-
matic methods to reconstitute large portions of both cultured and
uncultured microbial genomes. Organism diversity is analyzed in
relation to sampling locations and environmental pressures. Taken
together, these data and analyses serve as a foundation for greatly
expanding our understanding of individual microbial lineages and
their evolution, the nature of marine microbial communities, and
how they are impacted by and impact our world.
Introduction
The concept of microbial diversity is not well defined. It
can either refer to the genetic (taxonomic or phylogenetic)
diversity as commonly measured by molecular genetics
methods, or to the biochemical (physiological) diversity
measured in the laboratory with pure or mixed cultures.
However, we know surprisingly little about either the genetic
or biochemical diversity of the microbial world [1], in part
because so few microbes have been grown under laboratory
conditions [2,3], and also because it is likely that there are
immense numbers of low abundance ribotypes that have not
been detected using molecular methods [4]. Our under-
standing of microbial physiological and biochemical diversity
has come from studying the less than 1% of organisms that
can be maintained in enrichments or cultivated, while our
understanding of phylogenetic diversity has come from the
application of molecular techniques that are limited in terms
of identifying low-abundance members of the communities.
Historically, there was little distinction between genetic
and biochemical diversity because our understanding of
genetic diversity was based on the study of cultivated
microbes. Biochemical diversity, along with a few morpho-
logical features, was used to establish genetic diversity via an
approach called numerical taxonomy [5,6]. In recent years the
situation has dramatically changed. The determination of
genetic diversity has relied almost entirely on the use of gene
amplification via PCR to conduct taxonomic environmental
gene surveys. This approach requires the presence of slowly
evolving, highly conserved genes that are found in otherwise
very diverse organisms. For example, the gene encoding the
small ribosomal subunit RNA, known as 16S, based on
sedimentation coefficient, is most often used for distinguish-
ing bacterial and archaeal species [7–10]. The 16S rRNA
sequences are highly conserved and can be used as a
phylogenetic marker to classify organisms and place them
in evolutionary context. Organisms whose 16S sequences are
at least 97% identical are commonly considered to be the
PLoS Biology | www.plosbiology.org | S23
Sorcerer II GOS Expedition
same ribotype [11], otherwise referred to as species, opera-
tional taxonomic units, or phylotypes.
Although rRNA-based analysis has revolutionized our view
of genetic diversity, and has allowed the analysis of a large
part of the uncultivated majority, it has been less useful in
predicting biochemical diversity. Furthermore, the relation-
ship between genetic and biochemical diversity, even for
cultivated microbes, is not always predictable or clear. For
instance, organisms that have very similar ribotypes (97% or
greater homology) may have vast differences in physiology,
biochemistry, and genome content. For example, the gene
complement of Escherichia coli O157:H7 was found to be
substantially different from the K12 strain of the same species
[12].
In this paper, we report the results of the first phase of the
Sorcerer II Global Ocean Sampling (GOS) expedition, a
metagenomic study designed to address questions related to
genetic and biochemical microbial diversity. This survey was
inspired by the British Challenger expedition that took place
from 1872–1876, in which the diversity of macroscopic
marine life was documented from dredged bottom samples
approximately every 200 miles on a circumnavigation [13–15].
Through the substantial dataset described here, we identified
60 highly abundant ribotypes associated with the open ocean
and aquatic samples. Despite this relative lack of diversity in
ribotype content, we confirm and expand upon previous
observations that there is tremendous within-ribotype diver-
sity in marine microbial populations [4,7,8,16,17]. New
techniques and tools were developed to make use of the
sampling and sequencing metadata. These tools include: (1)
the fragment recruitment tool for performing and visualizing
comparative genomic analyses when a reference sequence is
available; (2) new assembly techniques that use metadata to
produce assemblies for uncultivated abundant microbial taxa;
and (3) a whole metagenome comparison tool to compare
entire samples at arbitrary degrees of genetic divergence.
Although there is tremendous diversity within cultivated and
uncultivated microbes alike, this diversity is organized into
phylogenetically distinct groups we refer to as subtypes.
Subtypes can occupy similar environments yet remain
genetically isolated from each other, suggesting that they are
adapted for different environmental conditions or roles
within the community. The variation between and within
subtypes consists primarily of nucleotide polymorphisms but
includes numerous small insertions, deletions, and hyper-
variable segments. Examination of the GOS data in these
terms sheds light on patterns of evolution and also suggests
approaches towards improving the assembly of complex
metagenomic datasets. At least some of this variation can be
associated with functional characters that are a direct
response to the environment. More than 6.1 million proteins,
including thousands of new protein families, have been
annotated from this dataset (described in the accompanying
paper [18]). In combination, these papers bring us closer to
reconciling the genetic and biochemical disconnect and to
understanding the marine microbial community.
We describe a metagenomic dataset generated from the
Sorcerer II expedition. The GOS dataset, which includes and
extends our previously published Sargasso Sea dataset [19],
now encompasses a total of 41 aquatic, largely marine
locations, constituting the largest metagenomic dataset yet
produced with a total of ;7.7 million sequencing reads. In
0399 Special Section from March 2007 | Volume 5 | Issue 3 | e77

Figure 1. Sampling Sites
Microbial populations were sampled from locations in the order shown. Samples were collected at approximately 200 miles (320 km) intervals along the
eastern North American coast through the Gulf of Mexico into the equatorial Pacific. Samples 00 and 01 identify sets of sites sampled as part of the
Sargasso Sea pilot study [19]. Samples 27 through 36 were sampled off the Galapagos Islands (see inset). Sites shown in gray were not analyzed as part
of this study.
doi:10.1371/journal.pbio.0050077.g001
the pilot Sargasso Sea study, 200 l surface seawater was
filtered to isolate microorganisms for metagenomic analysis.
DNA was isolated from the collected organisms, and genome
shotgun sequencing methods were used to identify more than
1.2 million new genes, providing evidence for substantial
microbial taxonomic diversity [19]. Several hundred new and
diverse examples of the proteorhodopsin family of light-
harvesting genes were identified, documenting their exten-
sive abundance and pointing to a possible important role in
energy metabolism under low-nutrient conditions. However,
substantial sequence diversity resulted in only limited
genome assembly. These results generated many additional
questions: would the same organisms exist everywhere in the
ocean, leading to improved assembly as sequence coverage
increased; what was the global extent of gene and gene family
diversity, and can we begin to exhaust it with a large but
achievable amount of sequencing; how do regions of the
ocean differ from one another; and how are different
environmental pressures reflected in organisms and com-
munities? In this paper we attempt to address these issues.
Results
Sampling and the Metagenomic Dataset
Microbial samples were collected as part of the Sorcerer II
expedition between August 8, 2003, and May 22, 2004, by the
S/V Sorcerer II, a 32-m sailing sloop modified for marine
research. Most specimens were collected from surface water
marine environments at approximately 320-km (200-mile)
intervals. In all, 44 samples were obtained from 41 sites
(Figure 1), covering a wide range of distinct surface marine
PLoS Biology | www.plosbiology.org | S24
Sorcerer II GOS Expedition
environments as well as a few nonmarine aquatic samples for
contrast (Table 1).
Several size fractions were isolated for every site (see
Materials and Methods). Total DNA was extracted from one
or more fractions, mostly from the 0.1–0.8-lm size range.
This fraction is dominated by bacteria, whose compact
genomes are particularly suitable for shotgun sequencing.
Random-insert clone libraries were constructed. Depending
on the uniqueness of each sampling site and initial estimates
of the genetic diversity, between 44,000 and 420,000 clones
per sample were end-sequenced to generate mated sequenc-
ing reads. In all, the combined dataset includes 6.25 Gbp of
sequence data from 41 different locations. Many of the clone
libraries were constructed with a small insert size (,2 kbp) to
maximize cloning efficiency. As this often resulted in mated
sequencing reads that overlapped one another, overlapping
mated reads were combined, yielding a total of ;6.4 M
contiguous sequences, totaling ;5.9 Gbp of nonredundant
sequence. Taken together, this is the largest collection of
metagenomic sequences to date, providing more than a 5-fold
increase over the dataset produced from the Sargasso Sea
pilot study [19] and more than a 90-fold increase over the
other large marine metagenomic dataset [20].
Assembly
Assembling genomic data into larger contigs and scaffolds,
especially metagenomic data, can be extremely valuable, as it
places individual sequencing reads into a greater genomic
context. A largely contiguous sequence links genes into
operons, but also permits the investigation of larger
biochemical and/or physiological pathways, and also connects
otherwise-anonymous sequences with highly studied ‘‘taxo-
0400 Special Section from March 2007 | Volume 5 | Issue 3 | e77
 Table 1. Sampling Locations and Environmental Data

ID Sample Country Date, Time Location Sample Water T (8C)a Sb Size Habitat Chl a Sample Good
Location mm/dd/yy Depth, Depth, (ppt) Fraction Type Month (Annual Sequences
m m (lm) 6 SE) mg/m3

GS00a Sargasso Stations 13 and 11 Bermuda (UK) 02/26/03 3:00 3183296 99 n; 63835942 99 w 5.0 .4,200 20.0 20.5 36.6 0.1–0.8 Open ocean 0.17 (0.0.9 6 0.02) 644,551
10:10 31810950 99 n; 64819927 99 w 36.7
GS00b Sargasso Stations 13 and 11 Bermuda (UK) 02/26/03 3:35 31832910 99 n; 63835970 99 w 5.0 .4,200 20.0 20.5 36.6 0.22–0.8 Open ocean 0.17 (0.0.9 6 0.02) 317,180
10:43 31810950n; 64819927 99 w 36.7
GS00c Sargasso Stations 3 Bermuda (UK) 02/25/03 13:00 32809930 99 n; 64800936 99 w 5.0 .4,200 19.8 36.7 0.22–0.8 Open ocean 0.17 (0.0.9 6 0.02) 368,835
GS00d Sargasso Stations 13 Bermuda (UK) 02/25/03 17:00 3183296 99 n; 63835942 99 w 5.0 .4,200 20.0 36.6 0.22–0.8 Open ocean 0.17 (0.0.9 6 0.02) 332,240
GS01a Hydrostation S Bermuda (UK) 05/15/03 11:40 32810900 99 n 64830900 99 w 5.0 .4,200 22.9 36.7 3.0–20.0 Open ocean 0.10 (0.10 6 0.01) 142,352
GS01b Hydrostation S Bermuda (UK) 05/15/03 11:40 32810900 99 n; 64830900 99 w 5.0 .4,201 22.9 36.7 0.8–3.0 Open ocean 0.10 (0.10 6 0.01) 90,905
GS01c Hydrostation S Bermuda (UK) 05/15/03 11:40 32810900 99 n; 64830900 99 w 5.0 .4,202 22.9 36.7 0.1–0.8 Open ocean 0.1 (0.1 6 0.01) 92,351
GS02 Gulf of Maine USA 08/21/03 6:32 42830911 99 n; 67814924 99 w 1.0 106 18.2 29.2 0.1–0.8 Coastal 1.4 (1.12 6 0.19) 121,590
GS03 Browns Bank, Gulf of Maine Canada 08/21/03 11:50 42851910 99 n; 6681392 99 w 1.0 119 11.7 29.9 0.1–0.8 Coastal 1.4 (1.12 6 0.19) 61,605
GS04 Outside Halifax, Nova Scotia Canada 08/22/03 5:25 4488914 99 n; 63838940 99 w 2.0 142 17.3 28.3 0.1–0.8 Coastal 0.4 (0.78 6 0.17) 52,959

PLoS Biology | www.plosbiology.org | S25

GS05 Bedford Basin, Nova Scotia Canada 08/22/03 16:21 44841925 99 n; 63838914 99 w 1.0 64 15.0 30.2 0.1–0.8 Embayment 6 (6.76 6 0.98) 61,131
GS06 Bay of Fundy, Nova Scotia Canada 08/23/03 10:47 4586942 99 n; 64856948 99 w 1.0 11 11.2 0.1–0.8 Estuary 2.8 (1.87 6 0.18) 59,679
GS07 Northern Gulf of Maine Canada 08/25/03 8:25 43837956 99 n; 66850950 99 w 1.0 139 17.9 31.7c 0.1–0.8 Coastal 1.4 (1.12 6 0.19) 50,980
GS08 Newport Harbor, RI USA 11/16/03 16:45 4182999 99 n; 7182194 99 w 1.0 12 9.4 26.5c 0.1–0.8 Coastal 2.2 (1.59 6 0.17) 129,655
GS09 Block Island, NY USA 11/17/03 10:30 4185928 99 n; 7183698 99 w 1.0 32 11.0 31.0c 0.1–0.8 Coastal 4.0 (2.72 6 0.24) 79,303
GS10 Cape May, NJ USA 11/18/03 4:30 38856924 99 n; 7484196 99 w 1.0 10 12.0 31.0c 0.1–0.8 Coastal 2.0 (2.75 6 0.33) 78,304
GS11 Delaware Bay, NJ USA 11/18/03 11:30 3982594 99 n; 75830915 99 w 1.0 8 11.0 0.1–0.8 Estuary 4.8 (9.23 6 1.02) 124,435
GS12 Chesapeake Bay, MD USA 12/18/03 11:32 38856949 99 n; 7682592 99 w 1.0 25 3.2 3.47c 0.1–0.8 Estuary 21.0 (15.0 6 1.01) 126,162
GS13 Off Nags Head, NC USA 12/19/03 6:28 3680914 99 n; 75823941 99 w 1.0 20 9.3 0.1–0.8 Coastal 3.0 (2.24 6 0.25) 138,033
GS14 South of Charleston, SC USA 12/20/03 17:12 32830925 99 n; 79815950 99 w 1.0 31 18.6 0.1–0.8 Coastal 1.70 (1.92 6 0.25) 128,885

0401
GS15 Off Key West, FL USA 01/08/04 6:25 24829918 99 n; 8384912 99 w 2.0 47 25.3 36.0 0.1–0.8 Coastal 0.2 (0.27 6 0.09) 127,362
GS16 Gulf of Mexico USA 01/08/04 14:15 24810929 99 n; 84820940 99 w 2.0 3,333 26.4 35.8 0.1–0.8 Coastal sea 0.16 (0.11 6 0.01) 127,122
GS17 Yucatan Channel Mexico 01/09/04 13:47 20831921 99 n; 85824949 99 w 2.0 4,513 27.0 35.8 0.1–0.8 Open ocean 0.13 (0.09 6 0.01) 257,581
GS18 Rosario Bank Honduras 01/10/04 8:12 1882912 99 n; 8384795 99 w 2.0 4,470 27.4 35.4 0.1–0.8 Open ocean 0.14 (0.09 6 0.01) 142,743
GS19 Northeast of Colón Panama 01/12/04 9:03 10842959 99 n; 80815916 99 w 2.0 3,336 27.7 35.4 0.1–0.8 Coastal 0.23 (0.15 6 0.02) 135,325
GS20 Lake Gatun Panama 01/15/04 10:24 989952 99 n; 79850910 99 w 2.0 4 28.5 0.06 0.1–0.8 Fresh water 296,355
GS21 Gulf of Panama Panama 01/19/04 16:48 887945 99 n; 79841928 99 w 2.0 76 27.6 30.7 0.1–0.8 Coastal 0.50 (0.73 6 0.22) 131,798
GS22 250 miles from Panama City Panama 01/20/04 16:39 6829934 99 n; 82854914 99 w 2.0 2,431 29.3 32.3 0.1–0.8 Open ocean 0.33 (0.28 6 0.02) 121,662
GS23 30 miles from Cocos Island Costa Rica 01/21/04 15:00 5838924 99 n; 86833955 99 w 2.0 1,139 28.7 32.6 0.1–0.8 Open ocean 0.07 (0.19 6 0.02) 133,051
GS25 Dirty Rock, Cocos Island Costa Rica 01/28/04 10:51 5833910 99 n; 8785916 99 w 1.1 30 28.3 31.4 0.8–3.0 Fringing reef 0.11 (0.19 6 0.01) 120,671
GS26 134 miles NE of Galapagos Ecuador 02/01/04 16:16 1815951 99 n; 90817942 99 w 2.0 2,376 27.8 32.6 0.1–0.8 Open ocean 0.22 (0.28 6 0.02) 102,708
GS27 Devil’s Crown, Floreana Ecuador 02/04/04 11:41 1812958 99 s; 90825922 99 w 2.0 2.3 25.5 34.9 0.1–0.8 Coastal 0.40 (0.38 6 0.03) 222,080
GS28 Coastal Floreana Ecuador 02/04/04 15:47 181391 99 s; 90819911 99 w 2.0 156 25.0c 0.1–0.8 Coastal 0.35 (0.35 6 0.02) 189,052
GS29 North James Bay, Santigo Ecuador 02/08/04 18:03 081290 99 s; 9085097 99 w 2.0 12 26.2 34.5 0.1–0.8 Coastal 0.40 (0.39 6 0.03) 131,529
GS30 Warm seep, Roca Redonda Ecuador 02/09/04 11:42 0816920 99 n; 9183890 99 w 19.0 19 26.9 0.1–0.8 Warm seep 359,152
GS31 Upwelling, Fernandina Ecuador 02/10/04 14:43 081894 99 s; 9183996 99 w 12.0 19 18.6 0.1–0.8 Coastal upwelling 0.35 (0.39 6 0.03) 436,401
GS32 Mangrove, Isabella Ecuador 02/11/04 11:30 0835938 99 s; 9184910 99 w 0.3 0.67 25.4 0.1–0.8 Mangrove 148,018
c
GS33 Punta Cormorant Lagoon, Floreana Ecuador 02/19/04 13:35 1813942 99 s; 90825945 99 w 0.2 0.33 37.6 46 0.1–0.8 Hypersaline 692,255
GS34 North Seamore Ecuador 02/19/04 17:06 0822959 99 s; 90816947 99 w 2.0 35 27.5 0.1–0.8 Coastal 0.36 (0.35 6 0.02) 134,347
GS35 Wolf Island Ecuador 03/01/04 16:44 1823921 99 n; 9184991 99 w 2.0 71 21.8 34.5 0.1–0.8 Coastal 0.28 (0.31 6 0.02) 140,814
GS36 Cabo Marshall, Isabella Ecuador 03/02/04 12:52 081915 99 s; 91811952 99 w 2.0 67 25.8 34.6 0.1–0.8 Coastal 0.65 (0.45 6 0.05) 77,538
GS37 Equatorial Pacific TAO Buoy International 03/17/04 16:38 1858926 99 s; 9580953 99 w 2.0 3,334 28.8 0.1–0.8 Open ocean 0.21 (0.24 6 0.02) 65,670
GS47 201 miles from French Polynesia International 03/28/04 15:25 1087953 99 s; 135826958 99 w 30.0 2,400 28.6 37.3 0.1–0.8 Open ocean 66,023
GS51 Rangirora Atoll French Polynesia 05/22/04 7:04 1588937 99 s; 14782696 99 w 1.0 10 27.3 34.2 0.1–0.8 Coral reef atoll 128,982
Total 7,697,926

a
Temperature.
b
Salinity.
Sorcerer II GOS Expedition

Special Section from March 2007 | Volume 5 | Issue 3 | e77

c
Measurements were acquired from nearby vessels and/or research stations.
doi:10.1371/journal.pbio.0050077.t001

Table 2. Summary Assembly Statistics
Category Statistic
Assembly Number of reads used for assembly
inputs
Total read length (bp)
Number of ‘‘intigs’’ used for assemblya
Total intig length (bp)a
Assembly Number of assembliesb
outputs
Total assembled consensus length (bp)
Percentage of unassembled reads
Percentage of assembly at .13 coverage
Base pairs in contigs  10 kb
Base pairs in contigs  50 kb
Base pairs in scaffolds  2.5 kb (consensus bases)
Base pairs in scaffolds  5 kb (consensus bases)
Base pairs in scaffolds  10 kb (consensus bases)
Base pairs in scaffolds  50 kb (consensus bases)
Base pairs in scaffolds  100 kb (consensus bases)
Base pairs in scaffolds  300 kb (consensus bases)
Percentage of assembly in scaffolds  10 kbc
Length of longest contig (bp)
Length of longest scaffold (bp)
N1d assembly bp
d would be fewer conflicts within a single sample (i.e., that
N10 assembly bp
N50d assembly bp
N1d contig bp
N10d contig bp
N50d contig bp
a
Intigs are overlapping mated reads that have been collapsed into a single sequence as
input into the assembler.
b
Assemblies refers to the total number of scaffolds, pairs of mated nonoverlapping
singletons, and singleton unmated reads.
c
For comparison purposes, 10 kb is the average contig size predicted for 4.13 coverage
for an idealized shotgun assembly of a repeat-free, clonal genome [22].
d
N1 indicates the length of the next largest assembled sequence or contig such that 1%
of the sequence data falls into longer assemblies or contigs. N10 and N50 indicate that
10% and 50% of the data fell into larger assemblies or contigs.
doi:10.1371/journal.pbio.0050077.t002
nomic markers’’ such as 16S or recA, thus clearly identifying
the taxonomic group with which they are associated. The
primary assembly of the combined GOS dataset was
performed using the Celera Assembler [21] with modifica-
tions as previously described [19] and as given in Materials
and Methods. The assembly was performed with quite
stringent criteria, beginning with an overlap cutoff of 98%
identity to reduce the potential for artifacts (e.g., chimeric
assemblies or consensus sequences diverging substantially
from the genome of any given cell). This assembly was the
substrate for annotation (see the accompanying paper by
Yooseph et al. [18]).
The degree of assembly of a metagenomic sample provides
an indication of the diversity of the sample. A few substantial
assemblies notwithstanding, the primary assembly was strik-
ingly fragmented (Table 2). Only 9% of sequencing reads
went into scaffolds longer than 10 kbp. A majority (53%) of
the sequencing reads remained unassembled singletons.
Scaffolds containing more than 50 kb of consensus sequence
totaled 20.7 Mbp; of these, .75% were produced from a
single Sargasso Sea sample and correspond to the Burkholderia
or Shewanella assemblies described previously [19]. These
results highlight the unusual abundance of these two
organisms in a single sample, which significantly affected
PLoS Biology | www.plosbiology.org | S26
Sorcerer II GOS Expedition
our expectations regarding the current dataset. Given the
large size of the combined dataset and the substantial amount
of sequencing performed on individual filters, the overall lack
Value of assembly provides evidence of a high degree of diversity in
surface planktonic communities. To put this in context,
7,697,926 suppose there were a clonal organism that made up 1% of
6,325,208,303
our data, or ;60 Mbp. Even a genome of 10 Mbp—enormous
6,389,523 by bacterial standards—would be covered ;6-fold. Such data
5,883,982,712 might theoretically assemble with an average contig ap-
3,081,849 proaching 50 kb [22]. While real assemblies generally fall
short of theory for various reasons, Shewanella data make up
4,460,027,783
53% ,1% of the total GOS dataset, and yet most of the relevant
15.3% reads assemble into scaffolds .50 kb. Thus, with few scaffolds
39,427,102 of significant length, we could conclude that there are very
15,723,513
few clonal organisms present at even 1% in the GOS dataset.
458,196,599
138,137,150 To investigate the nature of the implied diversity and to see
65,238,481 whether greater assembly could be achieved, we explored
20,738,836 several alternative approaches. Breaks in the primary
16,005,244
assembly resulted from two factors: incomplete sequence
8,805,668
1.5% coverage and conflicts in the data. Conflicts can break
977,960 assemblies when there is no consistent way to chain together
2,097,794 all overlapping sequencing reads. As it was possible that there
15,915
2,533
1,611 diversity within a single sample would be lower), assemblies
8,994 were attempted with individual samples. However, the results
2,447 did not show any systematic improvements even in those
(single reads) samples with greater coverage (unpublished data). Upon
manual inspection, most assembly-breaking conflicts were
found to be local in nature. These observations suggested that
reducing the degree of sequence identity required for
assembly could ameliorate both factors limiting assembly:
effective coverage would increase and many minor conflicts
would be resolved.
Accordingly, we produced a series of assemblies based on
98%, 94%, 90%, 85%, and 80% identity overlaps for two
subsets of the GOS dataset, again using the Celera Assembler.
Assembly lengths increased as the overlap cutoff decreased
from 98% to 94% to 90%, and then leveled off or even
dropped as stringency was reduced below 90% (Table 3).
Although larger assemblies could be generated using lower
identity overlaps, significant numbers of overlaps satisfying
the chosen percent identity cutoff still went unused in each
assembly. This is consistent with a high rate of conflicting
overlaps and in turn diagnostic of significant polymorphism.
In mammalian sequencing projects the use of larger insert
libraries is critical to producing larger assemblies because of
their ability to span repeats or local polymorphic regions [23].
The shotgun sequencing libraries from the GOS filters were
typically constructed from inserts shorter than 2 kb. Longer
plasmid libraries were attempted but were much less stable.
We obtained paired-end sequences from 21,419 fosmid clones
(average insert size, 36 kb; [24,25]) from the 0.1-micron
fraction of GS-33. The effect of these long mate pairs on the
GS-33 assembly was quite dramatic, particularly at high
stringency (e.g., improving the largest scaffold from 70 kb to
1,247 kb and the largest contig from 70 kb to 427 kb). At least
for GS-33 this suggests that many of the polymorphisms affect
small, localized regions of the genome that can be spanned
using larger inserts. This degree of improvement may be
greater than what could be expected in general, as the
diversity of GS-33 is by far the lowest of any of the currently
0402 Special Section from March 2007 | Volume 5 | Issue 3 | e77
 Sorcerer II GOS Expedition

from the National Center for Biotechnology Information

Table 3. Evaluation of Alternative Assembly Methods (NCBI; http://www.ncbi.nlm.nih.gov). At the time of this study,
we used 334 finished and 250 draft microbial genomes as
Dataset Type Percent Base Pairs in Base Pairs in references for comparison with the GOS sequencing reads.
Identity 10 k Contigs 100 k Contigs Comparisons were carried out in nucleotide-space using the
sequence alignment tool BLAST [26]. BLAST parameters
GS33 plasmids WGSa 98 13,669,678 0 were designed to be extremely lenient so as to detect even
94 19,536,324 2,749,543 distant similarities (as low as 55% identity). A large
90 20,996,826 3,729,765
85 20,327,989 3,505,324
proportion of the GOS reads, 70% in all, aligned to one or
80 19,245,637 4,195,959 more genomes under these conditions. However, many of the
E-asmb 98 22,000,579 5,604,857 alignments were of low identity and used only a portion of
94 22,781,462 7,302,801 the entire read. Such low-quality hits may reflect distant
90 22,702,764 7,600,441
evolutionary relationships, and therefore less information is
85 22,570,933 7,937,079
80 20,335,558 4,779,684 gained based on the context of the alignment. More stringent
GS33 with fosmids WGSa 98 15,031,557 1,306,992 criteria could be imposed requiring that the reads be aligned
94 22,310,335 4,449,710 over nearly their entire length without any large gaps. Using
90 22,944,278 5,585,959 this stringent criterion only about 30% of the reads aligned
85 22,251,738 5,485,013
80 21,088,975 5,684,925
to any of the 584 reference genomes. We refer to these fully
GS17,18,23,26 WGSa 98 185,058 0 aligned reads as ‘‘recruited reads.’’ Recruited reads are far
94 5,422,366 213,755 more likely to be from microbes closely related to the
90 10,694,783 373,822 reference sequence (same species) than are partial align-
85 11,514,421 800,290
ments. Despite the large number of microbial genomes
80 9,004,221 879,401
E-asmb 98 2,047,524 0 currently available, including a large number of marine
94 10,668,547 1,184,881 microbes, these results indicate that a substantial majority of
90 15,215,981 2,634,227 GOS reads cannot be specifically related to available micro-
85 15,786,515 3,132,152 bial genomes.
80 13,767,929 2,942,160
Combined GOS WGSa 98 39,427,102 11,488,828
The amount and distribution of reads recruited to any
94 98,887,937 12,376,236 given genome provides an indication of the abundance of
E-asmb 98 91,526,091 16,444,304 closely related organisms. Only genomes from the five
94 163,612,717 25,564,163 bacterial genera Prochlorococcus, Synechococcus, Pelagibacter,
90 186,614,813 28,752,198
Shewanella, and Burkholderia yielded substantial and uniform
85 181,887,218 27,154,335
80 161,160,091 23,794,832 recruitment of GOS fragments over most of a reference
genome (Table 4). These genera include multiple reference
a
genomes, and we observed significant differences in recruit-
Whole-genome shotgun (WGS) assembly performed with the Celera Assembler.
b
Assemblies performed using extreme assembly approach (E-asm). ment patterns even between organisms belonging to the same
doi:10.1371/journal.pbio.0050077.t003 species (Figure 2A–2I). Three genera, Pelagibacter (Figure 2A),
Prochlorococcus (Figure 2B–2F), and Synechococcus (Figure 2G–
sequenced GOS samples, yet it clearly indicates the utility of 2I), were found abundantly in a wide range of samples and
including larger insert libraries for assembly. together accounted for roughly 50% of all the recruited reads
(though only ;15% of all GOS sequencing reads). By
Fragment Recruitment contrast, although every genome tested recruited some GOS
In the absence of substantial assembly, direct comparison reads, most recruited only a small number, and these reads
of the GOS sequencing data to the genomes of sequenced clustered at lower identity to locations corresponding to large
microbes is an alternative way of providing context, and also highly conserved genes (for typical examples see Figure 2E–
allows for exploration of genetic variation and diversity. A 2F). We refer to this pattern as nonspecific recruitment as it
large and growing set of microbial genomes are available reflects taxonomically nonspecific signals, with the reads in

Table 4. Microbial Genera that Recruited the Bulk of the GOS Reads

Genus Read Count Best Strain

All Reads 80%þa 90%þb All Reads 80%þa 90%þb

Pelagibacter 922,677 195,539 36,965 HTCC1062 HTCC1062 HTCC1062

Prochlorococcus 208,999 159,102 84,325 MIT9312 MIT9312 MIT9312
Synechococcus 60,650 26,365 21,594 CC9902 RS9917 RS9917
Burkholderia 151,123 108,610 93,081 383 383 383
Shewanella 59,086 34,138 27,693 MR-1 MR-1 MR-1
Remaining 43,244 2,367 564 Buchnera aphidicola Str. Sg Buchnera aphidicola Str. APS Alteromonas macleodii

a
Reads aligned at or above 80% identity over the entire length of the read.
b
Reads aligned at or above 90% identity over the entire length of the read.
doi:10.1371/journal.pbio.0050077.t004

PLoS Biology | www.plosbiology.org | S27 0403 Special Section from March 2007 | Volume 5 | Issue 3 | e77
 Sorcerer II GOS Expedition

Figure 2. Fragment Recruitment Plots

The horizontal axis of each panel corresponds to a 100-kb segment of genomic sequence from the indicated reference microbial genome. The vertical
axis indicates the sequence identity of an alignment between a GOS sequence and the reference genomic sequence. The identity ranges from 100%
(top) to 50% (bottom). Individual GOS sequencing reads were colored to reflect the sample from which they were isolated. Geographically nearby
samples have similar colors (see Poster S1 for key). Each organism shows a distinct pattern of recruitment reflecting its origin and relationship to the
environmental data collected during the course of this study.
(A) P. ubique HTCC1062 recruits the greatest density of GOS sequences of any genome examined to date. The GOS sequences show geographic

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 Sorcerer II GOS Expedition

stratification into bands, with sequences from temperate water samples off the North American coast having the highest identity (yellow to yellow-
green colors). At lower identity, sequences from all the marine environments could be aligned to HTCC1062.
(B) P. marinus MIT9312 recruits a large number of GOS sequences into a single band that zigzags between 85%–95% identity on average. These
sequences are largely derived from warm water samples in the Gulf of Mexico and eastern Pacific (green to greenish-blue reads).
(C) P. marinus MED4 recruits largely the same set of reads as MIT9312 (B) though the sequences that form the zigzag recruit at a substantially lower
identity. A small number of sequences from the Sargasso Sea samples (red) are found at high identity.
(D) P. marinus NATL2A recruits far fewer sequences than any of the preceding panels. Like MED4, a small number of high-identity sequences were
recruited from the Sargasso samples.
(E) P. marinus MIT9313 is a deep-water low-light–adapted strain of Prochlorococcus. GOS sequences were recruited almost exclusively at low identity in
vertical stacks that correspond to the locations of conserved genes. On the left side of this panel is a very distinctive pattern of recruitment that
corresponds to the highly conserved 16S and 23S mRNA gene operon.
(F) P. marinus CCMP1375, another deep-water low-light–adapted strain, does not recruit GOS sequences at high identity. Only stacks of sequences are
seen corresponding to the location of conserved genes.
(G) Synechococcus WH8102 recruits a modest number of high-identity sequences primarily from the Sargasso Sea samples. A large number of moderate
identity matches from the Pacific and hypersaline lagoon (GS33) samples are also visible.
(H) Synechococcus CC9605 recruits largely the same sequences as does Synechococcus WH8102, but was isolated from Pacific waters. GOS sequences
from some of the Pacific samples recruit at high identity, while sequences from the Sargasso and hypersaline lagoon (bluish-purple) were recruited at
moderate identities.
(I) Synechococcus CC9902 is distantly related to either of the preceding Synechococcus strains. While this strain also recruits largely the same sequences
as the WH8102 and CC9902 strains, they recruit at significantly lower identity.
(J–O) Fragment recruitment plots to extreme assemblies seeded with phylogenetically informative sequences. Using this approach it is not only
possible to assemble contigs with strong similarities to known genomes but to identify contigs from previously uncultured genomes. In each case a
100-kb segment from an extreme assembly is shown. Each plot shows a distinct pattern of recruitment that distinguishes the panels from each other.
(J) Seeded from a Prochlorococcus marinus-related sequence, this contig recruits a broad swath of GOS sequences that correspond to the GOS
sequences that form the zigzag on P. marinus MIT9312 recruitment plots (see [B] or Poster S1 for comparison).
(K–L) Seeded from SAR11 clones, these contigs show significant synteny to the known P. ubique HTCC1062 genome. (K) is strikingly similar to previous
recruitment plots to the HTCC1062 genome (see [A] or Poster S1). In contrast, (L) identifies a different strain that recruits high-identity GOS sequences
primarily from the Sargasso Sea samples (red).
(M–O) These three panels show recruitment plots to contigs belonging to the uncultured Actinobacter, Roseobacter, and SAR86 lineages.
doi:10.1371/journal.pbio.0050077.g002

question often recruiting to distantly related sets of genomes.
Most microbial genomes, including many of the marine
microbes (e.g., the ubiquitous genus Vibrio), demonstrated this
nonspecific pattern of recruitment.
The relationship between the similarity of an individual
sequencing read to a given genome and the sample from which
the read was isolated can provide insight into the structure,
evolution, and geographic distribution of microbial popula-
tions. These relationships were assessed by constructing a
‘‘percent identity plot’’ [27] in which the alignment of a read to
a reference sequence is shown as a bar whose horizontal
position indicates location on the reference and whose vertical
position indicates the percent identity of the alignment. We
colored the plotted reads according to the samples to which
they belonged, thus indirectly representing various forms of
metadata (geographic, environmental, and laboratory varia-
bles). We refer to these plots that incorporate metadata as
fragment recruitment plots. Fragment recruitment plots of
GOS sequences recruited to the entire genomes of Pelagibacter
ubique HTCC1062, Prochlorococcus marinus MIT9312, and Syn-
echococcus WH8102 are presented in Poster S1.
Within-Ribotype Population Structure and Variation
Characteristic patterns of recruitment emerged from each
of these abundant marine microbes consisting of horizontal
bands made up of large numbers of GOS reads. These bands
seem constrained to a relatively narrow range of identities
that tile continuously (or at least uniformly, in the case when
abundance/coverage is lower) along ;90% of the reference
sequence. The uninterrupted tiling indicates that environ-
mental genomes are largely syntenic with the reference
genomes. Multiple bands, distinguished by degree of sim-
ilarity to the reference and by sample makeup, may arise on a
single reference (Poster S1D and S1F). Each of these bands
appears to represent a distinct, closely related population we
refer to as a subtype. In some cases, an abundant subtype is
highly similar to the reference genome, as is the case for P.
marinus MIT9312 (Poster S1) and Synechococcus RS9917
(unpublished data). P. ubique HTCC1062 and other Synecho-
coccus strains like WH8102 show more complicated banding
patterns (Poster S1D and S1F) because of the presence of
multiple subtypes that produce complex often overlapping
bands in the plots. Though the recruitment patterns can be
quite complex they are also remarkably consistent over much
of the reference genome. In these more complicated recruit-
ment plots, such as the one for P. ubique HTCC1062,
individual bands can show sudden shifts in identity or
disappear altogether, producing a gap in recruitment that
appears to be specific to that band (see P. ubique recruitment
plots on Poster S1B and S1E, and specifically between 130–
140 kb). Finally, phylogenetic analysis indicates that separate
bands are indeed evolutionarily distinct at randomly selected
locations along the genome.
The amount of sequence variation within a given band
cannot be reliably determined from the fragment recruit-
ment plots themselves. To examine this variation, we
produced multiple sequence alignments and phylogenies of
reads that recruited to several randomly chosen intervals
along given reference genomes to show that there can be
considerable within-subtype variation (Figure 3A–3B). For
example, within the primary band found in recruitment plots
to P. marinus MIT9312, individual pairs of overlapping reads
typically differ on average between 3%–5% at the nucleotide
level (depending on exact location in the genome). Very few
reads that recruited to MIT9312 have perfect (mismatch-free)
overlaps with any other read or to MIT9312, despite ;100-
fold coverage. While many of these differences are silent (i.e.,
do not change amino acid sequences), there is still consid-
erable variation at the protein level (unpublished data). The
amount of variation within subtypes is so great that it is likely
that no two sequenced cells contained identical genomes.

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 Sorcerer II GOS Expedition

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Figure 3. Population Structure and Variation as Revealed by Phylogeny
Phylogenies were produced using neighbor-joining. There is significant within-clade variation as well as an absence of strong geographic structure to
variants of SAR11 (P. ubique HTCC1062) and P. marinus MIT9312. Similar reads are not necessarily from similar locations, and reads from similar locations
are not necessarily similar.
(A) Geographic distribution of SAR11 proteorhodopsin variants. Keys to coloration: blue, Pacific; pink, Atlantic.
(B) Geographic distribution of Prochlorococcus variants. Keys to coloration: blue, Pacific; pink, Atlantic.
(C) Origins of spectral tuning of SAR11 proteorhodopsins. Reads are colored according to whether they contain the L (green) or Q (blue) variant at the
spectral tuning residue described in the text. The selection of tuning residue is lineage restricted, but each variant must have arisen on two separate
occasions.
doi:10.1371/journal.pbio.0050077.g003
Identifying Genomic Structural Variation with
Metagenomic Data
Variation in genome structure in the form of rearrange-
ments, duplications, insertions, or deletions of stretches of
DNA can also be explored via fragment recruitment. The use
of mated sequencing reads (pairs of reads from opposite ends
of a clone insert) provides a powerful tool for assessing
structural differences between the reference and the environ-
mental sequences. The cloning and sequencing process
determines the orientation and approximate distance be-
tween two mated sequencing reads. Genomic structural
variation can be inferred when these are at odds with the
way in which the reads are recruited to a reference sequence.
Relative location and orientation of mated sequences provide
a form of metadata that can be used to color-code a fragment
recruitment plot (Figure 4). This makes it possible to visually
identify and classify structural differences and similarities
between the reference and the environmental sequences
(Figure 5). For the abundant marine microbes, a high
Figure 4. Categories of Recruitment Metadata
The recruitment metadata distinguishes eight different general catego-
ries based on the relative placement of paired end sequencing reads
(mated reads) when recruited to a reference sequence in comparison to
their known orientation and separation on the clone from which they
were derived. Assuming orientation is correct, two mated reads can be
recruited closer together, further apart, or within expected distances
given the size of the clone from which the sequences were derived.
These sequences are categorized as ‘‘short,’’ ‘‘long,’’ or ‘‘good,’’
respectively. Alternately, the mated reads may be recruited in a mis-
oriented fashion, which trumps issues of separation. These reads can be
categorized as ‘‘normal,’’ ‘‘anti-normal,’’ or ‘‘outie.’’ In addition, there are
two other categories. ‘‘No mate’’ indicates that no mated read was
available for recruitment, possibly due to sequencing error. Perhaps most
useful of any of the recruitment categories, ‘‘missing’’ mates indicate
that while a mated sequence was available, it was not recruited to the
reference. ‘‘Missing’’ mates identify breaks in synteny between the
environmental data and the reference sequence.
doi:10.1371/journal.pbio.0050077.g004
PLoS Biology | www.plosbiology.org | S31
Sorcerer II GOS Expedition
proportion of mated reads in the ‘‘good’’ category (i.e., in
the proper orientation and at the correct distance) show that
synteny is conserved for a large portion of the microbial
population. The strongest signals of structural differences
typically reflect a variant specific to the reference genome
and not found in the environmental data. In conjunction with
the requirement that reads be recruited over their entire
length without interruption, recruitment plots result in
pronounced recruitment gaps at locations where there is a
break in synteny. Other rearrangements can be partially
present or penetrant in the environmental data and thus may
not generate obvious recruitment gaps. However, given
sufficient coverage, breaks in synteny should be clearly
identifiable using the recruitment metadata based on the
presence of ‘‘missing’’ mates (i.e., the mated sequencing read
that was recruited but whose mate failed to recruit; Figure 4).
The ratio of missing mates to ‘‘good’’ mates determines how
penetrant the rearrangement is in the environmental
population.
In theory, all genome structure variations that are large
enough to prevent recruitment can be detected, and all such
rearrangements will be associated with missing mates.
Depending on the type of rearrangement present other
recruitment metadata categories will be present near the
rearrangements’ endpoints. This makes it possible to distin-
guish among insertions, deletions, translocations, inversions,
and inverted translocations directly from the recruitment
plots. Examples of the patterns associated with different
rearrangements are presented in Figure 5. This provides a
rapid and easy visual method for exploring structural
variation between natural populations and sequenced repre-
sentatives (Poster S1A and S1B).
Genomic Structural Variation in Abundant Marine
Microbes
Variation in genome structure potentially results in func-
tional differences. Of particular interest are those differences
between sequenced (reference) microbes and environmental
populations. These differences can indicate how representa-
tive a cultivated microbe might be and shed light on the
evolutionary forces driving change in microbial populations.
Fragment recruitment in conjunction with the mate metadata
helped us to identify both the consistent and the rare
structural differences between the genomes of microbial
populations in the GOS data and their closest sequenced
relatives. Our analysis has thus far been confined to the three
microbial genera that were widespread in the GOS dataset as
represented by the finished genomes of P. marinus MIT9312,
P. ubique HTCC1062, and to a lesser extent Synechococcus
WH8102. Each of these genomes is characterized by large and
small segments where little or no fragment recruitment took
place. We refer to these segments as ‘‘gaps.’’ These gaps
0407 Special Section from March 2007 | Volume 5 | Issue 3 | e77
 Sorcerer II GOS Expedition

Figure 5. Fragment Recruitment at Sites of Rearrangements

Environmental sequences recruited near breaks in synteny have characteristic patterns of recruitment metadata. Indeed, each of five basic
rearrangements (i.e., insertion, deletion, translocation, inversion, and inverted translocation) produced a distinct pattern when examining the
recruitment metadata. Here, example recruitment plots for each type of rearrangement have been artificially generated. The ‘‘good’’ and ‘‘no mate’’
categories have been suppressed. In each case, breaks in synteny are marked by the presence of stacks of ‘‘missing’’ mate reads. The presence or
absence of other categories distinguishes each type of rearrangement from the others.
doi:10.1371/journal.pbio.0050077.g005

represent reference-specific differences that are not found in
the environmental populations rather than a cloning bias
that identifies genes or gene segments that are toxic or
unclonable in E. coli. The presence of missing mates flanking
these gaps indicates that the associated clones do exist, and
therefore that cloning issues are not a viable explanation for
the absence of recruited reads. Although the reference-
specific differences are quite apparent due to the recruitment
gaps they generate, there are also sporadic rearrangements
associated with single clones, mostly resulting from small
insertions or deletions.
Careful examination of the unrecruited mates of the reads
flanking the gaps allowed us to identify, characterize, and
quantify specific differences between the reference genome
and their environmental relatives. The results of this analysis
for P. ubique and P. marinus have been summarized in Table 5.
With few exceptions, small gaps resulted from the insertion
or deletion of only a few genes. Many of the genes associated
with these small insertions and deletions have no annotated
function. In some cases the insertions display a degree of
variability such that different sets of genes are found at these
locations within a portion of the population. In contrast,
many of the larger gaps are extremely variable to the extent
that every clone contains a completely unrelated or highly
divergent sequence when compared to the reference or to
other clones associated with that gap. These segments are
hypervariable and change much more rapidly than would be
expected given the variation in the rest of the genome. Sites
containing a hypervariable segment nearly always contained
some insert. We identified two exceptions both associated
with P. ubique. The first is approximately located at the 166-kb
position in the P. ubique HTCC1062 genome. Though no large
gap is present, the mated reads indicate that under many
circumstances a highly variable insert is often present. The
second is a gap on HTCC1062 that appears between 50 and
90 kb. This gap appears to be less variable than other
hypervariable segments and is occasionally absent based on
the large numbers of flanking long mated reads (Poster S1A).
Interestingly, the long mated reads around this gap seem to
be disproportionately from the Sargasso Sea samples,
suggesting that this segment may be linked to geographic
and/or environmental factors. Thus, hypervariable segments
are highly variable even within the same sample, can on
occasion be unoccupied, and the variation, or lack thereof,
can be sample dependent.
Hypervariable segments have been seen previously in a
wide range of microbes, including P. marinus [28], but their
precise source and functional role, especially in an environ-
mental context, remains a matter of ongoing research. For
clues to these issues we examined the genes associated with
the missing mates flanking these segments and the nucleotide
composition of the gapped sequences in the reference
genomes. In some rare cases the genes identified on reads
that should have recruited within a hypervariable gap were
highly similar to known viral genes. For example, a viral
integrase was associated with the P. ubique HTCC1062
hypervariable gap between 516 and 561 kb. However, in the
majority of cases the genes associated with these gaps were
uncharacterized, either bearing no similarity to known genes
or resembling genes of unknown function. If these genes were
indeed acquired through horizontal transfer then we might
expect that they would have obvious compositional biases.
Oligonucleotide frequencies along the P. ubique HTCC1062
and Synechococcus WH8102 genomes are quite different in the
large recruitment gaps in comparison to the well-represented
portions of the genome (Poster S1). Surprisingly, this was less
true for P. marinus MIT9312, where the gaps have been linked
to phage activity [28]. These results suggest that these
hypervariable segments of the genome are widespread among
marine microbial populations, and that they are the product
of horizontal transfer events perhaps mediated by phage or
transposable elements. These results are consistent with and
expand upon the hypothesis put forward by Coleman et al.
[28] suggesting that these segments are phage mediated, and
conflicts with initial claims that the HTCC1062 genome was
devoid of genes acquired by horizontal transfer [29].

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 Sorcerer II GOS Expedition

Table 5. Atypical Segments in P. marinus MIT9312 and P. ubique HTCC1062 (SAR11)

Reference Genome Begina Endb Size, bp Type of Variantc Description

MIT9312 36,401 38,311 2,132 Variable deletion 12 out of 66 clones support simple deletion. Remaining clones show considerable
sequence variation amongst themselves.
MIT9312 124,448 125,219 771 Variable insertion Associated with ASN tRNA gene; in the environment, half the reads identify pair
of small inserts with no similarity to known genes; the other half point to small
and large inserts of undetermined nature.
MIT9312 233,826 233,910 84 Insertion All clones support small insert (270 bp) with no clear sequence similarity to
known genes or sequences.
MIT9312 243,296 245,115 424 Variable deletion 24 of 42 clones support simple deletion of hypothetical protein. 13 support
slightly larger deletion. Remaining not clearly resolved.
MIT9312 296,818 300,888 4,344 Variable deletion 44 clones support deletion of 3,070 bp segment containing 4 genes (3 hypotheti-
cal; 1 carbamoyltransferase). 17 clones support alternative sequences with little or
no similarity to each other.
gMIT9312 342,404 342,662 326 Variable insert In environment is 93% chance of finding deoxyribodopyrimiden photolyase with
7% chance of finding an ABC type Fe3þ siderophore transport system permease
component.
MIT9312 345,933 365,351 19,418 Hypervariable Very limited similarity among clones indicates that this is a hypervariable seg-
ment. Note that it is closely associated with a site-specific integrase/recombinase.
MIT9312 551,347 552,025 678 Deletion Two small deletions within a hypothetical protein.
MIT9312 617,914 621,556 3,642 Hypervariable About 50% of the clones support small deletions among several hypothetical
proteins. Remaining support significant variability suggesting hypervariable seg-
ment. At least two of the missed mates contain integrase-like genes.
MIT9312 646,340 652,375 6,035 Deletion/Hypervariable Majority of clones support simple deletion of eight hypothetical and hypothetical
genes. Small number of clones indicate this may by hypervariable as well.
MIT9312 655,241 655,800 559 Deletion Small deletion between hypothetical proteins.
MIT9312 665,000 678000 12,000 Deletions Complex set of deletions and replacements that vary with geographic location.
MIT9312 665,824 666,380 556 Insertion Small inserted hypothetical protein.
MIT9312 670,747 671,933 1,186 Deletion Deletes hypothetical gene.
MIT9312 736,266 736,289 23 Insertion All clones support insertion of fructose-bisphosphate aldolase and fructose-1,6-bi-
sphosphate aldolase.
MIT9312 762,156 762,717 561 Deletion Small deletion between hypothetical proteins.
MIT9312 779,006 779,309 303 Insertion Small hypothetical protein inserted.
MIT9312 874,349 874,913 564 Insertion Small insertion including gene with similarity to RNA-dependent RNA-polymerase.
MIT9312 943,389 946,997 3,608 Variable Several small changes.
MIT9312 1,043,129 1,131,874 88,745 Hypervariable
MIT9312 1,140,922 1,141,412 490 Insertion Small insertion.
MIT9312 1,144,307 1,144,790 483 Insertion Small insertion of several genes.
MIT9312 1,155,123 1,156,440 1,317 Deletion Deletes high-light–inducible protein.
MIT9312 1,172,609 1,177,292 4,683 Variable Several genes have been replaced or deleted.
MIT9312 1,202,643 1,274,335 71,692 Hypervariable
MIT9312 1,288,481 1,290,367 1,886 Variable deletion Deletes polysaccharide export-related periplasmic protein (28 out of 55). Other
deletions are variable and may include replacement with alternate sequences.
MIT9312 1,323,606 1,324,523 917 Deletion Environmental sequences lack a small hypothetical protein.
MIT9312 1,369,637 1,369,996 359 Insertion NAD-dependent DNA ligase absent from MIT9312; has possible paralog
MIT9312 1,381,273 1,382,049 776 Deletion Small insert in MIT9312 not present in environment
MIT9312 1,384,664 1,385,110 446 Deletion Deletes delta(12)-fatty acid dehydrogenase and replaces gene with small (;100
bp) sequence. There is some variation in the exact location and replacement se-
quence.
MIT9312 1,388,430 1,389,718 1,288 Replacement Segment between two high-light–inducible proteins swapped for different se-
quence with no similarity.
MIT9312 1,392,865 1,392,976 111 Replacement Small replacement deletes hypothetical gene and replaces it with small, unknown
sequence. There is some variation in the precise boundaries of the deletion and
in the replacement sequences.
MIT9312 1,397,696 1,420,005 22,309 Hypervariable
MIT9312 1,486,145 1,487,971 231 Variable Small insertion of dolichyl-phosphate-mannose-protein mannosyltransferase; alter-
nately deletes glycosyl transferase (8 out of 56).
MIT9312 1,519,810 1,520,860 1,050 Variable deletion Deletes a single hypothetical protein; about half of the deletions contain variable
sequences of unknown origin.
MIT9312 1,568,049 1,569,121 1,072 Replacement Typically 928-bp portion of MIT9312 replaced by 175-bp stretch in environment;
some small amount of variation in environmental replacement sequence (11 out
of 51).
HTCC1062 50,555 93,942 43,387 Variable deletion Low recruitment segment containing many hypothetical, transporter, and secre-
tion genes.
HTCC1062 146,074 146,415 341 Deletion Often deleted segment containing DoxD-like and ferredoxin dependent gluta-
mate synthase peptide.
HTCC1062 166,600 166,700 100 Variable replacement GOS sequences indicate that variable blocks of genes are frequently inserted
here.
HTCC1062 308,720 309,633 913 Deletion Potential sulfotransferase domain deleted.
HTCC1062 339,545 339,951 406 Deletion Deletes a predicted O-linked N-acetylglucosamine transferase.
HTCC1062 385,348 386,224 876 Deletion SAM-dependent methyltransferase deleted.

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 Sorcerer II GOS Expedition

Table 5. Continued.

Reference Genome Begina Endb Size, bp Type of Variantc Description

HTCC1062 441,074 441,152 78 Deletion Deletes possible methyltransferase FkbM.

HTCC1062 516,041 561,604 45,563 Variable replacement Several high identity ‘‘missed’’ mates match phage genes, including an integrase.
HTCC1062 660,413 660,978 565 Deletion Deletes hypothetical protein.
HTCC1062 675,141 676,399 1,258 Deletion Deletes hypothetical protein.
HTCC1062 766,022 768,263 2,241 Deletion Deletes four hypothetical proteins.
HTCC1062 814,015 816,386 2,371 Deletion Deletes steroid monoxygenase and short-chain dehydrogenase.
HTCC1062 893,450 922,604 29,154 Deletion Deletes large segment including a 7317-aa hypothetical gene.
HTCC1062 941,203 942,403 1,200 Deletion Deletes hypothetical protein.
HTCC1062 991,461 997,861 6,400 Deletion Deletes several hypotheticals and mix of other genes; adjacent to recombinase.
HTCC1062 1,117,126 1,143,483 26,357 Deletion Large number of hypothetical and transporters that are deleted.
HTCC1062 1,160,908 1,166,589 5,681 Deletion Deletes a small cluster of peptides with various functions.
HTCC1062 1,188,887 1,189,231 344 Deletion Deletes portion of winged helix DNA-binding protein but inserts sequences with
similarity to gij71082757 sodium bile symporter family protein found in large gap
between 50555 and 93942.

a
Begin indicates the approximate bp position which marks the beginning of the gap in recruitment.
b
End indicates the approximate bp position which marks the ending of the gap in recruitment.
c
The type of change indicates what would have to happen to the reference genome to produce the sequences seen in the environment (e.g., a deletion indicates that the indicated
portion of the reference would have to be deleted to generate the variant(s) seen in the environment).
doi:10.1371/journal.pbio.0050077.t005

Though insertions and deletions accounted for many of the
obvious regions of structural variation, we also looked for
rearrangements. The high levels of local synteny associated
with P. ubique and P. marinus suggested that large-scale
rearrangements were rare in these populations. To inves-
tigate this hypothesis we used the recruitment data to
examine how frequently rearrangements besides insertions
and deletions could be identified. We looked for rearrange-
ments consisting of large (greater than 50 kb) inversions and
translocations associated with P. marinus; however, we did not
identify any such rearrangements that consistently distin-
guished environmental populations from sequenced cultivars.
Rare inversions and translocations were identified in the
dominant subtype associated with MIT9312 (Table 6). Based
on the amount of sequence that contributed to the analysis,
we estimate that one inversion or translocation will be
observed for every 2.6 Mbp of sequence examined (less than
once per P. marinus genome).
A further observation concerns the uniformity along a
genome of the evolutionary history among and within
subtypes. For instance, the similarity between GOS reads
and P. marinus MIT9312 is typically 85%–95%, while the
similarity between MIT9312 and P. marinus MED4 is generally
;10% lower. However, there are several instances where the
divergence of MIT9312 and MED4 abruptly decreases to no
more than that between the GOS sequences and MIT9312
(Poster S1G). These results are consistent either with
horizontal transfer (recombination) or with inhomogeneous

Table 6. Six Large-Scale Translocations and Inversions Were Identified in the Abundant P. marinus Subtype

Group Low Low High High Read ID Low Low Low High High High Read Inversion Sample
Genome Genome Genome Genome Read Read Read Read Read Read Length
Begin End Begin End Begin Breakpoint Strand Breakpoint End Strand

1 34,428 34,904 1,536,417 1,536,774 1092255385627 0 476 1 477 834 1 834 No 15

2 607,778 608,467 1,131,368 1,131,621 1093017685727 270 959 0 0 251 1 959 Yes 18
3 618,997 619,375 1,172,217 1,172,728 1092963065572 19 397 1 425 939 1 939 No 17
3 618,997 619,372 1,172,203 1,172,728 1095433012642 0 375 1 403 931 1 941 No 26
3 618,997 619,251 1,172,216 1,172,728 1091140913752 2 256 1 284 799 1 799 No 19
3 618,997 619,331 1,172,280 1,172,728 1641121 2 337 1 365 816 1 816 No 00d
3 619,007 619,375 1,172,223 1,172,728 1092963490951 19 387 1 425 933 1 933 No 17
4 652,933 653,483 1,369,774 1,370,077 200560 325 875 0 0 303 1 875 Yes 00a
4 652,979 653,350 1,369,774 1,370,200 1492647 0 371 1 439 865 0 867 Yes 00d
4 652,979 653,496 1,369,774 1,370,086 1092256128910 10 527 1 595 905 0 906 Yes 15
4 652,979 653,496 1,369,774 1,370,061 1092405979387 14 531 1 599 886 0 886 Yes 25
4 652,979 653,353 1,369,774 1,370,132 1093017637883 2 376 1 444 802 0 802 Yes 18
4 652,980 653,496 1,369,774 1,370,111 1092343389654 6 522 1 591 928 0 928 Yes 25
5 1,049,484 1,049,793 1,172,301 1,172,782 1400802 518 827 0 1 483 0 827 No 00d
6 1,219,993 1,220,519 1,485,834 1,486,222 1092256207885 2 528 1 532 919 1 919 No 17
6 1,219,993 1,220,496 1,485,925 1,486,222 380485 300 803 0 0 296 0 803 No 00a
6 1,219,993 1,220,477 1,485,782 1,486,204 1484583 0 484 1 506 927 1 927 No 00d
6 1,220,005 1,220,388 1,485,733 1,486,216 1682914 507 890 0 2 484 0 913 No 00d

doi:10.1371/journal.pbio.0050077.t006

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Figure 6. Examples of Chimeric Extreme Assemblies
(A) Fragment recruitment to an extreme assembly contig indicates the
assembly is chimeric between two organisms, based on dramatic shifts in
density of recruitment, level of conservation, and sample distribution.
(B) Fragment recruitment to a SAR11-related extreme assembly. Changes
in color, density, and vertical location toward the top of the figure
indicate transitions among multiple subtypes of SAR11.
doi:10.1371/journal.pbio.0050077.g006
selectional pressures. Similar patterns are present in the two
high-identity subtypes seen on the P. ubique HTCC1062
genome (Poster S1D). Other regions show local increases in
similarity between MIT9312 and the dominant subtype that
are not reflected in the MIT9312/MED4 divergence (e.g., near
positions 50 kb, 288 kb, 730 kb, 850 kb, and 954 kb on
MIT9312; also see Poster S1G). These latter regions might
reflect either regions of homogenizing recombination or
regions of higher levels of purifying selection. However, the
lengths of the intervals (several are 10 kb or more) are longer
than any single gene and correspond to genes that are not
extremely conserved over greater taxonomic distances (in
contrast to the ribosomal RNA operon). Equally, if widespread
horizontal transfer of an advantageous segment explains these
intervals, the transfers occurred long enough ago for
appreciable variation to accumulate (unpublished data).
PLoS Biology | www.plosbiology.org | S35
Sorcerer II GOS Expedition
Extreme Assembly of Uncultivated Populations
The analyses described above have been confined to those
organisms with representatives in culture and for which
genomes were readily available. Producing assemblies for
other abundant but uncultivated microbial genera would
provide valuable physiological and biochemical information
that could eventually lead to the cultivation of these
organisms, help elucidate their role in the marine commun-
ity, and allow similar analyses of their evolution and variation
such as those performed on sequenced organisms. Previous
assembly efforts and the fragment recruitments plots showed
that there is considerable and in many cases conflicting
variation among related organisms. Such variation is known
to disrupt whole-genome assemblers. This led us to try an
assembly approach that aggressively resolves conflicts. We call
this approach ‘‘extreme assembly’’ (see Materials and Meth-
ods). This approach currently does not make use of mate-
pairing data and, therefore produces only contigs, not
scaffolded sequences. Using this approach, contigs as large
as 900 kb could be aligned almost in their entirety to the P.
marinus MIT9312 and P. ubique HTCC1062 genomes (Figure
2J–2L). Consistent patterns of fragment recruitment (see
below) generally provided evidence of the correctness of
contigs belonging to otherwise-unsequenced organisms.
Accordingly, large contigs from these alternate assemblies
were used to investigate genetic and geographic population
structure, as described below. However, the more aggressive
assemblies demonstrably suffered from higher rates of
assembly artifacts, including chimerism and false consensus
sequences (Figure 6). Thus, the more stringent primary
assembly was employed for most assembly-based analyses, as
manual curation was not practical.
As just noted, many of the large contigs produced by the
more aggressive assembly methods described above did not
align to any great degree with known genomes. Some could
be tentatively classified based on contained 16S sequences,
but the potential for computationally generated chimerism
within the rRNA operon is sufficiently high that inspection of
the assembly or other means of confirming such classifica-
tions is essential. An alternative to an unguided assembly that
facilitates the association of assemblies with known organisms
is to start from seed fragments that can be identified as
belonging to a particular taxonomic group. We employed
fragments outside the ribosomal RNA operon that were
mated to a 16S-containing read, limiting extension to the
direction away from the 16S operon. This produced contigs
of 100 kb or more for several of the ribotypes that were
abundant in the GOS dataset. When evaluated via fragment
recruitment (Figure 2M–2O), these assemblies revealed
patterns analogous to those seen for the sequenced genomes
described above: multiple subtypes could be distinguished
along the assembly, differing in similarity to the reference
sequence and sample distribution, with occasional gaps.
Hypervariable segments by definition were not represented
in these assemblies, but they may help explain the termi-
nation of the extreme assemblies for P. marinus and SAR11
and provide a plausible explanation for termination of
assemblies of the other deeply sampled populations as well.
This directed approach to assembly can also be used to
investigate variation within a group of related organisms (e.g.,
a 16S ribotype). We explored the potential to assemble
0411 Special Section from March 2007 | Volume 5 | Issue 3 | e77

Figure 7. Fragment Recruitment Plots to 20-kb Segments of SAR11-Like Contigs Show That Many SAR11 Subtypes, with Distinct Distributions, Can Be
Separated by Extreme Assembly
Each segment is constructed of a unique set of GOS sequencing reads (i.e., no read was used in more than one segment). Segments are arbitrarily
labeled (A–X) for reference in Figure 8.
doi:10.1371/journal.pbio.0050077.g007
distinct subtypes of SAR11 by repeatedly seeding extreme
assembly with fragments mated to a SAR11-like 16S sequence.
Figure 7 compares the first 20 kb from each of 24
independent assemblies. Eighteen of these segments could
be aligned full-length to a portion of the HTCC1062 genome
just upstream of 16S, while six appeared to reflect rearrange-
ments relative to HTCC1062. The rearranged segments were
associated with more divergent 16S sequences (8%–14%
diverged from the 16S of HTCC1062), while those without
rearrangements corresponded to less divergent 16S (averag-
ing less than 3% different from HTCC1062). In each segment,
many reads were recruited above 90% identity, but different
samples dominated different assemblies. Phylogenetic trees
support the inference of evolutionarily distinct subtypes with
distinctive sample distributions (Figure 8).
Taxonomic Diversity
Environmental surveys provide a cultivation-independent
means to examine the diversity and complexity of an
environmental sample and serve as a basis to compare the
populations between different samples. Typically, these
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Sorcerer II GOS Expedition
surveys use PCR to amplify ubiquitous but slowly evolving
genes such as the 16S rRNA or recA genes. These in turn can
be used to distinguish microbial populations. Since PCR can
introduce various biases, we identified 16S genes directly
from the primary GOS assembly. In total, 4,125 distinct full-
length or partial 16S were identified. Clustering of these
sequences at 97% identity gave a total of 811 distinct
ribotypes. Nearly half (48%) of the GOS ribotypes and 88%
of the GOS 16S sequences were assigned to ribotypes
previously deposited in public databases. That is, more than
half the ribotypes in the GOS dataset were found to be novel
at what is typically considered the species level [30]. The
overall taxonomic distribution of the GOS ribotypes sampled
by shotgun sequencing is consistent with previously published
PCR based studies of marine environments (Table 7) [31]. A
smaller amount (16%) of GOS ribotypes and 3.4% of the GOS
16S sequences diverged by more than 10% from any publicly
available 16S sequence, thus being novel to at least the family
level.
A census of microbial ribotypes allows us to identify the
abundant microbial lineages and estimate their contribution
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 Sorcerer II GOS Expedition

Figure 8. Phylogeny of GOS Reads Aligning to P. ubique HTCC1062 Upstream of 16S Gene Indicates That the Extreme Assemblies in Figure 7
Correspond to Monophyletic Subtypes
Coloring of branches indicates that the corresponding reads align at .90% identity to the extreme assembly segments shown in Figure 7; colored
labels (A–X) correspond to the labels in Figure 7, indicating the segment or segments to which reads aligned.
doi:10.1371/journal.pbio.0050077.g008

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Table 7. Taxonomic Makeup of GOS Samples Based on 16S Data
from Shotgun Sequencing
Phylum or Class Fractiona
Alpha Proteobacteria 0.32
Unclassified Proteobacteria 0.155
Gamma Proteobacteria 0.132
Bacteroidetes 0.13
Cyanobacteria 0.079
Firmicutes 0.075
Actinobacteria 0.046
Marine Group A 0.022
Beta Proteobacteria 0.017
OP11 0.008
Unclassified Bacteria 0.008
Delta Proteobacteria 0.005
Planctomycetes 0.002
Epsilon Proteobacteria 0.001
a
Values shown are averages over all samples.
doi:10.1371/journal.pbio.0050077.t007
to the GOS dataset. Of the 811 ribotypes, 60 contain more
than 8-fold coverage of the 16S gene (Table 8); jointly, these 60
ribotypes accounted for 73% of all the 16S sequence data. All
but one of the 60 have been detected previously, yet only a few
are represented by close relatives with complete or nearly
complete genome sequencing projects (see Fragment Recruit-
ment for further details). Several other abundant 16S
sequences belong to well-known environmental ribotypes that
do not have cultivated representatives (e.g., SAR86, Roseobacter
NAC-1–2, and branches of SAR11 other than those containing
P. ubique). Interestingly, archaea are nearly absent from the list
of dominant organisms in these near-surface samples.
The distribution of these ribotypes reveals distinct micro-
bial communities (Figure 9 and Table 8). Only a handful of
the ribotypes appear to be ubiquitously abundant; these are
dominated by relatives of SAR11 and SAR86. Many of the
ribotypes that are dominant in one or more samples appear
to reside in one of three separable marine surface habitats.
For example, several SAR11, SAR86, and alpha Proteobacteria,
as well as an Acidimicrobidae group, are widespread in the
surface waters, while a second niche delineated by tropical
samples contains several different SAR86, Synechococcus and
Prochlorococcus (both cyanobacterial groups), and a Rhodospir-
illaceae group. Other ribotypes related to Roseobacter RCA,
SAR11, and gamma Proteobacteria are abundant in the
temperate samples but were not observed in the tropical or
Sargasso samples. Not surprisingly, samples taken from
nonmarine environments (GS33, GS20, GS32), estuaries
(GS11, GS12), and larger-sized fraction filters (GS01a,
GS01b, GS25) have distinguishing ribotypes. Furthermore,
as the complete genomes of these dominant members are
obtained, the capabilities responsible for their abundances
may well lend insight into the community metabolism in
various oceanic niches.
Sample Comparisons
The most common approach for comparing the microbial
community composition across samples has been to examine
the ribotypes present as indicated by 16S rRNA genes or by
analyzing the less-conserved ITS located between the 16S and
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Sorcerer II GOS Expedition
23S gene sequences [7,8,16,17]. However, a clear observation
emerging from the fragment recruitment views was that the
reference ribotypes recruit multiple subtypes, and that these
subtypes were distributed unequally among samples (Figures
2, 7, 8; Poster S1D, S1F, and S1I).
We developed a method to assess the genetic similarity
between two samples that potentially makes use of all
portions of a genome, not just the 16S rRNA region. This
similarity measure is assembly independent; under certain
circumstances, it is equivalent to an estimate of the fraction
of sequence from one sample that could be considered to be
in the other sample. Whole-metagenomic similarities were
computed for all pairs of samples. Results are presented for
comparisons at 98% and 90% identity. No universal cutoff
consistently divides sequences into natural subsets, but the
98% identity cutoff provides a relatively high degree of
resolution, while the 90% cutoff appears to be a reasonable
heuristic for defining subtypes. For instance, a 90% cutoff
treats most of the reads specifically recruited to P. marinus
MIT9312 as similar (those more similar to MED4 notably
excepted), while reasonably separating clades of SAR11
(Figures 7 and 8). Reads with no qualifying overlap alignment
to any other read in a pair of samples are uninformative for
this analysis, as they correspond to lineages that were so
lightly sequenced that their presence in one sample and
absence in another may be a matter of chance. For the 90%
cutoff, 38% of the sequence reads contributed to the analysis.
The resulting similarities reveal clear and consistent group-
ings of samples, as well as the outlier status of certain samples
(Figures 10 and 11).
The broadest contrast was between samples that could be
loosely labeled ‘‘tropical’’ (including samples from the
Sargasso Sea [GS00b, GS00c, GS00d] and samples that are
temperate by the formal definition but under the influence of
the Gulf Stream [GS14, GS15]) and ‘‘temperate.’’ Further
subgroups can be identified within each of these categories, as
indicated in Figures 10 and 11. In some cases, these groupings
were composed of samples taken from different ocean basins
during different legs of the expedition. A few pairs of samples
with strikingly high similarity were observed, including GS17
and GS18, GS23 and GS26, GS27 and GS28, and GS00b and
GS00d. In each case, these pairs of samples were collected
from consecutive or nearly consecutive samples. However,
the same could be said of many other pairs of samples that do
not show this same degree of similarity. Indeed, geographi-
cally and temporally separated samples taken in the Atlantic
(GS17, GS18) and Pacific (GS23, GS26) during separate legs of
the expedition are more similar to one another than were
most pairs of consecutive samples. The samples with least
similarity to any other sample were from unique habitats.
Thus, similarity cannot be attributed to geographic separa-
tion alone.
The groupings described above can be reconstructed from
taxonomically distinct subsets of the data. Specifically, the
major groups of samples visible in Figure 10 were reproduced
when sample similarities were determined based only on
fragments recruiting to P. ubique HTCC1062 (unpublished
data). Likewise, the same groupings were observed when the
fragments recruiting to either HTCC1062 or P. marinus
MIT9312, or both, were excluded from the calculations
(unpublished data). Thus, the factors influencing sample
similarities do not appear to rely solely on the most abundant
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 Sorcerer II GOS Expedition

Table 8. Most Abundant Ribotypes (97% Identity Clusters)

Ribotype Classificationa Depth of Coverageb Range Number of Matching GenBank Entriesc

SAR11 Surface 1 581 Widespread 100þ

SAR11 Surface 2 182 Sargasso and GS31d 100þ
Burkholderia 139 00a 100þ
Acidomicrobidae type a 133 Tropical and Sargassod 77
Prochlorococcus 112 Tropical and Sargasso 76
SAR11 Surface 3 109 Widespread 63
SAR86-like type a 108 Widespread 88
Shewanella 80 00a 49
Synechococcus 59 Tropicald 100þ
Rhodospirillaceae 50 Tropical and Sargassod 15
SAR86-like type b 47 Hypersaline pondd 24
SAR86-like type c 47 Tropical and Sargasso 75
Chlorobi-like 40 Hypersaline pond 1
Alpha Proteobacteria type a 38 Widespread 10
Roseobacter type a 35 Tropical and Sargassod 42
Cellulomonadaceae type a 34 Hypersaline pond 12
SAR86-like type d 28 Widespread 30
Alpha Proteobacteria type b 27 Widespread 43
SAR86-like type e 26 Widespread 71
Cytophaga type a 24 Tropical and Sargasso 21
Bacteroidetes type a 23 Widespread 17
Bdellovibrionales type a 21 Tropical and Sargasso 36
Acidomicrobidae type b 21 Temperate 41
SAR116-like 21 Widespread 28
Marine Group A type a 20 Tropical and Sargassod 9
Remotely SAR11-like type a 19 Sargasso and tropical 12
Frankineae type a 19 Fresh and estuary 6
Frankineae type b 18 Hypersaline pondd 71
SAR86-like type f 18 Tropical 15
SAR86-like type g 17 Tropical 13
Remotely SAR11-like type b 16 Tropical and Sargasso 4
Gamma Proteobacteria type a 16 Sargasso and GS14 18
Microbacteriaceae 15 Hypersaline pond 1
SAR102/122-like 15 Tropical and Sargasso 15
SAR86-like type h 15 Tropical and Sargasso 27
Bacteroidetes type b 14 Tropicald 14
Remotely SAR11-like type c 14 Tropical and Sargasso 18
SAR86-like type i 14 Sargassod 13
Rhodobium-like type a 14 Sargasso and GS31d 9
Marine Group A type b 13 Tropical and Sargassod 28
Gamma Proteobacteria type b 12 S. temperate and GS31 22
Oceanospirillaceae 12 Mangrove 1
Gamma Proteobacteria type c 12 Widespread 14
SAR11-like type a 12 Temperate 17
SAR11-like type b 11 Fresh and estuary 11
SAR11-like type c 11 Sargasso and GS31d 12
SAR86-like type j 11 Tropical and Sargasso 1
Roseobacter Algicola 11 Widespread 20
Remotely SAR102/122-like 11 Hypersaline pond 0
Frankineae type c 10 Fresh and estuary 11
Rhodobium-like type b 10 Widespread 9
Roseobacter RCA 10 Temperate 39
Remotely SAR11-like type d 10 Tropical 32
Acidobacteria 9 Fresh 4
Remotely SAR11-like type e 9 Tropical and Sargasso 8
Frankineae type d 9 Estuary and fresh 89
SAR11-like type d 9 Sargasso and GS31 4
Methylophilus 9 Temperated 41
Archaea C1 C1a 8 Sargasso and GS31 2
Cytophaga type b 8 Widespread 9

a
Taxonomic classifications based on Hugenholtz ARB database. Labels indicate the most specific taxonomic assignment that could be confidently assigned to each ribotype. ‘‘Type a,’’
‘‘type b,’’ etc., used to arbitrarily discriminate separate 97% ribotypes that would otherwise be given the same name.
b
Note that the 16S rRNA gene can be multicopy.
c
Matching GenBank entries required full-length matches at  98% identity.
d
Less than 13 coverage outside described range.
doi:10.1371/journal.pbio.0050077.t008

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 Sorcerer II GOS Expedition

Figure 9. Presence and Abundance of Dominant Ribotypes

The relative abundance of various ribotypes (rows) in each filter (columns) is represented by the area of the corresponding spot (if any). The listed
ribotypes each satisfied the following criteria in at least one filter: the ribotype was among the five most abundant ribotypes detected in the shotgun
data, and was represented by at least three sequencing reads. Relative abundance is based on the total number of 16S sequences in a given filter. Order
and grouping of filters is based on the clustering of genomic similarity shown in Figure 11. Ribotype order was determined based on similarity of
sample distribution. A marked contrast between temperate and tropical groups is visible. Estuarine samples GS11 and GS12 contained a mix of
ribotypes seen in freshwater and temperate marine samples, while samples from nonmarine habitats or larger filter sizes were pronounced outliers. The
presence of large amounts of Burkholderia and Shewanella in one Sargasso Sea sample (GS00a) makes this sample look much less like other Sargasso
and tropical marine samples than it otherwise would. Note that 16S is not a measure of cell abundance since 16S genes can be multicopy.
doi:10.1371/journal.pbio.0050077.g009

organisms but rather are reflected in multiple microbial
lineages.
It is tempting to view the groups of similar samples as
constituting community types. Sample similarities based on
genomic sequences correlated significantly with differences
in the environmental parameters (Table 1), particularly water
temperature and salinity (unpublished data). Samples that are
very similar to each other had relatively small differences in
temperature and salinity. However, not all samples that had
similar temperature and salinity had high community
similarities. Water depth, primary productivity, fresh water
input, proximity to land, and filter size appeared consistent
with the observed groupings. Other factors such as nutrients
and light for phototrophs and fixed carbon/energy for
chemotrophs may ultimately prove better predictors, but
these results demonstrate the potential of using metagenomic
data to tease out such relationships.
Examining the groupings in Figure 11 in light of habitat
and physical characteristics, the following may be observed.
The first two samples, a hypersaline pond in the Galapagos
Islands (GS33) and the freshwater Lake Gatun in the Panama
Canal (GS20) are quite distinct from the rest. Salinity—both
higher and lower than the remaining coastal and ocean
samples—is the simplest explanation.

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 Sorcerer II GOS Expedition

Twelve samples form a strong temperate cluster as seen in samples from Nova Scotia through the Gulf of Maine. This
the similarity matrix of Figure 11 as a darker square bounded is followed by a subcluster of four samples between Rhode
by GS06 and GS12. Embedded within the temperate cluster Island and North Carolina. The northern subcluster was
are three subclusters. The first subcluster includes five sampled in August, the southern subcluster in November and

Figure 10. Similarity between Samples in Terms of Shared Genomic Content

Genomic similarity, as described in the text, is an estimate of the amount of the genetic material in two filters that is ‘‘the same’’ at a given percent
identity cutoff—not the amount of sequence in common in a finite dataset, but rather in the total set of organisms present on each filter. Similarities are
shown for 98% identity.
(A) Hierarchical clustering of samples based on pairwise similarities.
(B) Pairwise similarities between samples, represented as a symmetric matrix of grayscale intensities; a darker cell in the matrix indicates greater
similarity between the samples corresponding to the row and column, with row and column ordering as in (A). Groupings of similar filters appear as
subtrees in (A) and as squares consisting of two or more adjacent rows and columns with darker shading. Colored bars highlight groups of samples
described in the text; labels are approximate characterizations rather than being strictly true of every sample in a group.
doi:10.1371/journal.pbio.0050077.g010

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Figure 11. Sample Similarity at 90% Identity
Similarity between samples in terms of shared genomic content similar to Figure 10, except that the plots were done using a 90% identity cutoff that
has proven reasonable for separating some moderately diverged subtypes
doi:10.1371/journal.pbio.0050077.g011
December. Though all samples were collected in the top few
meters, the southern samples were in shallower waters, 10 to
30 m deep, whereas most of the northern samples were in
waters greater than 100 m deep. Monthly average estimates of
chlorophyll a concentrations were typically higher in the
southern samples as well (Table 1). All of these factors—
temperature, system primary production, and depth of the
sampled water body—likely contribute to the differences in
microbial community composition that result in the two well-
defined clusters. The final temperate subgroup includes two
estuaries, Chesapeake Bay (GS12) and Delaware Bay (GS11),
distinguished by their lower salinity and higher productivity.
However, GS11 is markedly similar not only to GS12 but also
to coastal samples, whereas the latter appears much more
unique. Interestingly, the Bay of Fundy estuary sample (GS06)
clearly did not group with the two other estuaries, but rather
with the northern subgroup, perhaps reflecting differences in
the rate or degree of mixing at the sampling site.
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Sorcerer II GOS Expedition
Continuing to the right and downward in Figure 11, one
can see a large cluster of 25 samples from the tropics and
Sargasso Sea, bounded by GS47 and GS00b. This can be
further subdivided into several subclusters. The first sub-
cluster (a square bounded by GS47 and GS14) includes 14
samples, about half of which were from the Galapagos. The
second distinct subcluster (a square bounded by GS16 and
GS26) includes seven samples from Key West, Florida, in the
Atlantic Ocean to a sample close to the Galapagos Islands in
the Pacific Ocean. Loosely associated with this subcluster is a
sample from a larger filter size taken en route to the
Galapagos (GS25). The remaining samples group weakly with
the tropical cluster. GS32 was taken in a coastal mangrove in
the Galapagos. The thick organic sediment at a depth of less
than a meter is the likely cause for it being unlike the other
samples. Sample 00a was from the Sargasso Sea and contained
a large fraction of sequence reads from apparently clonal
Burkholderia and Shewanella species that are atypical. When this
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 Sorcerer II GOS Expedition

Table 9. Relative Abundance of TIGRFAMs Associated with a Specific Sample

TIGRFAM Number of Samplea Relative Major Minor Description

Peptides Abundanceb Category Category

TIGR01526 131 GS01a 3.4 Nicotinamide-nucleotide adenylyltransferase

TIGR00661 214 GS01a 2.6 Hypothetical Conserved Conserved hypothetical protein
TIGR01833 135 GS01a 2 Hydroxymethylglutaryl-CoA synthase
TIGR01408 267 GS01b 4.5 Ubiquitin-activating enzyme E1
TIGR01678 144 GS01b 2.6 Sugar 1,4-lactone oxidases
TIGR01879 758 GS01b 2.5 Amidase, hydantoinase/carbamoylase family
TIGR00890 131 GS01b 2.4 Transport Carbohydrates, Oxalate/Formate Antiporter
TIGR01767 112 GS01b 2.4 5-methylthioribose kinase
TIGR00101 495 GS01b 2.3 Central Nitrogen Urease accessory protein UreG
TIGR01659 186 GS01b 2.2 Sex-lethal family splicing factor
TIGR00313 455 GS01b 2.1 Biosynthesis Heme, Cobyric acid synthase CobQ
TIGR00317 306 GS01b 2.1 Biosynthesis Heme, Cobalamin 59-phosphate synthase
TIGR00601 186 GS01b 2.1 DNA DNA UV excision repair protein Rad23
TIGR01792 904 GS01b 2.1 Central Nitrogen Urease, alpha subunit
TIGR00749 483 GS01b 2 Energy Glycolysis/gluconeogenesis Glucokinase
TIGR01001 485 GS01b 2 Amino Aspartate Homoserine O-succinyltransferase
TIGR02238 165 GS32 5.1 Meiotic recombinase Dmc1
TIGR02239 159 GS32 3.5 DNA repair protein RAD51
TIGR02232 140 GS32 2.6 Myxococcus cysteine-rich repeat
TIGR02153 212 GS32 2.5 Protein tRNA Glutamyl-tRNA(Gln) amidotransferase, subunit D
TIGR00519 289 GS32 2.4 L-asparaginases, type I
TIGR00288 248 GS32 2 Hypothetical Conserved Conserved hypothetical protein TIGR00288
TIGR01681 136 GS32 2 HAD-superfamily phosphatase, subfamily IIIC
TIGR02236 143 GS32 2 DNA DNA DNA repair and recombination protein RadA
TIGR00028 110 GS33 14.7 Mycobacterium tuberculosis PIN domain family
TIGR01550 131 GS33 9.1 Unknown General Death-on-curing family protein
TIGR01552 200 GS33 5.3 Mobile Other Prevent-host-death family protein
TIGR00143 151 GS33 4.6 Protein Protein [NiFe] hydrogenase maturation protein HypF
TIGR01641 131 GS33 4.3 Mobile Prophage Phage putative head morphogenesis protein, SPP1 gp7 family
TIGR01710 1,926 GS33 3.9 Cellular Pathogenesis General secretion pathway protein G
TIGR01539 251 GS33 3.6 Mobile Prophage Phage portal protein, lambda family
TIGR00016 217 GS33 3.5 Energy Fermentation Acetate kinase
TIGR00942 117 GS33 3.5 Transport Cations Multicomponent Naþ:Hþ antiporter
TIGR01836 174 GS33 3.3 Fatty Biosynthesis Poly(R)-hydroxyalkanoic acid synthase, class III, PhaC subunit
TIGR02110 135 GS33 3.2 Biosynthesis Other Coenzyme PQQ biosynthesis protein PqqF
TIGR01106 902 GS33 3.1 Energy ATP-proton Na,H/K antiporter P-type ATPase, alpha subunit
TIGR01497 726 GS33 3.1 Transport Cations Kþ-transporting ATPase, B subunit
TIGR01524 603 GS33 3.1 Transport Cations Magnesium-translocating P-type ATPase
TIGR02140 166 GS33 3.1 Transport Anions Sulfate ABC transporter, permease protein CysW
TIGR01409 122 GS33 3 Tat (twin-arginine translocation) pathway signal sequence
TIGR02195 282 GS33 3 Cell Biosynthesis Lipopolysaccharide heptosyltransferase II
TIGR01522 696 GS33 2.9 Calcium-transporting P-type ATPase, PMR1-type
TIGR01523 766 GS33 2.9 Potassium/sodium efflux P-type ATPase, fungal-type
TIGR00202 228 GS33 2.8 Regulatory RNA Carbon storage regulator
TIGR01116 798 GS33 2.8 Transport Cations Calcium-translocating P-type ATPase, SERCA-type
TIGR02094 204 GS33 2.8 Alpha-glucan phosphorylases
TIGR02051 296 GS33 2.7 Regulatory DNA Hg(II)-responsive transcriptional regulator
TIGR01005 259 GS33 2.6 Transport Carbohydrates, Exopolysaccharide transport protein family
TIGR01222 129 GS33 2.6 Cellular Cell Septum site-determining protein MinC
TIGR01334 157 GS33 2.6 Unknown General modD protein
TIGR01554 152 GS33 2.6 Mobile Prophage Phage major capsid protein, HK97 family
TIGR00640 1,059 GS33 2.5 Methylmalonyl-CoA mutase C-terminal domain
TIGR01708 801 GS33 2.5 Cellular Pathogenesis General secretion pathway protein H
TIGR02018 373 GS33 2.5 Regulatory DNA Histidine utilization repressor
TIGR00554 182 GS33 2.4 Biosynthesis Pantothenate Pantothenate kinase
TIGR01202 152 GS33 2.4 Biosynthesis Chlorophyll Chlorophyll synthesis pathway, bchC
TIGR01583 102 GS33 2.4 Energy Electron Formate dehydrogenase, gamma subunit
TIGR02092 241 GS33 2.4 Energy Biosynthesis Glucose-1-phosphate adenylyltransferase, GlgD subunit
TIGR00052 263 GS33 2.3 Hypothetical Conserved Conserved hypothetical protein TIGR00052
TIGR00824 166 GS33 2.3 Signal PTS PTS system, mannose/fructose/sorbose family, IIA component
TIGR01003 321 GS33 2.3 Transport Carbohydrates, Phosphocarrier, HPr family
TIGR01439 191 GS33 2.3 Regulatory DNA Transcriptional regulator, AbrB family
TIGR01457 220 GS33 2.3 HAD-superfamily subfamily IIA hydrolase, TIGR01457
TIGR01517 684 GS33 2.3 Calcium-translocating P-type ATPase, PMCA-type
TIGR02028 632 GS33 2.3 Biosynthesis Chlorophyll Geranylgeranyl reductase
TIGR00452 217 GS33 2.2 Unknown Enzymes Methyltransferase, putative
TIGR00609 2,555 GS33 2.2 DNA DNA Exodeoxyribonuclease V, beta subunit
TIGR00876 265 GS33 2.2 Energy Pentose Transaldolase

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 Sorcerer II GOS Expedition

Table 9. Continued.

TIGRFAM Number of Samplea Relative Major Minor Description

Peptides Abundanceb Category Category

TIGR00996 302 GS33 2.2 Cellular Pathogenesis Virulence factor Mce family protein
TIGR01254 419 GS33 2.2 Transport Other ABC transporter periplasmic binding protein, thiB subfamily
TIGR01278 508 GS33 2.2 Biosynthesis Chlorophyll Light-independent protochlorophyllide reductase, B subunit
TIGR01512 1,820 GS33 2.2 Transport Cations Cadmium-translocating P-type ATPase
TIGR01525 2,037 GS33 2.2 Heavy metal translocating P-type ATPase
TIGR01543 418 GS33 2.2 Protein Other Phage prohead protease, HK97 family
TIGR01857 1,495 GS33 2.2 Purines Purine Phosphoribosylformylglycinamidine synthase
TIGR02015 183 GS33 2.2 Energy Photosynthesis Chlorophyllide reductase subunit Y
TIGR02072 1,052 GS33 2.2 Biosynthesis Biotin Biotin biosynthesis protein BioC
TIGR02099 273 GS33 2.2 Hypothetical Conserved Conserved hypothetical protein TIGR02099
TIGR00203 168 GS33 2.1 Energy Electron Cytochrome d ubiquinol oxidase, subunit II
TIGR00218 141 GS33 2.1 Energy Sugars Mannose-6-phosphate isomerase, class I
TIGR00915 4,834 GS33 2.1 Transport Other Transporter, hydrophobe/amphiphile efflux-1 (HAE1) family
TIGR01315 319 GS33 2.1 FGGY-family pentulose kinase
TIGR01330 253 GS33 2.1 39(29),59-bisphosphate nucleotidase
TIGR01508 848 GS33 2.1 Diaminohydroxyphosphoribosylaminopyrimidine reductase
TIGR01511 1,928 GS33 2.1 Transport Cations Copper-translocating P-type ATPase
TIGR01764 387 GS33 2.1 Unknown General DNA binding domain, excisionase family
TIGR02014 298 GS33 2.1 Energy Photosynthesis Chlorophyllide reductase subunit Z
TIGR02047 222 GS33 2.1 Cd(II)/Pb(II)-responsive transcriptional regulator
TIGR00586 990 GS33 2 DNA DNA Mutator mutT protein
TIGR00853 114 GS33 2 Signal PTS PTS system, lactose/cellobiose family IIB component
TIGR00937 364 GS33 2 Transport Anions Chromate transporter, chromate ion transporter (CHR) family
TIGR01030 221 GS33 2 Protein Ribosomal Ribosomal protein L34
TIGR01214 2,834 GS33 2 Cell Biosynthesis dTDP-4-dehydrorhamnose reductase
TIGR01698 608 GS33 2 Purine nucleotide phosphorylase
TIGR02190 465 GS33 2 Glutaredoxin-family domain

a
Reads associated with Shewanella and Burkholderia have been excluded.
b
TIGRFAM is this many times more abundant than in the next most abundant sample.
doi:10.1371/journal.pbio.0050077.t009

sample is reanalyzed to exclude reads identified as belonging
to these two groups, sample GS00a groups loosely with
GS00b, GS00c, and GS00d (unpublished data). Finally, three
subsamples from a single Sargasso sample (GS01a, GS01b,
GS01c) group together, despite representing three distinct
size fractions (3.0–20, 0.8–3.0, and 0.1–0.8 lm, respectively;
Table 1).
The complete set of sample similarities is more complex
than described above, and indeed is more complex than can
be captured by a hierarchical clustering. For instance, the
southern temperate samples are appreciably more similar to
the tropical cluster than are the northern temperate samples.
GS22 appears to constitute a mix of tropical types, showing
strong similarity not only to the GS47–GS14 subcluster to
which it was assigned, but also to the other tropical samples.
These results may be compared to the more traditional
view of community structure afforded by 16S sequences
(Figure 9). Some of the same groupings of samples are visible
using both analyses. Several ribotypes recapitulated the
temperate/tropical clustering described above. Others were
restricted to the single instances of nonmarine habitats.
Several of the most abundant organisms from the coastal
mangrove, hypersaline lagoon, and freshwater lake were
found exclusively in these respective samples. However, while
several ribotypes recapitulated the temperate/tropical dis-
tinction revealed by the genomic sequence, others crosscut it.
A few dominant 16S ribotypes, related to SAR11, SAR86, and
SAR116, were found in every marine sample. The brackish
waters from two mid-Atlantic estuaries (GS11 and GS12)
contained a mixture of otherwise exclusively marine and
freshwater ribotypes; similarity of these sites to the fresh-
water sample (GS20) was minimal at the metagenomic level,
while the greater similarity of GS11 to coastal samples visible
at the metagenomic level was not readily visible here. A fuller
comparison of metagenome-based measurements of diversity
based on a large dataset of PCR-derived 16S sequences will be
presented in another paper (in preparation).
Variation in Gene Abundance
Differences in gene content between samples can identify
functions that reflect the lifestyles of the community in the
context of its local environment [20,32]. We examined the
relative abundance of genes belonging to specific functional
categories in the distinct GOS samples. Genes were binned
into functional categories using TIGRFAM hidden Markov
models [18], which are well annotated and manually curated
[33].
The results can be filtered in various ways to highlight
genes associated with specific environments. One catalog of
possible interest is genes that were predominantly found in a
single sample. We identified 95 TIGRFAMs that annotated
large sets of genes (100 or more) that were significantly more
frequent (greater than 2-fold) in one sample than in any other
sample (Table 9). Not surprisingly, this approach dispropor-
tionately singles out genes from the samples collected on
larger filters (GS01a, GS01b, and GS25) and from the
nonmarine environments, particularly the hypersaline pond
(sample GS33). Another contrast might be between the

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 Sorcerer II GOS Expedition

Table 10. Relative Abundance of TIGRFAM Matches in Temperate and Tropical Waters

TIGRFAM Number of Sample(s) Relative Major Minor Description

Peptides Abundancea Category Category

TIGR01153 729 GS15–GS19 32.7 Energy Photosynthesis Photosystem II 44 kDa subunit reaction center protein
TIGR02093 673 GS15–GS19 29.6 Energy Biosynthesis Glycogen/starch/alpha-glucan phosphorylases
TIGR01335 813 GS15–GS19 26.6 Energy Photosynthesis Photosystem I core protein PsaA
TIGR01336 806 GS15–GS19 26.5 Energy Photosynthesis Photosystem I core protein PsaB
TIGR00975 648 GS15–GS19 11 Transport Anions Phosphate ABC transporter, phosphate-binding protein
TIGR00297 261 GS15–GS19 8.6 Hypothetical Conserved Conserved hypothetical protein TIGR00297
TIGR00992 302 GS15–GS19 8.5 Transport Amino Chloroplast envelope protein translocase, IAP75 family
TIGR02030 560 GS15–GS19 7.5 And Chlorophyll Magnesium chelatase ATPase subunit I
TIGR02041 359 GS15–GS19 6 Central Sulfur Sulfite reductase (NADPH) hemoprotein, beta-component
TIGR01151 2,095 GS15–GS19 4.7 Energy Photosynthesis Photosystem q(b) protein
TIGR01152 1,865 GS15–GS19 4.7 Energy Photosynthesis Photosystem II D2 protein (photosystem q(a) protein)
TIGR02031 800 GS15–GS19 4.2 Biosynthesis Chlorophyll Magnesium chelatase ATPase subunit D
TIGR01790 629 GS15–GS19 4 Lycopene cyclase family protein
TIGR02100 512 GS15–GS19 4 Energy Biosynthesis Glycogen debranching enzyme GlgX
TIGR00073 284 GS15–GS19 3.4 Protein Protein Hydrogenase accessory protein HypB
TIGR00159 497 GS15–GS19 3 Hypothetical Conserved Conserved hypothetical protein TIGR00159
TIGR01515 594 GS15–GS19 3 Energy Biosynthesis 1,4-alpha-glucan branching enzyme
TIGR00217 601 GS15–GS19 2.7 Energy Biosynthesis 4-alpha-glucanotransferase
TIGR01486 505 GS15–GS19 2.7 Mannosyl-3-phosphoglycerate phosphatase family
TIGR01098 720 GS15–GS19 2.6 Transport Carbohydrates Phosphonate ABC transporter, periplasmic phosphonate-binding protein
TIGR00101 495 GS15–GS19 2.5 Central Nitrogen Urease accessory protein UreG
TIGR01273 567 GS15–GS19 2.4 Central Polyamine Arginine decarboxylase
TIGR01470 179 GS5–GS10 25.7 Biosynthesis Heme Siroheme synthase, N-terminal domain
TIGR00361 374 GS5–GS10 12.1 Cellular DNA DNA internalization-related competence protein ComEC/Rec2
TIGR01537 333 GS5–GS10 6.2 Mobile Prophage Phage portal protein, HK97 family
TIGR00201 291 GS5–GS10 6 Cellular DNA comF family protein
TIGR00879 420 GS5–GS10 5 Transport Carbohydrates Sugar transporter
TIGR02018 373 GS5–GS10 4.1 Regulatory DNA Histidine utilization repressor
TIGR02183 294 GS5–GS10 4.1 Glutaredoxin, GrxA family
TIGR00427 602 GS5–GS10 4 Hypothetical Conserved Conserved hypothetical protein TIGR00427
TIGR01109 219 GS5–GS10 3.6 Energy Other Sodium ion-translocating decarboxylase, beta subunit
TIGR01262 840 GS5–GS10 2.8 Energy Amino Maleylacetoacetate isomerase

a
Average abundance of TIGRFAM is that many times more abundant the average abundance in the given samples than in the other set of samples (in this case, GS15–GS19 were
compared with GS5–GS10).
doi:10.1371/journal.pbio.0050077.t010

temperate and tropical clusters (Figures 10 and 11). We
identified 32 proteins that were more than 2-fold more
frequent in one or the other group (Table 10). The presence
of various Prochlorococcus-associated genes in this list high-
lights some of the potential challenges with this sort of
approach. Overrepresentation may reflect: a direct response
to particular environmental pressures (as the excess of salt
transporters plausibly do in the hypersaline pond); a lineage-
restricted difference in functional repertoire (as exemplified
by the excess of photosynthesis genes in samples containing
Prochlorococcus); or a more incidental ‘‘hitchhiking’’ of a
protein found in a single organism that happens to be
present.
We explored whether clearer and more informative
differences could be discovered between communities by
focusing on groups of samples that are highly similar in
overall taxonomic/genetic content. Two pairs of samples
provide a particularly nice illustration of this approach.
Samples GS17 and GS18 from the western Caribbean Sea and
samples GS23 and GS26 from the eastern Pacific Ocean were
all very similar based on the presence of abundant ribotypes
and overall similarity in genetic content (Figures 9–11).
Despite these similarities, several genes are found to be up to
seven times more common in the pair of Caribbean samples
than the Pacific pair (Table 11). No genes are more than 2-
fold higher in the Pacific than the Caribbean pair of samples.
Several of the most differentially abundant genes are related
to phosphate transport and utilization. It is very plausible
that this is a reflection of a functional adaptation: these
differences correlate well with measured differences in
phosphate abundance between the Atlantic and eastern
Pacific samples [34,35], and phosphate abundance plays a
critical role in microbial growth [36,37]. Indeed, the ability to
acquire phosphate, especially under conditions where it is
limited, is thought to determine the relative fitness of
Prochlorococcus strains [38].
The single greatest difference between GS17 and GS18 on
the one hand and GS23 and GS26 on the other was attributed
to a set of genes annotated by the hidden Markov model
TIGR02136 as a phosphate-binding protein (PstS). This
TIGRFAM identified a single gene in both P. marinus
MIT9312 and P. ubique HTCC1062. In P. marinus MIT9312,
this gene is located at 672 kb lying roughly in the middle of a
15-kb segment of the genome that recruits almost no GOS
sequences from the Pacific sampling sites (Poster S1H). In P.
ubique HTCC1062, the PstS gene is found at 1,133 kb in a 5-kb
segment that also recruited far fewer GOS sequences from all
the Pacific samples except for GS51 (Poster S1E). These
genomic segments differ structurally among isolates but they
are no more variable than the flanking regions, and thus are

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 Sorcerer II GOS Expedition

Table 11. Relative Abundance of TIGRFAM Matches in Atlantic and Pacific Open Ocean Waters

TIGRFAM Number of Sample(s) Relative Major Minor Description

Peptides Abundancea Category Category

TIGR02136 1,130 GS17, GS18 7.2 Transport Anions Phosphate-binding protein

TIGR00974 2,122 GS17, GS18 3.5 Transport Anions Phosphate ABC transporter, permease protein PstA
TIGR00975 648 GS17, GS18 3.5 Transport Anions Phosphate ABC transporter, phosphate-binding protein
TIGR02138 2,139 GS17, GS18 3.4 Transport Anions Phosphate ABC transporter, permease protein PstC
TIGR00206 459 GS17, GS18 2.8 Cellular Chemotaxis Flagellar M-ring protein FliF
TIGR01782 1,297 GS17, GS18 2.4 Transport Unknown TonB-dependent receptor
TIGR00642 862 GS17, GS18 2.3 Central Other Methylmalonyl-CoA mutase, small subunit
TIGR02135 899 GS17, GS18 2.3 Transport Anions Phosphate transport system regulatory protein PhoU

a
Relative Abundance: average abundance of TIGRFAM is at least that many times more abundant the average abundance in the given samples than in the other set of samples (in this
case, GS17–GS18 were compared to GS23 and GS26).
doi:10.1371/journal.pbio.0050077.t011

not hypervariable in the sense used previously (unpublished
data). Nor are they particularly conserved when present,
indicating that they are not the result of a recent lateral
transfer. Phylogenetic analyses outside these segments did not
produce any evidence of a Pacific versus Caribbean clade of
either Prochlorococcus or SAR11 (Figure 3A–3B). The presence
or absence of phosphate transporters is not limited to these
two types of organisms. The number of phosphate trans-
porters that were found in the Caribbean far exceeds the
number that can be attributed to HTCC1062- and MIT9312-
like organisms. However, these results indicate that within
individual strains or subtypes the ability to acquire phosphate
(in one or more of its forms) can vary without detectable
differences in the surrounding genomic sequences.
Biogeographic Distribution of Proteorhodopsin Variants
Variation in gene content is only one aspect of the
tremendous diversity in the GOS data. The functional
significance of all the polymorphic differences between
homologous proteins remains largely unknown. To look for
functional differences, we analyzed members of proteorho-
dopsin gene family. Proteorhodopsins are fast, light-driven
proton pumps for which considerable functional information
is available though their biological role remains unknown.
Proteorhodopsins were highly abundant in the Sargasso Sea
samples [19] and continue to be highly abundant and evenly
distributed (relative to recA abundance) in all the GOS
samples. A total of 2,674 putative proteorhodopsin genes
were identified in the GOS dataset. Although many of the
sequences are fragmentary, 1,874 of these genes contain the
residue that is primarily responsible for tuning the light-
absorbing properties of the protein [39–41], and these
properties have been shown to be selected for under different
environmental conditions [42]. Variation at this residue is
strongly correlated with sample of origin (Figure 12). The
leucine (L) or green-tuned variant was highly abundant in the
North Atlantic samples and in the nonmarine environments
like the fresh water sample from Lake Gatun (GS20). The
glutamine (Q) or blue-tuned variant dominated in the
remaining mostly open ocean samples.
Given our limited understanding of the biological role for
proteorhopsin, the reason for this differential distribution is
not immediately clear. In coastal waters where nutrients are
more abundant, phytoplankton is dominant. Phytoplankton
absorbs primarily in the blue and red spectra; consequently,
the water appears green [43]. Conversely, in the open ocean
nutrients are rare and phytoplanktonic biomass is low, so
waters appear blue because in the absence of impurities the
red wavelengths are absorbed preferentially [44]. It may be
that proteorhodopsin-carrying microbes have simply adapted
to take advantage of the most abundant wavelengths of light
in these systems.
Proteorhodopsins encoded on reads that were recruited to
P. ubique HTCC1062 account for a fraction (;25%) of all the
proteorhodopsin-associated reads, suggesting that the re-
mainder must be associated with a variety of marine micro-
bial taxa (see also [45–47]). Phylogenetic analysis of the
SAR11-associated proteins revealed that each variant has
arisen independently at least two times in the SAR11 lineage
(Figure 3C). Consistent with other findings that proteorho-
dopsins are widely distributed throughout the microbial
world [48], we conclude that multiple microbial lineages are
responsible for proteorhodopsin spectral variation and that
the abundance of a given variant reflects selective pressures
rather than taxonomic effects. Similar mechanisms seem to
be involved in the evolution and diversification of opsins that
mediate color vision in vertebrates [49].
Discussion
Our results highlight the astounding diversity contained
within microbial communities, as revealed through whole-
genome shotgun sequencing carried out on a global scale.
Much of this microbial diversity is organized around
phylogenetically related, geographically dispersed popula-
tions we refer to as subtypes. In addition, there is tremendous
variation within subtypes, both in the form of sequence
variation and in hypervariable genomic islands. Our ability to
make these observations derived from not only the large
volumes of data but also from the development of new tools
and techniques to filter and organize the information in
manageable ways.
Variation and Diversity
Our data demonstrate to an unprecedented degree the
nature and evolution of genetic variation below the species
level. Variation can be analyzed in several ways, including
observed differences in sequence, genomic structure, and

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 Sorcerer II GOS Expedition

Figure 12. Distribution of Common Proteorhodopsin Variants across GOS Samples

The leucine (L) and methionine (M) variants absorb maximally in the green spectrum (Oded Beja, personal communication) while the glutamine (Q)
variant absorbs maximally in the blue spectrum. The relative abundance of each variant is shown as a percentage (x-axis) per sample (y-axis). Total
abundance for all variants in read equivalents normalized by the abundance of recA protein are shown on the right side of the y-axis. The L and Q
variants show a nonrandom distribution. The L variant is abundant in temperate Atlantic waters close to the U.S. and Canadian coast. The Q variant is
abundant in warmer waters further from land. The M variant is moderately abundant in a wide range of samples with no obvious geographic/
environmental association.
doi:10.1371/journal.pbio.0050077.g012

gene complement. The observed patterns of variation shed
light on the mechanisms by which marine prokaryotes evolve.
Gene synteny seems to be more highly conserved than the
nucleotide and protein sequences. This variation is seen over
essentially the entire genome in every abundant group of
organisms sufficiently related for us to recognize a popula-
tion by fragment recruitment. (These include, but are not
limited to, the organisms shown in Figure 2 and Poster S1.)
Notably, we found no evidence of widespread low-diversity
organisms such as B. anthracis [50].
Phylogenetic trees and fragment recruitment plots (Figures
7 and 8) indicate that the variation within a species is not an
unstructured swarm or cloud of variants all equally diverged
from one another. Instead, there are clearly distinct subtypes,
in terms of sequence similarity, gene content, and sample
distribution. Similar findings have been shown for specific
organisms, based on evaluation of one or a few loci [2,51–53].
These results rule out certain trivial models of population
history and evolution for what is commonly considered a
bacterial ‘‘species.’’ For instance, it argues against a recent
explosive population growth from a single successful indi-
vidual (selective sweep) [54]. Equally, it argues against a
perfectly mixed population, suggesting instead some barriers
to competition and exchange of genetic material.
In principle, this variation could reflect some combination of
physical barriers (true biogeography), short-term stochastic
effects, and/or functional differentiation. Given the confound-
ing variables of geography, time, and environmental conditions
in the current collection of samples, it is difficult to definitively
separate these effects, but various observations argue for
functional differentiation between subtypes (i.e., they con-
stitute distinct ecotypes). First, individual subtypes may be
found in a wide range of locations; P. ubique HTCC1062 was
isolated in the Pacific Ocean off the coast of Oregon [55], but
closely related sequences are relatively abundant in our samples
taken in the Atlantic Ocean. Second, geography per se cannot
fully explain differences in subtype distributions, as multiple
subtypes are found simultaneously in a single sample. Third, the
collection of samples in which a given subtype was found
generally exhibits similar environmental conditions. A strong
independent illustration of this comes from the correlation of
temperature with the distribution of Prochlorococcus subtypes
[56]. Fourth, the extensive variation within each subtype (i.e.,
the fact that subtypes are not clonal populations) indicates that
it cannot be chance alone that makes genetically similar
organisms have similar observed distributions.
Taken together, these results argue that subtype classifica-
tion is more informative for categorizing microbial popula-

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tions than classification using 16S-based ribotypes, or finger-
printing techniques based on length polymorphism, such as T-
RFLPs [57] or ARISA [58]. For example, the grouping of such
disparate microbial populations under the umbrella P. marinus
dilutes the significance of the term ‘‘species.’’ Indeed,
numerous papers have been devoted to comparing and
contrasting the differences and variability in P. marinus isolates
to better understand how this particularly abundant group of
organisms has evolved and adapted within the dynamic marine
environment [28,52,56,59–66]. Prior to the widespread use of
marker-based phylogenetic approaches, microbial systematics
relied on a wide range of variables to distinguish microbial
populations [67]. Subtypes bring us back to these more
comprehensive approaches since they reflect the influences
of a wide range of factors in the context of an entire genome.
Although subtypes are a salient feature of our data,
variation within a ribotype does not stop at the level of
subtypes. Variation within subtypes is so extensive that few
GOS reads can be aligned at 100% identity to any other GOS
read, despite the deep coverage of several taxonomic groups.
Related findings have been shown for the ITS region in
various organisms [2,51,52], and in a limited number of
organisms for individual protein coding and intergenic
regions [2,53,68]. High levels of diversity within the ribotype
can be convincingly demonstrated in the 16S gene itself [69].
The applicability of these results over the entire genome were
recently shown for P. marinus [28] using data from the
Sargasso Sea samples taken as a pilot project for the
expedition reported here [19]. We have definitively demon-
strated the generality of these findings, greatly increased our
understanding of the minimum number of variants of a given
organism, and shown that these observations apply to the
entire genome for a wide range of abundant taxonomic
groups and across a wide range of geographic locations.
Average pairwise differences of several percent between
overlapping P. marinus or SAR11 reads imply that this
variation did not arise recently. If one uses substitution rates
estimated for E. coli [70], one could conclude that on average
any two P. marinus cells must have diverged millions of years
ago. Mutational rates are notoriously variable and hard to
estimate, and assumptions of molecular clocks are equally
chancy, but clearly within-subtype variants have persisted
side by side for quite some time. This raises a question related
to the classic ‘‘Paradox of the Plankton’’: how can so many
similar organisms have coexisted for so long [71,72]? One
explanation, which we favor, is that not only subtypes but also
individual variants are sufficiently different phenotypically to
prevent any one strain from completely replacing all others
(discussed further below; see [71] for a recent theoretical
treatment). An alternative is that recombination might
prevent selective sweeps within ecotypes, as proposed by
Cohan (reviewed in [73]).
The Significance of Within-Subtype Variation
Given the apparent generality of subtypes and intra-
subtype variation, it is important to understand if and how
these subpopulations are functionally distinct. At the level of
DNA sequence, a substantial fraction of substitutions are
silent in terms of amino acid sequence, and others may be
nonsynonymous but functionally neutral. However, two
organisms that differ by 5% in their genetic sequence (e.g.,
100,000 substitutions in 2 Mbp of shared sequence) will
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Sorcerer II GOS Expedition
inevitably have at least minor functional differences such as
in the optimal temperature or pH for the activity of some
enzyme. At the level of gene content, the observation of
hypervariable segments ([28] and here) implies that there is
an additional dimension to functional variability. Hyper-
variable genomic islands with preferential insertion sites
could potentially be associated with a wide range of
functions, though to date they have been most closely
examined for their role in pathogenicity (for a review, see
[74]). However, given their apparent variability within even a
single sampling site, it seems unlikely that these elements
reflect a specific adaptive advantage to the local population.
Identifying the source(s), diversity, and range of functionality
associated with these islands by fully sequencing a large
number of these segments and understanding how their
individual abundances fluctuate should be quite informative.
Some might still argue that these differences must be moot
for the purpose of understanding the role these organisms play
in an ecosystem. Yet even small differences in optimal
conditions may have profound effects. They may prevent any
single genotype from being universally fittest, allowing and/or
necessitating the coexistence of multiple variants [2,51,69].
Moreover, variation within subtype might afford a form of
functional ‘‘buffering,’’ such that the population as a whole
may be more stable in its ecosystem role than any one clone
could be (see also [51]). That is, while any one strain of
Prochlorococcus might thrive and provide energy input to the
rest of the community at a limited range of temperatures, light
conditions, etc., the ensemble might provide such inputs over a
wider range of environmental conditions. In this way, micro-
diversity might provide system stability or robustness through
functional redundancy and the ‘‘insurance effect’’ (reviewed in
[75]). Thus, while the extent of microdiversity suggests that
knowing the behavior of any one isolate in exquisite detail
might not be as useful to reductionist modeling as one might
hope, this buffering could afford a more stable ensemble
behavior, facilitating the development and maintenance of an
ecosystem and allowing for system-level modeling.
A direct equation of subtypes with ecotypes is tempting,
but not entirely clear-cut. The correlation of PstS distribution
with phosphate abundance suggests a functional adaptation,
but within Prochlorococcus and SAR11 the presence or absence
of PstS subdivides subtypes without apparent respect for
phylogenetic structure. This contrasts markedly with the
distribution of proteorhodopsin-tuning variants within
SAR11, which, despite a few convergent substitutions, are
strongly congruent with phylogeny. It is interesting to ask
what distinguishes pressures or adaptations that respect (or
that lead to) lineage splits from those that show little or no
phylogenetic structuring. These two specific examples plau-
sibly reflect two different mechanisms (i.e., convergent but
independent mutation in proteorhodopsin genes and the
acquisition by horizontal transfer of genes involved in
phosphate uptake). Yet, we must wonder: given the evidence
that proteorhodopsin has been transferred laterally [48], and
that only a small number of mutations, in some circumstances
even a single base-pair change, are required to switch
between the blue-absorbing and green-absorbing forms
[39,40], why should proteorhodopsin variants show any
lineage restriction? Perhaps this relates to the modularity of
the system in question: proteorhodopsin tuning may be part
of a larger collection of synergistic adaptations that are
0424 Special Section from March 2007 | Volume 5 | Issue 3 | e77

collectively not easily evolved, acquired, or lost, while the PstS
and surrounding genes may represent a functional unit that
can be readily added and removed over relatively short
evolutionary time scales. If so, perhaps subtypes are indeed
ecotypes, but rapidly evolving characters can lead to
phenotypes that crosscut or subdivide ecotypes.
Phage provide one possible mechanism for rapid evolution
of microbial populations or strains, and have been found in
abundance with this and other marine metagenomic datasets
[18,20]. It has been proposed that hypervariable islands are
phage mediated [28]. However, there are reasons to be
cautious about invoking phage as an explanation for rapidly
evolving characteristics. While we see variability of PstS and
neighboring genes in both SAR11 and P. marinus populations,
this variation does not seem to be linked to recent phage
activity. Initially, the distribution of PstS seems similar to the
variation associated with the hypervariable islands, which
may be phage mediated [28]. Indeed, phosphate-regulating
genes including PstS have been identified in phage genomes
[64], presumably because enhanced phosphate acquisition is
required during the replication portion of their life cycle.
However, the regions containing the PstS genes in both
SAR11 and Prochlorococcus do not behave in the same fashion
as clearly hypervariable regions, being effectively bimorphic
(modulo the level of sequence variation observed elsewhere in
the genome), whereas clearly hypervariable regions are so
diverse that nearly every sampled clone falling in such a
region appears completely unrelated to every other. Nor do
the other genes in PstS-containing regions appear to be phage
associated. These observations suggest that differences in PstS
presence or absence arose in the distant past, or that
different mechanisms are at work. It seems likely that phage
may mediate lateral transfer of PstS and other phosphate
acquisition genes, but it is unclear whether these genes then
can become fixed within the population. Phage require
enhanced phosphate acquisition as part of their life cycle
[64], so regulatory or functional differences in these genes
may limit their suitability for being acquired by the host cell
for its own purposes. The rate of phage-mediated horizontal
transfer of genes may reflect a combination of the gene’s
value to the host and to the agent mediating the transfer (e.g.,
phage), suggesting that PstS may have much greater immedi-
ate value than do proteorhodopsin genes and their variants.
In practical terms, these results highlight the limitations
associated with marker-based analysis and the use of these
approaches to infer the physiology of a particular microbial
population. At the resolution used here, marker-based
approaches are not always informative regarding differences
in gene content (e.g., the PstS gene as well as neighboring
genes), especially those associated with hypervariable seg-
ments. Though phosphate acquisition is known to vary within
different strains of P. marinus [64,76], our results clearly show
that this variability can happen within a single subtype (as
represented by MIT9312), effectively identifying distinct
ecotypes. Given the correct samples from the appropriate
environments, other core genes might also show similar
variation and allow us to more fully assess the reliability of
reference genomes as indicators of physiological potential.
Tools and Techniques
Analysis of the GOS dataset has benefited from the
development of new tools and techniques. Many of these
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Sorcerer II GOS Expedition
approaches rely on fairly well-known techniques but have
been modified to take greater advantage of the metadata.
The technique of fragment recruit and the corresponding
fragment recruitment plots have proven highly useful for
examining the biogeography and genomic variation of
abundant marine microbes when a close reference genome
exists. Ultimately, this approach derives from the percent
identity plots of PipMaker [27]. Similar approaches have been
used to examine variation with respect to metagenomic
datasets. For example, hypervariable segments and sequence
variation have been visualized in P. marinus MIT9312 using
the Sargasso Sea data [28] and in human gut microbes
Bifidobacterium longum and Methanobrevibacter smithii [77].
Our primary advance associated with fragment recruit-
ment plots is the incorporation of metadata associated with
the isolation or production of the sequencing data. While
simple in nature, the resulting plots can be extremely
informative due to the volume of data being presented.
Being able to present the sequence similarity and metadata
visually allows a researcher to quickly identify interesting
portions of the data for further examination. This is one of
the first tools to make extensive use of the metadata collected
during a metagenomic sequencing project. The use of sample
and recruitment metadata is just the beginning. It is not
difficult to imagine displaying other variations such as water
temperature, salinity, phosphate abundance, and time of year
with this approach. Even sample independent metadata such
as phylogenetic information may produce informative views
of the data. The usefulness of this and related approaches will
only grow as the robust collection of metadata becomes
routine and the variables that are most relevant to microbial
communities are further elucidated.
The greatest limitation of fragment recruitment is the lack
of appropriate reference sequences, particularly finished
genomes. Using a series of modifications to the Celera
Assembler referred to as ‘‘extreme assembly,’’ we have
produced large assemblies for cultivated and uncultivated
marine microbes. On its own, the extreme assembly approach
would be excessively prone to producing chimeric sequences.
However, when extreme assemblies are used as references for
fragment recruitment, the metadata provides additional
criteria to validate the sampling consistency along the length
of the scaffold. Chimeric joins can be rapidly detected and
avoided. This argues that future metagenomic assemblers
could be specifically designed to make use of the metadata to
produce more accurate assemblies, and that metagenomic
assemblies will be improved by using data from multiple
sources. Finding ways to represent the full diversity in these
assemblies remains a pressing issue.
Extreme assembly can produce much larger assemblies but
it is still limited by overall coverage. While many ribotypes are
presumably present in sufficient quantities that reasonable
assemblies of these genomes might be expected, this did not
occur even for the most abundant organisms, including
SAR11 and P. marinus. Many of the problems can be
attributed to the diversity associated with the hypervariable
segments where the effective coverage drops precipitously. If
these are indeed commonplace in the microbial world, it is
unlikely that complete genomes will be produced using the
small insert libraries presented here. However, the ability to
bin the larger sequences based on their coverage profiles
across multiple samples, oligonucleotide frequency profiles,
0425 Special Section from March 2007 | Volume 5 | Issue 3 | e77

and phylogenetic markers suggests that large portions of a
microbial genome can be reconstructed from the environ-
mental data. This in turn should provide critical insights into
the physiology and biochemistry of these microbial lineages
that will inform culture techniques to allow cultivation of
these recalcitrant organisms under laboratory conditions.
Not every technique described herein relies on metadata.
The marker-less, overlap-based metagenomic comparison
provides a quantitative approach to comparing the overall
genetic similarity of two samples (Figures 10 and 11). In
essence, genomic similarity acts as a proxy for community
similarity. Marker-based approaches such as ARISA including
the use of 16S sequences described herein can also be used to
infer community similarity, though these approaches more
aptly generate a census of the community members
[51,69,78,79]. This census is biased to the extent that 16S
genes can vary in copy number and relies on linkage of the
marker gene to infer genome composition. While our
metagenome comparison does not directly provide a census,
the sensitivity can be tuned by restricting the identity of
matches. This means that even subtype-level differences can
be detected across samples. It would also identify the
substantial gene content differences between the K12 and
O157:H7 E. coli strains [12]. Such large-scale gene content
differences have yet to be seen between closely related marine
microbes, but may be a factor in other environments.
Although the requisite amount of data will vary with the
complexity of the environment or the degree of resolution
required, we have found that 10,000 sequencing reads is
sufficient to reliably measure the similarity of two surface
water samples (unpublished data). This analysis may become a
general tool for allocating sequencing resources by allowing a
shallow survey of many samples followed by deep sequencing
of a select number of ‘‘interesting’’ ones.
The application of this technique for comparing samples
along with detailed analysis of fragments recruiting to a given
reference sequence can also help explicate differences among
communities in gene content or sequence variation. For
example, recent metagenomic studies have reported differ-
ences in abundance of various gene families or differing
functional roles between samples. Some of these differences
correspond to plausible differences in physiology and
biochemistry, such as the relative overabundance of photo-
synthetic or light-responsive genes in surface water samples
[20,32]. Other differences however are less obvious, such as
the abundance of ribosomal proteins at 130 m or the
abundance of tranposase at 4,000 m [20]; some of these may
reflect ‘‘taxonomic hitchhiking,’’ such that a sample rich in
Archaea or Firmicutes or Cyanobacteria, etc., has an over-
representation of genes more reflective of their recent
evolutionary history than of a response to environmental
conditions. Being able to control or account for these
taxonomic effects is crucial to understanding how microbial
populations have adapted to environmental conditions and
how they may behave under changing conditions. The
metagenomic comparison method described here provides
a new tool to more accurately measure the impact of
taxonomic effects.
In conclusion, this study reveals the wealth of biological
information that is contained within large multi-sample
environmental datasets. We have begun to quantify the
amount and structure of the variation in natural microbial
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Sorcerer II GOS Expedition
populations, while providing some information about how
these factors are structured along phylogenetic and environ-
mental factors. At the same time, many questions remain
unanswered. For example, although microbial populations
are structured and therefore genetically isolated, we do not
understand the mechanisms that lead to this isolation. Their
isolation seems contradictory given overwhelming evidence
that horizontal gene transfer associated with hypervariable
islands is a common phenomenon in marine microbial
populations. Whatever the mechanism, the role and rate at
which gene exchange occurs between populations will be
crucial to understanding population structure within micro-
bial communities and whether these communities are chance
associations or necessary collections. The hypervariable
islands could be a source for tremendous genetic innovation
and novelty as evidenced by the rate of discovery of novel
protein families in the GOS dataset [18]. However, it is not
clear whether these entities are the main source of this
novelty or whether this novelty resides in the vast numbers of
rare microbes [4] that cannot be practically accessed using
current metagenomic approaches. Altogether, this research
reaffirms our growing wealth and complexity of data and
paucity of understanding regarding the biological systems of
the oceans.
Materials and Methods
Sampling sites. A more detailed description of the sampling sites
provides additional context in which to understand the individual
samples. The northernmost site (GS05) was at Compass Buoy in the
highly eutrophic Bedford Basin, a marine embayment encircled by
Halifax, Nova Scotia, that has a 15-y weekly record of biological,
physical, and chemical monitoring (http://www.mar.dfo-mpo.gc.ca/
science/ocean/BedfordBasin/index.htm). Other temperate sites in-
cluded a coastal station sample near Nova Scotia (GS4), a station in
the Bay of Fundy estuary at outgoing tide (GS06), and three Gulf of
Maine stations (GS02, GS03, and GS07). These were followed by
sampling coastal stations from the New England shelf region of the
Middle Atlantic Bight (Newport Harbor through Delaware Bay;
GS08–GS11). The Delaware Bay (GS11) was one of several estuary
samples along the Global Expedition path. Estuaries are complex
hydrodynamic environments that exhibit strong gradients in oxygen,
nutrients, organic matter, and salinity and are heavily impacted by
anthropogenic nutrients. The Chesapeake Bay (GS12) is the largest
estuary in the United States and has microbial assemblages that are
diverse mixtures of freshwater and marine-specific organisms [80].
GS13 was collected near Cape Hatteras, North Carolina, inside and
north of the Gulf Stream, and GS14 was taken along the western
boundary frontal waters of the Gulf Stream off the coast of
Charleston, South Carolina. The vessel stopped at five additional
stations as it transited through the Caribbean Sea (GS15–GS19) to the
Panama Canal. In Panama, we sampled the freshwater Lake Gatun,
which drains into the Panama Canal (GS20). The first of the eastern
Pacific coastal stations GS21, GS22, and GS23 were sampled on the
way to Cocos Island (;500 km southwest of Costa Rica), followed by a
coastal Cocos Island sample (GS25). Near the island, ocean currents
diverge and nutrient rich upwellings mix with warm surface waters to
support a highly productive ecosystem. Cocos Island is distinctive in
the eastern Pacific because it belongs to one of the first shallow
undersea ridges in the region encountered by the easterly flowing
North Equatorial Counter/Cross Current in the Far Eastern Pacific
[81,82]. After departing Cocos Island, the vessel continued southwest
to the Galapagos Islands, stopping for an open ocean station (GS26).
An intensive sampling program was then conducted in the Galapagos.
The Galapagos Archipelago straddles the equator 960 km west of
mainland Ecuador in the eastern Pacific. These islands are in a
hydrographically complex region due to their proximity to the
Equatorial Front and other major oceanic currents and regional
front systems [83]. The coastal and marine parts of the Galapagos
Islands ecosystem harbor an array of distinctive habitats, processes,
and endemic species. Several distinct zones were targeted including a
shallow-water, warm seep (GS30), below the thermocline in an
0426 Special Section from March 2007 | Volume 5 | Issue 3 | e77

upwelling zone (GS31), a coastal mangrove (GS32), and a hypersaline
lagoon (GS33). The last stations were collected from open ocean sites
(GS37 and GS47) and a coral reef atoll lagoon (GS51) in the immense
South Pacific Gyre. The open ocean samples come from a region of
lower nutrient concentrations where picoplankton are thought to
represent the single most abundant and important factor for
biogeochemical structuring and nutrient cycling [84–87]. In the atoll
systems, ambient nutrients are higher, and bacteria are thought to
constitute a large biomass that is one to three times as large as that of
the phytoplankton [88–90].
Sample collection. A YSI (model 6600) multiparameter instrument
(http://www.ysi.com) was deployed to determine physical character-
istics of the water column, including salinity, temperature, pH,
dissolved oxygen, and depth. Using sterilized equipment [91], 40–200 l
of seawater, depending on the turbidity of the water, was pumped
through a 20-lm nytex prefilter into a 250-l carboy. From this sample,
two 20-ml subsamples were collected in acid-washed polyethylene
bottles and frozen (20 8C) for nutrient and particle analysis. At each
station the biological material was size fractionated into individual
‘‘samples’’ by serial filtration through 20-lm, 3-lm, 0.8-lm, and 0.1-
lm filters that were then sealed and stored at 20 8C until transport
back to the laboratory. Between 44,160 and 418,176 clones per station
were picked and end sequenced from short-insert (1.0–2.2 kb)
sequencing libraries made from DNA extracted from filters [19].
Data from these six Sorcerer II expedition legs (37 stations) were
combined with the results from samples in the Sargasso Sea pilot
study (four stations; GS00a–GS00d and GS01a–GS01c; [19]. The
majority of the sequence data presented came from the 0.8- to 0.1-lm
size fraction sample that concentrated mostly bacterial and archaeal
microbial populations. Two samples (GS01a, GS01b) from the
Sargasso Sea pilot study dataset and one GOS sample (GS25) came
from other filter size fractions (Table 1).
Filtration and storage. Microbes were size fractionated by serial
filtration through 3.0-lm, 0.8-lm, and 0.1-lm membrane filters
(Supor membrane disc filter; Pall Life Sciences, http://www.pall.com),
and finally through a Pellicon tangential flow filtration (Millipore,
http://www.millipore.com) fitted with a Biomax-50 (polyethersulfone)
cassette filter (50 kDa pore size) to concentrate a viral fraction to 100
ml. Filters were vacuum sealed with 5 ml sucrose lysis buffer (20 mM
EDTA, 400 mM NaCl, 0.75 M sucrose, 50 mM Tris-HCl [pH 8.0]) and
frozen to 20 8C on the vessel until shipment back to the Venter
Institute, where they were transferred to a 80 8C freezer until DNA
extraction. Glycerol was added (10% final concentration) as a
cryoprotectant for the viral/phage sample.
DNA isolation. In the laboratory, the impact filters were
aseptically cut into quarters for DNA extraction. Unused quarters
of the filter were refrozen at 80 8C for storage. Quarters used for
extraction were aseptically cut into small pieces and placed in
individual 50-ml conical tubes. TE buffer (pH 8) containing 50 mM
EGTA and 50 mM EDTA was added until filter pieces were barely
covered. Lysozyme was added to a final concentration of 2.5 mg/
ml1, and the tubes were incubated at 37 8C for 1 h in a shaking
water bath. Proteinase K was added to a final concentration of 200
lg/ml1, and the samples were frozen in dry ice/ethanol followed by
thawing at 55 8C. This freeze–thaw cycle was repeated once. SDS
(final concentration of 1%) and an additional 200 lg/ml1 of
proteinase K were added to the sample, and samples were incubated
at 55 8C for 2 h with gentle agitation followed by three aqueous
phenol extractions and one phenol/chloroform extraction. The
supernatant was then precipitated with two volumes of 100%
ethanol, and the DNA pellet was washed with 70% ethanol. Finally,
the DNA was treated with CTAB to remove enzyme inhibitors. Size
fraction samples not utilized in this study were archived for future
analysis.
Library construction. DNA was randomly sheared via nebulization,
end-polished with consecutive BAL31 nuclease and T4 DNA
polymerase treatments, and size-selected using gel electrophoresis
on 1% low-melting-point agarose. After ligation to BstXI adapters,
DNA was purified by three rounds of gel electrophoresis to remove
excess adapters, and the fragments were inserted into BstXI-
linearized medium-copy pBR322 plasmid vectors. The resulting
library was electroporated into E. coli. To ensure construction of
high-quality random plasmid libraries with few to no clones with no
inserts, and no clones with chimeric inserts, we used a series of
vectors (pHOS) containing BstXI cloning sites that include several
features: (1) the sequencing primer sites immediately flank the BstXI
cloning site to avoid excessive resequencing of vector DNA; (2)
elimination of strong promoters oriented toward the cloning site;
and (3) the use of BstXI sites for cloning facilitates the preparation of
libraries with a low incidence of no-insert clones and a high
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Sorcerer II GOS Expedition
frequency of single inserts. Clones were sequenced from both ends
to produce pairs of linked sequences representing ;820 bp at the end
of each insert.
Template preparation. Libraries were transformed, and cells were
plated onto large format (16 3 16cm) diffusion plates prepared by
layering 150 ml of fresh molten, antibiotic-free agar onto a previously
set 50-ml layer of agar containing antibiotic. Colonies were picked for
template preparation using the Qbot or QPix colony-picking robots
(Genetix, http://www.genetix.com), inoculated into 384-well blocks
containing liquid media, and incubated overnight with shaking. High-
purity plasmid DNA was prepared using the DNA purification
robotic workstation custom-built by Thermo CRS (http://www.
thermo.com) and based on the alkaline lysis miniprep [92]. Bacterial
cells were lysed, cell debris was removed by centrifugation, and
plasmid DNA was recovered from the cleared lysate by isopropanol
precipitation. DNA precipitate was washed with 70% ethanol, dried,
and resuspended in 10 mM Tris HCl buffer containing a trace of blue
dextran. The typical yield of plasmid DNA from this method is
approximately 600–800 ng per clone, providing sufficient DNA for at
least four sequencing reactions per template.
Automated cycle sequencing. Sequencing protocols were based on
the di-deoxy sequencing method [93]. Two 384-well cycle-sequencing
reaction plates were prepared from each plate of plasmid template
DNA for opposite-end, paired-sequence reads. Sequencing reactions
were completed using the Big Dye Terminator chemistry and
standard M13 forward and reverse primers. Reaction mixtures,
thermal cycling profiles, and electrophoresis conditions were
optimized to reduce the volume of the Big Dye Terminator mix
(Applied Biosystems, http://www.appliedbiosystems.com) and to ex-
tend read lengths on the AB3730xl sequencers (Applied Biosystems).
Sequencing reactions were set up by the Biomek FX (Beckman
Coulter, http://www.beckmancoulter.com) pipetting workstations.
Robots were used to aliquot and combine templates with reaction
mixes consisting of deoxy- and fluorescently labeled dideoxynucleo-
tides, DNA polymerase, sequencing primers, and reaction buffer in a
5 ll volume. Bar-coding and tracking promoted error-free template
and reaction mix transfer. After 30–40 consecutive cycles of
amplification, reaction products were precipitated by isopropanol,
dried at room temperature, and resuspended in water and trans-
ferred to one of the AB3730xl DNA analyzers. Set-up times were less
than 1 h, and 12 runs per day were completed with average trimmed
sequence read length of 822 bp.
Fosmid end sequencing. Fosmid libraries [24] were constructed
using approximately 1 lg DNA that was sheared using bead beating to
generate cuts in the DNA. The staggered ends or nicks were repaired
by filling with dNTPs. A size selection process followed on a pulse
field electrophoresis system with lambda ladder to select for 39–40 Kb
fragments. The DNA was then recovered from a gel, ligated to the
blunt-ended pCC1FOS vector, packaged into lambda packaging
extracts, incubated with the host cells, and plated to select for the
clones containing an insert. Sequencing was performed as described
for plasmid ends.
Metagenomic assembly. Assembly was conducted with the Celera
Assembler [21], with modifications as follows. The ‘‘genome length’’
was artificially set at the length of the dataset divided by 50 to allow
unitigs of abundant organisms to be treated as unique, as previously
described [19]. Several distinct assemblies were computed. In the
primary assembly, all pairs of mated reads were tested to see whether
the paired reads overlapped one another; if so, they were merged into
a single pseudo-read that replaced the two original reads; further,
only overlaps of 98% identity or higher were used to construct
unitigs. A second assembly was conducted in the same fashion with
the exception of using a 94% identity cutoff to construct unitigs.
Finally, series of assemblies at various stringencies were computed for
subsets of the GOS data; in these assemblies, overlapping mates were
not preassembled and the Celera Assembler code was modified
slightly to allow for overlapping and multiple sequence alignment at
lower stringency.
Construction of a low-identity overlap database. An all-against-all
comparison of unassembled (but merged and duplicate-stripped)
sequences from the combined dataset was performed using a
modified version of the overlapper component of the Celera
Assembler [21]. The code was modified to find overlap alignments
(global alignments allowing free end gaps) starting from pairs of reads
that share an identical substring of at least 14 bp. An alignment
extension was then performed with match/mismatch scores set to
yield a positive outcome if an overlap alignment was found with
65% identity. Overlaps involving alignments of 40 bp were
retained for various analyses. For the GOS dataset described here,
this process resulted in a dataset of 1.2 billion overlaps. Due to the 14-
0427 Special Section from March 2007 | Volume 5 | Issue 3 | e77

bp requirement and certain heuristics for early termination of
apparently hopeless extensions, not all alignments at 65% were
found. In addition, some of the lowest-identity overlaps are bound to
be chance matches; however, this was a relatively uncommon event.
Approximately one in 5 3 106 pairs of 800-bp random sequences (all
sites independent, A ¼ C ¼ G ¼ T ¼ 25%) can be aligned to overlap
40 bp at 65% identity using the same procedure. At a 70% cutoff,
the value is reduced to one in 4 3 107, and one in 5 3 108 at a 75% cut
off.
Extreme assembly. Like many assembly algorithms, the extreme
assembler proceeds in three phases: overlap, layout, and consensus.
The overlap phase is provided by the all-against-all comparison
described above. The consensus phase is performed by a version of
the Celera Assembler, modified to accept higher rates of mismatch.
The layout phase begins with a single sequencing read (‘‘seed’’) that is
chosen at random or specified by the user and is considered the
‘‘current’’ read. The following steps are performed off one or both
ends of the seed. (1) Starting from the current fragment end, add the
fragment with the best overlap off that end and mark the current
fragment as ‘‘used,’’ thus making the added fragment the new current
fragment. (2) Mark as used any alternative overlap that would have
resulted in a shorter extension. The simplest notion of ‘‘best overlap’’
is simply the one having the highest identity alignment, but more
complicated criteria have certain advantages. A simple but useful
refinement is to favor fragments whose other ends have overlaps over
those which are dead ends. For an unsupervised extreme assembly,
when the sequence extension terminates because there are no more
overlaps, a new unused fragment is chosen as the next seed and the
process is repeated until all fragments have been marked used.
Construction of multiple SAR11 variants. Sequencing reads mated
to SAR11-like 16S sequences but themselves outside of the ribosomal
operon (n ¼ 348) were used as seeds in independent extreme
assemblies. Since the assemblies were independent, the results were
highly redundant, with a given chain of overlapping fragments
typically being used in multiple assemblies. A subset of 24 assemblies
that shared no fragments over their first 20 kb was identified as
follows. (1) Connected components were determined in a graph
defined by nodes corresponding to extreme assemblies. If the
assemblies shared at least one fragment in the first 20 kb of each
assembly, the two nodes were connected by an edge. (2) A single
assembly was chosen at random from each of the connected
components. The consensus sequence over the 20-kb segment of
each such representative was used as the reference for fragment
recruitment.
Phylogeny. Phylogenies of sequences homologous to a given
portion of a reference sequence (typically 500 bp) were determined
in the following manner. A set of homologous fragments was
identified based on fragment recruitment to the reference as
described above. Fragments that fully spanned the segment of
interest and had almost full-length alignments to the reference
sequence of a user-defined percent identity (typically, 70%) were
used for further analysis. A preliminary master–slave multiple
sequence alignment of the recruited reads (slaves) to the reference
segment (master) was performed with a modified version of the
consensus module of the Celera Assembler. Based on this alignment,
reads were trimmed to the portion aligning to the reference segment
of interest. A refined multiple sequence alignment was then
computed with MUSCLE [94]. Distance based phylogenies were
computed using the programs DNADIST and NEIGHBOR from the
PHYLIP package [95] using default settings. Trees were visualized
using HYPERTREE [96].
Measurement of library-to-library similarity. Based on the low-
identity overlap database described above, the similarity of a library i
to another library j at a given percent identity cutoff was computed as
follows. For each sequence s of i, let ns,i ¼ the number of overlaps to
other fragments of i satisfying the cutoff; ns,j ¼ the number of overlaps
to fragments of j satisfying the cutoff; and fs,i ¼ ns,i/(ns,i þ ns,j) ¼ fraction
of reads overlapping s from i or j that are from i.
X approach successfully mimics the types of reads found in the GOS
ri;i ¼ fs;i
s Abundance of proteorhodopsin variants. A total of 2,644 proteo-
X frames derived from the GOS assembly [18]. These genes could
ri;j ¼ 1  fs;i
s
pffiffiffiffiffiffiffiffiffiffiffiffiffi
si;j ¼ 0:5 ðri;j þ rj;i Þ= ri;i  rj;j
Si;j ¼ Sj;i ¼ 2si;j =ð1 þ si;j Þ
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Sorcerer II GOS Expedition
A read that can be overlapped to another at sufficiently high-
sequence identity was taken to indicate that they were from similar
organisms, and, relatedly, that similar genes were present in the
samples. Only reads with such overlaps contributed to the calcu-
lation. Other reads reflect genes or segments of genomes that were so
lightly sampled (i.e., at such low abundance) that they were not
informative regarding the similarity of two samples. Consequently,
the analysis automatically corrects for differences in the amount of
sequencing, and can be computed over sets of samples that vary
considerably in diversity. The resulting measure of similarity Si,j takes
on a value between 0 and 1, where 0 implies no overlaps between i
and j, and 1 implies that a fragment from i and a fragment from j are
as likely to overlap one another as are two fragments from i or two
fragments from j. As with the Bray-Curtis coefficient [97], abundance
of categories affects the computation. In an idealized situation where
two libraries can each be divided into some number k of ‘‘species’’ at
equal abundance, and the libraries have l of the species in common,
the similarity statistic will approach l/k for large samplings; in this
sense, Si,j ¼ x indicates that the two samples share approximately a
fraction x of their genetic material. It is frequently useful to define Di,j
¼ 1  Si,j, the ‘‘dissimilarity’’ or distance between two samples.
Ribotype clustering and identification of representatives. An all-
against-all comparison of predicted 16S sequences was performed to
determine the alignment between pairs of overlapping sequences
using a version of an extremely fast bit-vector algorithm [98]. A
hierarchical clustering was determined using percent-mismatch in
the resulting alignments as the distance between pairs of sequences.
Order of clustering and cluster identity scores were based on the
average-linkage criterion, with distances between nonoverlapping
partial sequences treated as missing data. Ribotypes were the
maximal clusters with an identity score above the cutoff (typically
97%). Representative sequences were chosen for each cluster based
on both length and highest average identity to other sequences in the
cluster.
Taxonomic classification. Taxonomic classification of 16S sequen-
ces was conducted using phylogenetic techniques based on clade
membership of similar sequences with 16S sequences with defined
taxonomic membership. Representative sequences from clustered
sequences were analyzed as described previously [19,99] and by
addition into an ARB database of small subunit rDNAs [100,101].
Results were spot-checked against the Ribosomal Database Project II
Classifier server [102] and the taxonomic labels of the best BLASTN
hits against the nonredundant database at NCBI.
Fragment recruitment. Global ocean sequences were aligned to
genomic sequences of different bacteria and phage using NCBI
BLASTN [26]. The following blast parameters were designed to
identify alignments as low as 55% identity that could contain large
gaps: -F ‘‘m L’’ -U T -p blastn -e 1e-4 -r 8 -q -9 -z 3000000000 -X 150.
Reads were filtered in several steps to identify the reads that were
aligned over more or less their entire length. Reads had to be aligned
for more than 300 bp at .30% identity with less than 25 bp of
unaligned bases on either end, or reads had to be aligned over more
than 100 bp at .30% identity with less than 20 bp of overhang off
either end. Identity was calculated ignoring gaps. In some instances a
read might be placed, but the mate would not be placed under these
criteria. In such cases, if 80% or more of the mate were successfully
aligned, then the mate would be rescued and considered successfully
aligned.
Generation of shredded artificial reads from finished genomes.
Random pieces of DNA from the genome in question with a length
between 1,800 to 2,500 bp were selected. For each piece a read length
N1 was selected from the distribution of lengths using the GOS
dataset. If that GOS sequence had a mate pair, then a second length
N2 was again randomly selected. The length N1 was used to generate
a read from the 59 end of the DNA. The piece of DNA was then
reverse complemented and if appropriate, a second length N2 was
used to generate a second read. The relationship between these two
reads was then recorded and used to produce a fasta file. This
ð1Þ data with similar rates of missing mates.
rhodopsin genes were identified from the clustered open reading
ð2Þ
be linked back to 3,608 GOS clones. Open reading frames were
predicted from these clones as described in [18]. The peptide
sequences were aligned with NCBI blastpgp with the following
ð3Þ parameters: -j 5 -U T -e 10 -W 2 -v 5 -b 5000 -F ‘‘m L’’ -m 3. The
search was performed with a previously described blue-absorbing
proteorhodopsin protein BPR (gij32699602) as the query. The amino
ð4Þ acid associated with light absorption is found within a short
0428 Special Section from March 2007 | Volume 5 | Issue 3 | e77

conserved motif RYVDWLLTVPL*IVEF, where the asterisk indicates
tuning amino acid [39–41]. In total, 1,938 clones were found to
contain this motif. Clones and the sample metadata were then
associated with the tuning amino acid to determine the relative
abundance of the different amino acids at these positions. Clones
could be associated with SAR11 if both mated sequencing reads
(when available) were recruited to P. ubique HTCC1062.
Site abundance estimates and comparisons. Given a set of genes
identified on the GOS sequences, we can identify the scaffolds on
which these genes were annotated. A vector indicating the number of
sequences contributed by every sample is determined for every gene.
This vector reflects the number of sequences from every sample that
assembled into the scaffold on which the gene was identified after
normalizing for the proportion of scaffold covered by the gene. For
example, if a 10-kb scaffold contains a 1-kb gene, then each sample
will contribute one GOS sequence for every ten GOS sequences it
contributed to the entire scaffold. The vectors are then summed and
normalized to account for either the total number of GOS sequences
obtained from each sample or based on the number of typically single
copy recA genes (identified as in [18]). Unless stated otherwise, recA
was used to normalize abundance across samples. When comparisons
using groups of samples were performed, the average value for the
samples was compared.
Oligonucleotide composition profile. A 1-D profile representing
oligonucleotide frequencies was computed as follows. A sequence was
converted into a series of overlapping 10,000-bp segments, each
segment offset by 1,000 bp from the previous one, using perl and shell
scripting. Dinucleotide frequencies are computed on each segment
using a C program written for this purpose. Higher-order oligonu-
cleotides were examined and gave similar results for the genomes of
interest. Remaining calculations were performed using the R package
[103]. Principle component analysis (function princomp with default
settings) was applied to the matrix of frequencies per window
position. The value of the first component for each position was
normalized by the standard deviation of these values, and truncated
to the range [5, 5]. For visualizations, the resulting values were
plotted at the center of each window.
Estimating frequency of large-scale translocations and inversions.
The unrecruited mated sequencing reads of reads recruited to P.
marinus MIT9312 at or above 80% identity were examined. An
unrecruited mate indicated a potential translocation or inversion if it
aligned to the MIT9312 genome in two and only two distinct
alignments separated by at least 50 kb, if each aligned portion was at
least 250 bp long, if there was less than 100 bp of unaligned sequence
and no more than 100 bp of overlapping sequence between the two
aligned portions in read coordinates, and if each aligned portion was
anchored to one end of the sequencing read with less than 25 bp of
unaligned sequence from each end. In total, 18 rearrangements were
identified, six of which appear to be unique events.
The rate of discovery was estimated by determining the number of
rearrangements in a given volume of sequence. We estimated the
volume of sequence that was potentially examined by identifying
recruited mated sequencing reads that fit the ‘‘good’’ category (i.e.,
which were recruited in the correct orientation at the expected
distance from each other). For a given read, if the mate was recruited
at greater than or equal to 80% identity, then the expected amount of
sequence examined should be the current (as opposed to mate) read
length minus 500 bp. This produces an estimate of the search space to
be ;47 Mbp. Given 18 rearrangements, this leads to an estimate of
one rearrangement per 2.6 Mbp.
Quantification and assessment of sequences associated with gaps.
GOS reads assigned to the ‘‘missing mate’’ category that were
recruited at greater than 80% identity outside the gap in question
were identified. The mates of these reads were then identified and
clustering was attempted with Phrap (http://www.phrap.org). Reads
that were incorporated end to end into the Phrap assemblies were
identified. For most small gaps a single assembly included all the
missing mate reads and identified the precise difference between the
reference and the environmental sequences. For the hypervariable
segments, most of the reads failed to assemble at all, and those that
did show greater sequence divergence than typically seen. In the case
of SAR11-recruited reads, to increase the number of reads associated
with the hypervariable gaps we identified reads that did not recruit to
the P. ubique HTCC1062 but aligned in a single HSP (high-scoring
pair) over at least 500 bp with one end unaligned because it extended
into the hypervariable gap.
Data and tool release. To facilitate continued analysis of this and
other metagenomic datasets, the tools presented here along with their
source code will be available via the Cyberinfrastructure for Advanced
Marine Microbial Ecology Research and Analysis (CAMERA) website
PLoS Biology | www.plosbiology.org | S53
Sorcerer II GOS Expedition
(http://camera.calit2.net). The dataset and associated metadata will be
accessible via CAMERA (using the dataset tag CAM_PUB_Rusch07a).
Given the exceptional abundance of Burkholderia and Shewanella
sequences in the first Sargasso Sea sample and the feeling that these
may be contaminants, we are also providing a list of the scaffold IDs
and sequencing read IDs associated with these organisms to facilitate
analyses with or without the sequences. In addition to CAMERA, the
GOS scaffolds and annotations will be available via the public sequence
repositories such as NCBI (http://www.ncbi.nlm.nih.gov/entrez/
query.fcgi?db¼genomeprj&cmd¼Retrieve&dopt¼Overview&list_
uids¼13694), and the reads will be available via the Trace Archive
(http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?).
Supporting Information
Poster S1. Fragment Recruitment of GOS Data to Finished Microbial
Genomes
Found at doi:10.1371/journal.pbio.0050077.sd001 (21 MB PDF).
Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession num-
ber for proteorhodopsin protein BPR is gij32699602.
Acknowledgments
We acknowledge the Department of Energy (DOE), Office of Science,
and Office of Biological and Environmental Research (DE-FG02-
02ER63453), the Gordon and Betty Moore Foundation, the Discovery
Channel, and the J. Craig Venter Science Foundation for funding to
undertake this study. We are also indebted to a large group of
individuals and groups for facilitating our sampling and analysis. We
thank the governments of Canada, Mexico, Honduras, Costa Rica,
Panama, Ecuador, French Polynesia, and France for facilitating
sampling activities. All sequencing data collected from waters of the
above-named countries remain part of the genetic patrimony of the
country from which they were obtained. Canada’s Bedford Institute
of Oceanography provided a vessel and logistical support for
sampling in Bedford Basin. The Universidad Nacional Autónoma
de México (UNAM) facilitated permitting and logistical arrangements
and identified a team of scientists for collaboration. The scientists
and staff of the Smithsonian Tropical Research Institute (STRI)
hosted our visit in Panama. Representatives from Costa Rica’s
Organization for Tropical Studies (Jorge Arturo Jimenez and
Francisco Campos Rivera), the University of Costa Rica (Jorge
Cortés), and the National Biodiversity Institute (INBio) provided
assistance with planning, logistical arrangements, and scientific
analysis. Our visit to the Galapagos Islands was facilitated by
assistance from the Galapagos National Park Service Director,
Washington Tapia, and the Charles Darwin Research Institute,
especially Howard Snell and Eva Danulat. We especially thank Greg
Estes (guide), Héctor Cháuz Campo (Institute of Oceanography of the
Ecuador Navy), and a National Park Representative, Simon Ricardo
Villemar Tigrero, for field assistance while in the Galapagos Islands.
Martin Wilkalski (Princeton University) and Rod Mackie (University
of Illinois) provided planning advice for the Galapagos sampling plan.
We thank Matthew Charette (Woods Hole Oceanographic Institute)
for nutrient data analysis. We also acknowledge the help of Michael
Ferrari and Jennifer Clark for remote sensing data. The U.S.
Department of State facilitated Governmental communications on
multiple occasions. John Glass (J. Craig Venter Institute [JCVI])
provided valuable assistance in methods development. The dedicated
efforts of the quality systems, library construction, template, and
sequencing teams at the JCVI Joint Technology Center produced the
high quality sequence data that was the basis of this paper. We thank
Matthew LaPointe, Creative Director of JCVI, for assistance with
figure design, and the JCVI information technology support team
who facilitated many of the vessel related technical needs. Special
thanks are due for Charles H. Howard, captain of the Sorcerer II, and
fellow crew members Cyrus Foote and Brooke A. Dill for their time
and effort in support of this research. We gratefully acknowledge Dr.
Michael Sauri, who oversaw medical related issues for the crew of the
Sorcerer II.
Author contributions. DBR, ALH, KB, HS, CAP, JFH, MF, and JCV
conceived and designed the experiments. DBR, ALH, JMH, KB, BT,
HBT, CS, JT, JF, CAP, and JCV performed the experiments. DBR,
ALH, GS, KBH, SW, DW, JAE, KR, JEV, TU, YHR, MRF, KN, and RF
analyzed the data. DBR, ALH, GS, KBH, SY, JMH, KR, KB, BT, HS,
0429 Special Section from March 2007 | Volume 5 | Issue 3 | e77

HBT, CS, JT, JF, CAP, KL, SK, JFH, TU, YHR, LIF, VS, GBR, LEE,
DMK, SS, TP, EB, VG, GTC, MRF, RLS, MF, and JCV contributed
reagents/materials/analysis tools. DBR, ALH, GS, KBH, SW, SY, JAE,
RLS, KN, RF, MF, and JCV wrote the paper.
Funding. Funding for this study was received from the US
Department of Energy, Office of Science, and Office of Biological
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0431 Special Section from March 2007 | Volume 5 | Issue 3 | e77
 PLoS BIOLOGY

The Sorcerer II Global Ocean Sampling

Expedition: Expanding the Universe
of Protein Families
Shibu Yooseph1*, Granger Sutton1, Douglas B. Rusch1, Aaron L. Halpern1, Shannon J. Williamson1, Karin Remington1,
Jonathan A. Eisen1,2, Karla B. Heidelberg1, Gerard Manning3, Weizhong Li4, Lukasz Jaroszewski4, Piotr Cieplak4,
Christopher S. Miller5, Huiying Li5, Susan T. Mashiyama6, Marcin P. Joachimiak6, Christopher van Belle6,
John-Marc Chandonia6,7, David A. Soergel6, Yufeng Zhai3, Kannan Natarajan8, Shaun Lee8, Benjamin J. Raphael9,
Vineet Bafna8, Robert Friedman1, Steven E. Brenner6, Adam Godzik4, David Eisenberg5, Jack E. Dixon8,
Susan S. Taylor8, Robert L. Strausberg1, Marvin Frazier1, J. Craig Venter1
1 J. Craig Venter Institute, Rockville, Maryland, United States of America, 2 University of California, Davis, California, United States of America, 3 Razavi-Newman Center for
Bioinformatics, Salk Institute for Biological Studies, La Jolla, California, United States of America, 4 Burnham Institute for Medical Research, La Jolla, California, United States of
America, 5 University of California Los Angeles–Department of Energy Institute for Genomics and Proteomics, Los Angeles, California, United States of America, 6 University
of California Berkeley, Berkeley, California, United States of America, 7 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United
States of America, 8 University of California San Diego, San Diego, California, United States of America, 9 Brown University, Providence, Rhode Island, United States of
America

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein
families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of
sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million
Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total
of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no
detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of
sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in
the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously
categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans)
from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset
is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins,
the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their
evolution. These observations are illustrated using several protein families, including phosphatases, proteases,
ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS
data has implications for choosing targets for experimental structure characterization as part of structural genomics
efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the
addition of new sequences, implying that we are still far from discovering all protein families in nature.
Citation: Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, et al. (2007) The Sorcerer II Global Ocean Sampling expedition: Expanding the universe of protein families.
PLoS Biol 5(3): e16. doi:10.1371/journal.pbio.0050016

Academic Editor: Sean Eddy, Washington University St. Louis, United States of
America

Received March 24, 2006; Accepted August 15, 2006; Published March 13, 2007
Copyright: Ó 2007 Yooseph et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: aa, amino acid; ENS, Ensembl; EST, expressed sequence tag; GO,
Gene Ontology; GOS, Global Ocean Sampling; GS, glutamine synthetase; HMM,
hidden Markov model; IDO, indoleamine 2,3-dioxygenase; NCBI, National Center for
Biotechnology Information; ORF, open reading frame; PDB, Protein Data Bank; PG,
prokaryotic genomes; PP2C, protein phosphatase 2C; PSI, Protein Structure
Initiative; RLP, RuBisCO-like protein; TGI, TIGR gene indices; TC, trusted cutoff;
UVDE, UV dimer endonuclease
* To whom correspondence should be addressed. E-mail: Shibu.Yooseph@
venterinstitute.org
This article is part of Global Ocean Sampling collection in PLoS Biology. The full
collection is available online at http://collections.plos.org/plosbiology/gos-2007.
php.

PLoS Biology | www.plosbiology.org | S56 0432 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Author Summary
The rapidly emerging field of metagenomics seeks to examine the
genomic content of communities of organisms to understand their
roles and interactions in an ecosystem. Given the wide-ranging roles
microbes play in many ecosystems, metagenomics studies of
microbial communities will reveal insights into protein families
and their evolution. Because most microbes will not grow in the
laboratory using current cultivation techniques, scientists have
turned to cultivation-independent techniques to study microbial
diversity. One such technique—shotgun sequencing—allows ran-
dom sampling of DNA sequences to examine the genomic material
present in a microbial community. We used shotgun sequencing to
examine microbial communities in water samples collected by the
Sorcerer II Global Ocean Sampling (GOS) expedition. Our analysis
predicted more than six million proteins in the GOS data—nearly
twice the number of proteins present in current databases. These
predictions add tremendous diversity to known protein families and
cover nearly all known prokaryotic protein families. Some of the
predicted proteins had no similarity to any currently known proteins
and therefore represent new families. A higher than expected
fraction of these novel families is predicted to be of viral origin. We
also found that several protein domains that were previously
thought to be kingdom specific have GOS examples in other
kingdoms. Our analysis opens the door for a multitude of follow-up
protein family analyses and indicates that we are a long way from
sampling all the protein families that exist in nature.
Introduction
Despite many efforts to classify and organize proteins [1–6]
from both structural and functional perspectives, we are far
from a clear understanding of the size and diversity of the
protein universe [7–9]. Environmental shotgun sequencing
projects, in which genetic sequences are sampled from
communities of microorganisms [10–14], are poised to make
a dramatic impact on our understanding of proteins and
protein families. These studies are not limited to culturable
organisms, and there are no selection biases for protein
classes or organisms. These studies typically provide a gene-
centric (as opposed to an organism-centric) view of the
environment and allow the examination of questions related
to protein family evolution and diversity. The protein
predictions from some of these studies are characterized
both by their sheer number and diversity. For instance, the
recent Sargasso Sea study [10] resulted in 1.2 million protein
predictions and identified new subfamilies for several known
protein families.
Protein exploration starts by clustering proteins into
groups or families of evolutionarily related sequences. The
notion of a protein family, while biologically very relevant, is
hard to realize precisely in mathematical terms, thereby
making the large-scale computational clustering and classi-
fication problem nontrivial. Techniques for these problems
typically rely on sequence similarity to group sequences.
Proteins can be grouped into families based on the highly
conserved structural units, called domains, that they contain
[15,16]. Alternatively, proteins are grouped into families
based on their full sequence [17,18]. Many of these classi-
fications, together with various expert-curated databases [19]
such as Swiss-Prot [20], Pfam [15,21], and TIGRFAM [22,23],
or integrated efforts such as Uniprot [24] and InterPro [25],
PLoS Biology | www.plosbiology.org | S57
Expanding the Protein Family Universe
provide rich resources for protein annotation. However, a
vast number of protein predictions remain unclassified both
in terms of structure and function. Given varying rates of
evolution, there is unlikely to be a single similarity threshold
or even a small set of thresholds that can be used to define
every protein family in nature. Consequently, estimates of the
number of families that exist in nature vary considerably
based on the different thresholds used and assumptions made
in the classification process [26–29].
In this study, we explored proteins using a comprehensive
dataset of publicly available sequences together with environ-
mental sequence data generated by the Sorcerer II Global
Ocean Sampling (GOS) expedition [30]. We used a novel
clustering technique based on full-length sequence similarity
both to predict proteins and to group related sequences. The
goals were to understand the rate of discovery of protein
families with the increasing number of protein predictions,
explore novel families, and assess the impact of the environ-
mental sequences from the expedition on known proteins
and protein families. We used hidden Markov model (HMM)
profiling to examine the relative biases in protein domain
distributions in the GOS data and existing protein databases.
This profiling was also used to assess the impact of the GOS
data on target selection for protein structure character-
ization efforts. We carried out in-depth analyses on several
protein families to validate our clustering approach and to
understand the diversity and evolutionary information that
the GOS data added; the families included ultraviolet (UV)
irradiation DNA damage repair enzymes, phosphatases,
proteases, and the metabolic enzymes glutamine synthetase
and RuBisCO.
Results/Discussion
Data Generation, Sequence Clustering, and HMM Profiling
We used the following publicly available datasets in this
study (Table 1)—the National Center for Biotechnology
Information (NCBI)’s nonredundant protein database
(NCBI-nr) [31,32], NCBI Prokaryotic Genomes (PG) [31,33],
TIGR Gene Indices (TGI-EST) [34], and Ensembl (ENS)
[35,36]. The rationale for including these datasets is discussed
in Materials and Methods. All datasets were downloaded on
February 10, 2005.
None of the above-mentioned databases contained sequen-
ces from the Sargasso Sea study [10], the largest environ-
mental survey to date, and so we pooled reads from the
Sargasso Sea study with the reads from the Sorcerer II GOS
expedition [30], creating a combined set that we call the GOS
dataset. The GOS dataset was assembled using the Celera
Assembler [37] as described in [30] (see Materials and
Methods). The GOS dataset was primarily generated from
the 0.1 lm to 0.8 lm size filters and thus is expected to be
mostly microbial [30]. The data also included a small set of
sequences from a viral size (,0.1 lm) fraction (Table 1).
We identified open reading frames (ORFs) from the DNA
sequences in the PG, TGI-EST, and GOS datasets. An ORF is
commonly defined as a translated DNA sequence that begins
with a start codon and ends with a stop codon. To
accommodate partial DNA sequences, we extended this
definition to allow an ORF to be bracketed by either a start
codon or the start of the DNA sequence, and by either a stop
codon or the end of the DNA sequence. ORFs were generated
0433 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Table 1. The Complete Dataset Consisted of Sequences from NCBI-nr, ENS, TGI-EST, PG, and GOS, for a Total of 28,610,944 Sequences
Dataset Source Number of Amino Mean Sequence
Acid Sequences Length
NCBI-nr NCBI 2,317,995 339
PG ORFs NCBI 3,049,695 160
TGI-EST ORFs TIGR Gene Index 5,458,820 119
ENS Ensembl 361,668 466
GOS ORFs J. Craig Venter 17,422,766 134
Institute
doi:10.1371/journal.pbio.0050016.t001
by considering translations of the DNA sequence in all six
frames. For ORFs from the PG and TGI-EST datasets, we used
the appropriate codon usage table for the known organism.
For GOS ORFs from the assembled sequences, we used
translation table 11 (the code for bacteria, archaea, and
prokaryotic viruses) [31]. We did not include alternate codon
translations in this analysis. For all datasets, only ORFs
containing at least 60 amino acids (aa) were considered. Not
all ORFs are proteins. In this paper, ORFs that have
reasonable evidence for being proteins are called predicted
proteins; other ORFs are called spurious ORFs.
In summary, the total input data for this study (Table 1)
consisted of 28,610,994 sequences from NCBI-nr, PG, TGI-
EST, ENS, and GOS. All data and analysis results will be made
publicly available (see Materials and Methods).
We used a sequence similarity clustering to group related
sequences and subsequently predicted proteins from this
grouping. This approach of protein prediction was adopted
for two reasons. First, the GOS data make up a major portion
of the dataset being analyzed, and a large fraction of GOS
ORFs are fragmentary sequences. Traditional annotation
pipelines/gene finders, which presume complete or near-
complete genomic data, perform unsatisfactorily on this type
of data. Second, protein prediction based on the comparison
of ORFs to known protein sequences imposes limits on the
protein families that can be explored. In particular, novel
proteins that belong to known families will not be detected if
they are sufficiently distant from known members of that
family. This is the case even though there may be other novel
proteins that can transitively link them to the known
proteins. Similarly, truly novel protein families will also not
be detected.
As the primary input to our clustering process, we
computed the pairwise sequence similarity of the 28.6 million
aa sequences in our dataset using an all-against-all BLAST
search [38]. This required more than 1 million CPU hours on
two large compute clusters (see Materials and Methods). The
sequences were clustered in four steps (see Materials and
Methods). In the first step, we identified a nonredundant set
of sequences from the entire dataset using only pairwise
matches with 98% similarity and involving 95% of the
PLoS Biology | www.plosbiology.org | S58
Expanding the Protein Family Universe
Brief
Description
Consists of protein sequences submitted to SWISS-PROT, PDB, PIR, and PRF, and
also predicted proteins from both finished and unfinished genomes in GenBank,
EMBL, and DDBJ.
ORFs identified from 222 prokaryotic genome projects. Organisms are listed in
Protocol S1.
ORFs identified from 72 datasets in which each dataset consists of EST assem-
blies. Organisms are listed in Protocol S1.
Sequences from 12 species, including human, mouse, rat, chimp, zebrafish, fruit
fly, mosquito, honey bee, dog, two species of puffer fish, chicken, and worm.
ORFs identified from an assembly of 7.7 million reads. These reads include both
the reads from the Sorcerer II GOS Expedition and the reads from the earlier Sar-
gasso Sea study. Also included are 36,318 ORFs identified from an assembly of
sequences collected from the viral size (, 0.1 lm) fraction of one sample.
length of the shorter sequence. This step served the dual role
of identifying highly conserved groups of sequences (where
each group was represented by a nonredundant sequence) and
removing redundancy in the dataset due to identical and
near-identical sequences. Only nonredundant sequences were
considered for further steps in our clustering procedure. In
the second step, we identified core sets of similar sequences
using only matches between two sequences involving 80%
of the length of the longer sequence. We used a graph-
theoretic procedure to identify dense subgraphs (the core
sets) within a graph defined by these matches. While the
match parameters we used in this step were more relaxed
than those in the first step, we chose them to reduce the
grouping of unrelated sequences while simultaneously re-
ducing the unnecessary splitting of families. In the third step,
these core sets were transformed into profiles, and we used a
profile–profile method [39] to merge related core sets into
larger groups. In the final step, we recruited sequences to
core sets using sequence-profile matching (PSI-BLAST [40])
and BLAST matches to core set members. We required the
match to involve 60% of the length of the sequence being
recruited.
We identified and removed clusters containing likely
spurious ORFs using two filters (see Materials and Methods).
The first filter identified clusters containing shadow ORFs.
The second filter identified clusters containing conserved but
noncoding sequences, as indicated by a lack of selection at the
codon level. Only clusters that remained after the two
filtering steps and contained at least two nonredundant
sequences are reported in this analysis.
We examined the distribution of known protein domains
in the full dataset using profile HMMs [41] from the Pfam [15]
and TIGRFAM [22] databases (see Materials and Methods).
We labeled sequences that end up in clusters (containing at
least two nonredundant sequences) or that have HMM
matches as predicted proteins. The inclusion of the PG ORF
set allowed for the evaluation of protein prediction using our
clustering approach. A comparison of proteins predicted in
the PG ORF set by our clustering against PG ORFs annotated
as proteins by whole-genome annotation techniques revealed
that our protein prediction method via clustering has a
0434 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Table 2. Clustering and HMM Profiling Results Showing the Number of Predicted Proteins (Including Both Redundant and
Nonredundant Sequences) in Each Dataset
Dataset Original Set Clustering (A) HMM
Profiling (B)
NCBI-nr 2,317,995 1,939,056 1,645,146
PG ORFs 3,049,695 575,729 448,159
TGI-EST ORFs 5,458,820 1,097,083 606,779
ENS 361,668 319,855 253,007
GOS ORFs 17,422,766 6,046,914 3,701,388
Total 28,610,944 9,978,637 6,654,479
A \ B denotes the number of predicted proteins common to both the clustering and the HMM profiling; A  B, the number of predicted proteins in clusters but not in the HMM profile set;
B  A, the number of predicted proteins in the HMM profile set but not in clusters; and A [ B, the total number of predicted proteins in each dataset.
doi:10.1371/journal.pbio.0050016.t002
sensitivity of 83% and a specificity of 86% (see Materials and
Methods). The HMM profiling allowed for the evaluation of
our clustering technique’s grouping of sequences. We used
Pfam models in two different ways for this assessment (see
Materials and Methods) and make three observations. First,
using a simple Pfam domain architecture-based evaluation,
these clusters are mostly consistent as reflected by 93% of
clusters having less than 2% unrelated pairs of sequences in
them. Second, these clusters are quite conservative and can
split domain families, with 58% of domain architectures
being confined to single clusters and 88% of domain
architectures having more than half of their occurrences in
a single cluster. Third, the size distribution of these clusters is
quite similar to the size distribution of clusters induced by
Pfams.
Protein Prediction
Of the initial 28,610,944 sequences, we labeled 9,978,637
sequences (35%) as predicted proteins based on the cluster-
ing, of which nearly 60% are from GOS (Table 2). The HMM
profiling labeled only an additional 226,743 (0.8%) sequences
as predicted proteins, for a total of 10,205,380 predicted
proteins. This indicates that our clustering method captures
most of the sequences found by profile HMMs. For sequences
both in clusters and with HMM matches, (on average) 73.5%
of their length is covered by HMM matches. For sequences
not in clusters but with HMM matches, this value is only
45.3%. Furthermore, while 64% of sequences in clusters have
HMM matches, there are 3,550,901 sequences that are
grouped into clusters but do not have HMM matches. Most
of these clusters correspond either to families lacking profile
HMMs or contain sequences that are too remote to match
above the cutoffs used. The latter is an indication of the
diversity added to known families that is not picked up by
current profile HMMs.
Using our method, the predicted proteins constitute
different fractions of the totals for the five datasets, with
87% for NCBI-nr, nearly 20% for both PG ORFs and TGI-
EST ORFs, 92% for ENS, and 35% for GOS. The high rate of
prediction for ENS is a reflection of the high degree of
conservation of proteins across the metazoan genomes,
whereas the prediction rates for PG ORFs and TGI-EST
ORFs are similar to rates seen in other protein prediction
approaches. The 13% of NCBI-nr sequences that we marked
as spurious may constitute contaminants in the form of false
PLoS Biology | www.plosbiology.org | S59
Expanding the Protein Family Universe
A\B AB BA Total Predicted Mean Length
Proteins A [ B of Sequence
1,566,123 372,933 79,023 2,018,079 359
418,503 157,226 29,656 605,385 325
576,532 520,551 30,247 1,127,330 207
241,671 78,184 11,336 331,191 489
3,624,907 2,422,007 76,481 6,123,395 199
6,427,736 3,550,901 226,743 10,205,380 —
predictions or organism-specific proteins. Nearly two-thirds
of these sequences are labeled ‘‘hypotheticals,’’ ‘‘unnamed,’’
or ‘‘unknown.’’ This is more than twice the fraction of
similarly labeled sequences (30%) in the full NCBI-nr dataset.
Of the remaining one-third, half of them are less than 100 aa
in length. This suggests that they are either fast-evolving short
peptides, spurious predictions, or proteins that failed to meet
the length-based thresholds in the clustering.
Based on the clustering and the HMM profiling, there is
evidence for 6,123,395 proteins in the GOS dataset (Table 2).
Given the fragmentary nature of the GOS ORFs (as a result of
the GOS assembly [10,30]), it is not surprising that the average
length of a GOS-predicted protein (199 aa) is smaller than the
average length of predicted proteins in NCBI-nr (359 aa), PG
ORFs (325 aa), TGI-EST ORFs (207 aa), and ENS (489 aa). The
ratio of clustered ORFs to total ORFs is significantly higher
for the GOS ORFs (34%) compared to PG ORFs (19%). This
could be due to a large number of false-positive protein
predictions in the GOS dataset. However, this is unlikely for a
variety of reasons. Nearly 4.64 million GOS ORFs (26.6%)
have significant BLAST matches (with an E-value  13 1010)
to NCBI-nr sequences. The PG ORFs do not have a high false-
positive rate compared to the submitted annotation for the
prokaryotic genomes (see Materials and Methods). Most
importantly, based on the fragmentary nature of GOS
sequencing compared to PG sequencing, the number of
shadow (spurious) ORFs 60 aa is significantly reduced (see
Materials and Methods).
Some pairs of GOS-predicted proteins that belong to the
same cluster are adjacent in the GOS assembly. While some of
them correspond to tandem duplicate genes, an overwhelm-
ing fraction of the pairs are on mini-scaffolds [10], indicating
that they are potentially pieces of the same protein (from the
same clone) that we split into fragments. We estimate that this
effect applies to 3% of GOS-predicted proteins. Sequencing
errors and the use of the wrong translation table can also
result in the ORF generation process producing split ORF
fragments.
The combined set of predicted proteins in NCBI-nr, PG,
TGI-EST, and ENS, as expected, has a lot of redundancy. For
instance, most of the PG protein predictions are in NCBI-nr.
Removing exact substrings of longer sequences (i.e., 100%
identity) reduces this combined set to 3,167,979 predicted
proteins. When we perform the same filtering on the GOS
dataset, 5,654,638 predicted proteins remain. Thus, the GOS-
0435 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

largest clusters correspond to families that have functionally

diversified and expanded (Table 4). While some large families,
such as the HIV envelope glycoprotein family and the
immunoglobulins, also reflect biases in sequence databases,
many more, including ABC transporters, kinases, and short-
chain dehydrogenases, reflect their expected abundance in
nature.

Rate of Discovery of Protein Families

Figure 1. Proportion of Sequences for Each Kingdom
(A) The combined set of NCBI-nr, PG, TGI-EST, and ENS has 3,167,979
sequences. The eukaryotes account for the largest portion and is more
than twice the bacterial fraction.
(B) Predicted kingdom proportion of sequences in GOS. Out of the
5,654,638 GOS sequences, 5,058,757 are assigned kingdoms using a
BLAST-based scheme. The bacterial kingdom forms by far the largest
fraction in the GOS set.
doi:10.1371/journal.pbio.0050016.g001
predicted protein set is 1.8 times the size of the predicted
protein set from current publicly available datasets. We used
a simple BLAST based scheme to assign kingdoms for the
GOS sequences (see Materials and Methods). Of the sequences
that we could annotate by kingdom, 63% of the sequences in
the public datasets are from the eukaryotic kingdom, and
90.8% of the sequences in the GOS set are from the bacterial
kingdom (Figure 1).
Protein Clustering
The 9,978,637 protein sequences predicted by our cluster-
ing method are grouped into 297,254 clusters of size two or
more, where size of a cluster is defined to be the number of
nonredundant sequences in the cluster. There are 280,187
small clusters (size , 20), 12,992 medium clusters (size
between 20 and 200), and 4,075 large clusters (size . 200).
While the 17,067 medium- and large-sized clusters constitute
only 6% of the total number of clusters, they account for 85%
of all the sequences that are clustered (Table 3). Many of the
We examined the rate of discovery of protein families using
our clustering method to determine whether our sampling of
the protein universe is reaching saturation. We find that for
the present number of sequences there is an approximately
linear trend in the rate of discovery of clusters with the
addition of new (i.e., nonredundant) sequences (Figure 2).
Moreover, the observed distribution of cluster sizes is well
approximated by a power law [42,43], and this observed
power law can be used to predict the rate of growth of the
number of clusters of a given size (see Materials and
Methods). This rate is dependent on the value of the power
law exponent and decreases with increasing cluster sizes. We
find good agreement between the observed and predicted
growth rates for different cluster sizes. The approximately
linear relationship between the number of clusters and the
number of protein sequences indicates that there are likely
many more protein families (either novel or subfamilies
distantly related to known families) remaining to be
discovered.
GOS versus Known Prokaryotic versus Known
Nonprokaryotic
We also examined the GOS coverage of known proteins
and protein families. Based on the cell-size filtering
performed while collecting the GOS samples, we expected
that the sample would predominantly be a size-limited subset
of prokaryotic organisms [30]. We studied the content of the
17,067 medium- and large-sized clusters across three group-
ings: (1) GOS, (2) known prokaryotic (PG together with
bacterial and archaeal portions of NCBI-nr), and (3) known
nonprokaryotic (TGI-EST and ENS together with viral and
eukaryotic portions of NCBI-nr). The Venn diagram in Figure
3 shows the breakdown of these clusters by content (see
Materials and Methods). The largest section contains GOS-

Table 3. Cluster Size Distribution and the Distribution of Sequences in These Clusters

Cluster Size Number of Clusters Total Sequences NCBI-nr PG TGI-EST ENS GOS

2–4 214,033 756,269 194,297 87,699 149,687 32,920 291,666

5–9 48,348 415,166 97,759 30,565 71,414 14,828 200,600
10–19 17,806 350,918 90,682 19,904 60,783 23,493 156,056
20–49 7,255 310,770 78,153 13,809 58,496 26,486 133,826
50–99 3,086 337,296 80,470 14,342 55,190 26,150 161,144
100–199 2,631 595,903 165,846 28,100 107,490 40,465 254,002
200–499 2,134 1,036,567 218,940 57,131 164,581 49,797 546,118
500–999 799 914,207 148,084 54,077 90,020 24,047 597,979
1,000–2,000 620 1,503,116 205,196 79,348 105,866 21,883 1,090,823
2,000 542 3,758,425 659,629 190,754 233,556 59,786 2,614,700
Total 297,254 9,978,637 1,939,056 575,729 1,097,083 319,855 6,046,914

The size of a cluster is the number of nonredundant sequences in it. Column three shows the total number of sequences (both redundant and nonredundant) in these clusters. The
succeeding columns show their breakdown by the five datasets. There are 17,067 medium- and large-size clusters.
doi:10.1371/journal.pbio.0050016.t003

PLoS Biology | www.plosbiology.org | S60 0436 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Table 4. List of the Top 25 Clusters from the Clustering Process

Cluster ID Cluster Nonredundant Total NCBI-nr PG TGI-EST ENS GOS

Annotation Sequences Sequences

3510 Immunoglobulin 37,227 51,944 49,206 0 1,649 1,089 0

2568 ABC transporter 34,130 69,010 8,886 6,248 150 13 53,713
49 Short chain dehydrogenase 33,406 56,266 7,607 3,055 2,852 747 42,005
4294 NAD dependent epimerase/dehydratase 29,445 35,555 2,745 1,265 1,500 111 29,934
1239 AMP-binding enzyme 22,111 37,598 3,838 1,614 2,246 613 29,287
2630 Envelope glycoprotein 21,161 41,205 41,189 2 10 0 4
157 Glycosyl transferases group 1 20,366 27,012 2,766 1,446 557 42 22,201
183 Integral membrane protein 17,627 33,079 2,154 1,298 1,198 95 28,334
530 Aldehyde dehydrogenase 15,851 30,929 3,116 1,349 1,589 388 24,487
1308 Aminotransferase class-V and 15,757 22,484 1,849 1,086 413 71 19,065
DegT/DnrJ/EryC1/StrS aminotransferase
244 Kinase family, including pknb, epk, c6 15,112 21,641 6,384 83 10,809 2,761 1,604
336 Histidine kinase–, DNA gyrase B–, and HSP90-like ATPase 14,724 23,355 3,809 2,469 54 4 17,019
357 Tetratricopeptide repeat 14,323 17,058 1,598 609 1,320 315 13,216
4325 Alpha/Beta hydrolase fold 13,806 20,886 2,828 1,334 1,625 196 14,903
113 Aminotransferase class I and II 13,006 22,186 2,931 1,534 1,239 120 16,362
333 Zinc-binding dehydrogenase 12,737 22,298 4,055 1,370 2,383 269 14,221
1315 tRNA synthetases class I (I, L, M, and V) 12,545 19,992 1,152 600 472 131 17,637
26 Acyl-CoA dehydrogenase 12,150 22,340 2,081 1,152 541 179 18,387
159 ABC transporter and ABC transporter transmembrane 11,984 17,650 2,697 1,442 797 170 12,544
3357 Cytochrome P450 11,929 17,302 5,355 249 6,994 1,399 3,305
4556 Response regulator 11,928 21,903 5,387 3,320 348 5 12,843
1720 TonB-dependent receptor 11,890 17,080 1,789 1,090 34 2 14,165
514 NADH dehydrogenase (various subunits) 11,224 25,068 11,624 635 253 10 12,546
4235 Glycosyl transferase family 2 10,954 13,593 1,236 724 74 14 11,545
186 7 transmembrane receptor 10,654 22,252 13,943 0 1,475 6,829 5

Clusters were annotated using the most commonly matching Pfam domains. Many of these clusters correspond to families that have expanded and functionally diversified.
doi:10.1371/journal.pbio.0050016.t004

only clusters (23.40%) emphasizing the significant novelty
provided by the GOS data. The next section consists of
clusters containing sequences from only the known non-
prokaryotic grouping (20.78%), followed closely by the
section containing clusters with sequences from all three
groupings (20.23%). The large known nonprokaryotic–only
grouping shows that our current GOS sampling methodology
will not cover all protein families, and perhaps misses some
protein families that are exclusive to higher eukaryotes. The
large section of clusters that include all three groupings
indicates a large core of well-conserved protein families
across all domains of life. In contrast, the known prokaryotic
protein families are almost entirely covered by the GOS data.
Novelty Added by GOS Data
There are 3,995 medium and large clusters that contain
only sequences from the GOS dataset. Some are divergent
members of known families that failed to be merged by the
clustering parameters used, or are too divergent to be
detected by any current homology detection methods. The

Figure 2. Rate of Discovery of Clusters as (Nonredundant) Sequences Are Added

The x-axis denotes the number of sequences (in millions) and the y-axis denotes the number of clusters (in thousands). Seven datasets with increasing
numbers of (nonredundant) sequences are chosen as described in the text. The blue curve shows the number of core sets of size 3 for the seven
datasets. Curves for core set sizes 5, 10, and 20 are also shown. Linear regression gives slopes 0.027 (R2 ¼ 0.999), 0.011 (R2 ¼ 0.999), 0.0053 (R2 ¼
0.999), and 0.0024 (R2 ¼ 0.996) for size 3, size 5, size 10, and size 20, respectively.
doi:10.1371/journal.pbio.0050016.g002

PLoS Biology | www.plosbiology.org | S61 0437 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Figure 3. Venn Diagram Showing Breakdown of the 17,067 Medium and
Large Clusters by Three Categories—GOS, Known Prokaryotic, and
Known Nonprokaryotic
doi:10.1371/journal.pbio.0050016.g003
remaining clusters are completely novel families. In exploring
the 3,995 GOS-only clusters, 44.9% of them contain
sequences that have HMM matches, or BLAST matches to
sequences in a more recent snapshot of NCBI-nr (down-
loaded in August 2005) than was used in this study. The recent
NCBI-nr matches include phage sequences from cyanophages
(P-SSM2 and P-SSM4) [44] and sequences from the SAR-11
genome (Candidatus pelagibacter ubique HTCC1062) [45]. We
used profile–profile searches [39] to show that an additional
12.5% of the GOS-only clusters can be linked to profiles built
from Protein Data Bank (PDB), COG, or Pfam. The 2,295
clusters with detected homology are referred to as Group I
clusters. The remaining 1,700 (42.6%) GOS-only clusters with
no detectable homology to known families are labeled as
Group II clusters.
We applied a guilt-by-association operon method to
annotate the GOS-only clusters with a strategy that did not
rely on direct sequence homology to known families.
Function was inferred for the GOS-only clusters by examin-
ing their same-strand neighbors on the assembly (see
Materials and Methods). Similar strategies have been success-
fully used to infer protein function in finished microbial
genomes [46–48]. Despite minimal assembly of GOS reads,
many scaffolds and mini-scaffolds contain at least partial
fragments of more than one predicted ORF, thereby making
this approach feasible. For 90 (5.3%) of the Group II clusters,
and for 214 (9.3%) of the Group I clusters, at least one Gene
Ontology (GO) [49] biological process term at p-value 0.05
can be inferred. The inferred functions and neighbors of
some of these GOS-only clusters are highlighted in Table 5.
We observed that for Group I clusters, the neighbor-inferred
function is often bolstered by some information from weak
homology to known sequences. While neighboring clusters as
a whole are of diverse function, a number of GOS-only
clusters seem to be next to clusters implicated in photosyn-
PLoS Biology | www.plosbiology.org | S62
Expanding the Protein Family Universe
thesis or electron transport. These GOS-only clusters could
be of viral origin, as cyanophage genomes contain and
express some photosynthetic genes that appear to be derived
from their hosts [44,50,51]. In support of these observations,
we identified five photosynthesis-related clusters containing
hundreds to thousands of viral sequences, including psbA,
psbD, petE, SpeD, and hli in the GOS data; furthermore, our
nearest-neighbor analysis of these sequences reveals the
presence of multiple viral proteins (unpublished data).
Although the majority of GOS-only sequences are bacterial,
a higher than expected proportion of the GOS-only clusters
are predicted to be of viral origin, implying that viral
sequences and families are poorly explored relative to other
microbes. To assign a kingdom to the GOS-only clusters, we
first inferred the kingdom of neighboring sequences based on
the taxonomy of the top four BLAST matches to the NCBI-nr
database (see Materials and Methods). A possible kingdom was
assigned to the GOS-only cluster if more than 50% of
assignable neighboring sequences belong to the same king-
dom. Viewed in this way, 11.8% of Group I clusters and
17.3% of Group II clusters with at least one kingdom-assigned
neighbor have more than 50% viral neighbors (Figure 4).
Only 3.3% and 3.4% of random samples of clusters with size
distributions matching that of Group I and Group II clusters
have more than 50% viral neighbors, while 7.7% of all
clusters pass this criterion. A total of 547 GOS-only clusters
contain sequences collected from the viral size fraction
included in the GOS dataset. For these clusters, 38.9% of the
Group I subset and 27.5% of the Group II subset with one or
more kingdom-assigned neighbors would be inferred as viral,
based on the conservative criteria of having more than 50%
viral assignable neighbors. Several alternative kingdom
assignment methods were tried (see Materials and Methods)
and provide for a similar conclusion.
The GOS-only clusters also tend to be more AT-rich than
sequences from a random size-matched sample of clusters
(35.9% 6 8% GC content for Group II clusters versus 49.5%
6 11% GC content for sample). Phage genomes with a
Prochlorococcus host [44] are also AT rich (37% average GC
content). Our analysis of the graph constructed based on
inferred operon linkages between all clusters indicates that
the GOS-only clusters may constitute large sets of cotran-
scribed genes (see Materials and Methods).
The high proportion of potentially viral novel clusters
observed here is reasonable, as 60%–80% of the ORFs in
most finished marine phage genomes are not homologous to
known protein sequences [52]. Viral metagenomics projects
have reported an equally high fraction of novel ORFs [53],
and a recent marine metagenomics project estimated that up
to 21% of photic zone sequences could be of viral origin [51].
It has also been reported that 40% of ORFans (sequences that
lack similarity to known proteins and predicted proteins)
exist in close spatial proximity to each other in bacterial
genomes, and this combined with proximity to integration
signals has been used to suggest a viral horizontally trans-
ferred origin for many bacterial ORFans [54]. Others have
noted a clustering of ORFans in genome islands and
suggested they derive from a phage-related gene pool [55].
A recent analysis of genome islands from related Prochlor-
ococcus found that phage-like genes and novel genes cohabit
these dynamic areas of the genome [56]. In our GOS-only
clusters, 37 of the 1,700 clusters with no detectable similarity
0438 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Table 5. Neighbor-Based Inference of Function for Novel Clusters of GOS Sequences

Novel Inferred Function p-Valuea Neighboring Clusters with Other Neighbors Comments
Cluster Contributing GO Annotation of Interestb
ID
GO ID Biological process Cluster ID GO Annotation

8837 GO:0006260 DNA replication 4.70 3 104 812 ATPase involved in DNA Phage Mu Mom DNA Profile–profile match: DNA polymerase
replication modification enzyme processivity factor
2,655 DNA polymerase family B DNA methylase
12519 GO:0006118 Electron transport 4.54 3 103 1,362 Cytochrome c oxidase Profile–profile match: PF03626— cytochrome c oxidase
subunit III subunit IV; 3 predicted transmembrane helices
1,771 SCO1/SenC—biogenesis of
photosynthetic systems
11010151 GO:0017004 Cytochrome complex 1.00 3 105 8,136 Thioredoxin .20 diverse profile–profile matches, one of which is
assembly cytochrome c biogenesis factor ccmH_2

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9,364 Cytochrome c biogenesis protein
1,317 Cytochrome c assembly protein
18456 GO:0009252 Peptidoglycan biosynthesis 1.00 3 105 1,252 FAD binding domain Extracytoplasmic function One predicted TM helix
(ECF) sigma factor 24
10,764 UDP-N-acetylenolpyruvoylglucos- Viral RNA helicase
aminereductase
14219 GO:0009628 Response to abiotic stimulus 3.10 3 104 5,936 Colicin V production protein Predicted soluble; exclusively neighbors to just
two clusters.
MatE multidrug efflux pump
11480 GO:0015031 Protein transport 3.00 3 104 4,177 MotA/TolQ/ExbB proton channel family Four predicted TM helices; Tol proteins facilitate
transport of

0439
colicins, iron, and phage DNA
9,569 Biopolymer transport protein
ExbD/TolR
14360 GO:0006777 Mo-molybdopterin cofactor 1.00 3 105 9,745 MoaC family Sulfite oxidase SAR11 blast match annotated as probable moaD;
biosynthesis profile–profile matches to ThiS and molybdopterin
converting factor; ,.05% of sequences have PFAM
match to ThiS family
9,948 MoaE protein Predicted thioesterase
255 Radical SAM superfamily
8397 GO:0017004 Cytochrome complex 1.00 3 105 8,136 Thioredoxin SMC superfamily (homologous to Blast match to ‘‘periplasmic or inner membrane–
assembly ABC family) associated protein’’; two predicted TM helices;
0.7% of sequences have PFAM match to
cytochrome c biogenesis protein
9,364 Uncharacterized cytochrome c
biogenesis protein
5
13909 GO:0015979 Photosynthesis 1.00 3 10 13,990 Photosystem II reaction centre Predicted soluble; single blast match to cyanophage
N protein (psbN) P-SSM2 hypothetical protein; many phage proteins
as minor neighbors
5,184 Photosynthetic reaction centre
protein D1 (psbA)
7,664 Ferredoxin-dependent bilin reductase

a
p-Values were computed by simulating 100,000 neighbor cluster sets of equivalent size.
b
Not all clusters could be mapped to a GO term.
doi:10.1371/journal.pbio.0050016.t005

Special Section from March 2007 | Volume 5 | Issue 3 | e16

Expanding the Protein Family Universe
 Expanding the Protein Family Universe

Table 6. Functions Skewed in Domain Representation between

PG and GOS

Process Number Number Number GOS

of HMMs in PG in GOS Enrichment

Sarcosine oxidase 6 686 19,295 4.766

Oxidative stress 5 524 9,804 3.170
Ubiquinone synthesis 4 245 4,035 2.790
RecA 1 215 3,728 2.938
Topoisomerase IV 4 2,163 33,472 2.622
Photosynthesis 41 919 13,889 2.561
DNA polymerase 20 3,682 51,224 2.357
tRNA synthetases 11 5,499 71,294 2.197
Transketolase 4 2,127 26,440 2.106
DNA gyrase 7 4,146 49,677 2.030
TCA cycle 30 12,057 135,294 1.901
Shikimate metabolism 8 2,393 24,316 1.722
DnaJ 3 1,103 12,389 1.891
Universal ribosomal 39 8,555 80,321 1.591
components (found in
all three kingdoms)
UVR exonuclease operon 6 4,108 38,223 1.577
Figure 4. Enrichment in the GOS-Only Set of Clusters for Viral Neighbors
ABC transporter 39 193,689 727,314 0.636
Cluster sets from left to right are: I, GOS-only clusters with detectable Flagellum 38 3,771 12,988 0.584
BLAST, HMM, or profile-profile homology (Group I); II, GOS-only clusters Sugar transport 7 3,601 4,453 0.210
with no detectable homology (Group II); I-S, a sample from all clusters Transposase 13 4,354 4,365 0.170
chosen to have the same size distribution as Group I; II-S, a sample from Che operon (chemotaxis) 7 1,142 1,119 0.166
all clusters chosen to have the same size distribution as Group II; I-V, a
Ethanolamine 9 231 218 0.160
subset of clusters in Group I containing sequences collected from the
Hydrogenases 16 1,179 1,061 0.152
viral size fraction; II-V, a subset of clusters in Group II from the viral size
fraction; and all clusters. Notice that although predominantly bacterial, Pilus 14 700 623 0.151
GOS-only clusters are assigned as viral based on their neighbors more PTS phosphotransferase 32 11,439 6,661 0.099
often than the size-matched samples and the set of all clusters. system
doi:10.1371/journal.pbio.0050016.g004 Gas vesicle 6 49 19 0.066
Grþ nonspore 22 1,063 52 0.008
Grþ spores 15 503 0 0.000
(2.2%) have at least ten bacterial-classified and ten viral-
classified neighboring ORFs. This is 6.2-fold higher than the Functionally related families of domains were grouped by GO terms or by inspection to
rate seen for the size-matched sample of all clusters (six sum up total domain counts in GOS and PG. There were 8,935,364 domain matches in the
GOS data (corresponding to 3,701,388 sequences) and 1,513,880 domain matches in the
clusters, 0.35%). This would seem to add more support to a PG data (corresponding to 448,159 sequences). The GOS enrichment ratio is computed
phage origin for at least some ORFans found in bacterial from columns three and four, and then normalized to account for the 5.9 times the
genomes. number of domain matches in GOS compared to PG.
doi:10.1371/journal.pbio.0050016.t006
If a sizable portion of the novel families in the GOS data
are in fact of viral origin, it suggests that we are far from fully
exploring the molecular diversity of viruses, a conclusion ences reflect several factors, including differing biochemical
echoed in previous studies of viral metagenomes [53,57,58]. In needs of oceanic life and taxonomic biases in the two
studies of bacterial genomes, discovery of new ORFans shows datasets. An initial comparison of these domain profiles
no sign of reaching saturation [59]. Coverage of many phage helps shed light on these factors. 91% (964/1,056) of GOS-only
families in the GOS data may be low, given that there are domains are viral and/or eukaryotic specific (by Pfam
inherent differences in the abundance of their presumed annotation). Most of the remaining 92 domains are rare (63
bacterial hosts. These GOS-only clusters were operationally domains have less than ten copies in GOS), are predom-
defined as having at least 20 nonredundant sequences. inantly eukaryotic/viral, or are specific to narrow bacterial
Reducing this threshold to ten nonredundant sequences adds taxa without completed genome sequences. Most of the 879
7,241 additional clusters. Whether this vast diversity repre- PG-only domains are also rare (444 have ten or less members),
sents new families or is a reflection of the inability to detect and/or are restricted to tight lineages, such as Mycoplasma (104
distant homology will require structural and biochemical matches to five domains) or largely extremeophile archaeal-
studies, as well as continued development of computational specific domains (1,254 matches to 99 domains). Highly PG-
methods to identify remotely related sequences. enriched domains also tend to belong in these categories.
Many moderately skewed domains reflect the taxonomic skew
Comparison of Domain Profiles in GOS and PG Datasets between PG and GOS. For instance, we found that a set of six
We used HMM profiling to address the question of which sarcosine oxidase-related domains are 4.8-fold enriched in
biochemical and biological functions are expanded or GOS (Table 6). They are mostly found in a- and c-
contracted in GOS compared to the largely terrestrial proteobacteria, which are widespread in GOS. Normalizing
genomes in PG. Significant differences are seen in 68% of to the taxonomic class level predicts a 1.8-fold enrichment in
domains (4,722 out of the 6,975 domains that match either GOS, indicating that taxonomy alone cannot fully explain the
GOS or PG; p-value ,0.001, chi-square test). These differ- prevalence of these proteins in oceanic bacteria.

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 Expanding the Protein Family Universe

Figure 5. Coverage of GOS-100 and Public-100 by Pfam and Relative Sizes of Pfam Families by Kingdom, Sorted by Size
The public-100 sequences are annotated using the NCBI taxonomy and the source public database annotations. GOS-100 sequences were given
kingdom weights as described in Materials and Methods. For each kingdom, the fraction of sequences with 1 Pfam match are shown, while the ten
largest Pfam families shown as discrete sections whose size is proportional to the number of matches between that family and GOS-100 or public-100
sequences. Pfam families that are smaller than the ten largest are binned together in each column’s bottom section. Pfam covers public-100 better than
GOS-100 in all kingdoms, with the greatest difference occurring in the viral kingdom, where 89.1% of public-100 viral sequences match a Pfam domain,
while only 27.5% of GOS-100s have a sequence match.
doi:10.1371/journal.pbio.0050016.g005

Mysterious Lack of Characteristic Gram-Positive Domains
Gram-positive bacteria (Firmicutes and Actinobacteria) repre-
sent 26.7% of PG and ;12% of GOS [30]. Given the larger
size of the GOS dataset, one might predict Gram-positive–
specific domains to be ;2.4-fold enriched in GOS. Instead,
the opposite is consistently seen. Of 15 firmicute-specific
spore-associated domains, PG has 503 members, but GOS has
none. For another 22 firmicute-restricted domains of varying
or unknown function, the PG/GOS ratio is 1797:77 (Table 6).
Hence, it appears that GOS Gram-positive lineages lack most
of their characteristic protein domains. Two sequenced
marine Gram-positives (Oceanobacillus iheyensis [60] and Bacillus
sp. NRRL B-14911) have a large complement of these
domains. However, another recently assembled genome from
Sargasso sea surface waters, the actinomycete Janibacter sp.
HTCC2649, has just two of these domains, and may reveal a
whole-genome context for this curious loss of characteristic
domains.
Flagellae and Pili Are Selectively Lost from Oceanic
Species
Flagellum components from both eubacteria and archaea
are significantly underrepresented in the GOS dataset by
about 2-fold (Table 6). Ironically, at a bacterial scale,
swimming may be worthwhile on an almost dry surface, but
not in open water. The chemotaxis (che) operon that often
directs flagellar activity is also rare in GOS. Another direc-
tional appendage, the pilus, is even more reduced, though its
taxonomic distribution (mostly in proteobacteria, predom-
inantly c-proteobacteria) would have predicted enrichment.
Skew in Core Cellular Pathways
While taxonomically specialized domains are likely to be
skewed by taxonomic differences, core pathways found in
many or all organisms paint a different picture. We used GO
term mapping and text mining to group domains into major
functions and to look for consistent skews across several
domains. Several core functions, including DNA-associated
proteins (DNA polymerase, gyrase, topoisomerase), ribosomal
subunits shared by all three kingdoms, marker proteins such
as recA and dnaJ, and TCA cycle enzymes all tend to be GOS
enriched. This suggests that oceanic genomes may be more
compact than sequenced genomes and so have a higher
proportion of core pathways.
Characteristics and Kingdom Distribution of Known
Protein Domains
A decade ago, databases were highly biased towards
proteins of known function. Today, whole-genome sequenc-
ing and structural genomics efforts have presumably reduced
the biases that are a result of targeted protein sequencing. We
used the Pfam database to compare the characteristics and
kingdom distribution of known protein domains in the GOS
dataset to that of proteins in the publicly available datasets
(NCBI-nr, PG, TGI-EST, and ENS). Such an effort can be used
to assess biases in these datasets, help direct future sampling
efforts (of underrepresented organisms, proteins, and protein
families), make more informed generalizations about the
protein universe, and provide important context for deter-
mination of protein evolutionary relationships (as biased
sampling could indicate expected but missing sequences).
For this analysis we used the nonredundant datasets (at
100% identity) discussed in Figure 1. We refer to the set of
3,167,979 nonredundant sequences from NCBI-nr, PG, TGI-
EST, and ENS as the public-100 set and the similarly filtered set
of 5,654,638 sequences from the GOS data as the GOS-100 set.
About 70% of public-100 sequences and 56% of GOS-100
sequences significantly match at least one Pfam model. The
most obvious difference between the sets is that the vast
majority of GOS sequences are bacterial, and this has to be
taken into account when comparing the numbers. Since
different Pfam families appear with different frequencies in
the kingdoms, we considered the results for each kingdom
separately (Figure 5). We then evaluated all kingdoms
together, with results normalized by relative abundance of
members from the different kingdoms. A domain found
commonly and exclusively in eukaryotes and abundant in

PLoS Biology | www.plosbiology.org | S65 0441 Special Section from March 2007 | Volume 5 | Issue 3 | e16

public-100 would be expected to be found rarely in GOS-100.
We used a conservative BLAST-based kingdom assignment
method to assign kingdoms to the GOS sequences (see
Materials and Methods).
In each kingdom, sequences in GOS-100 are less likely to
match a Pfam family than those in public-100 (Figure 5). For
the cellular kingdoms, these differences are comparatively
modest. While diversity of the GOS data accounts for some of
this difference, it might also be explained in part by the
fragmentary nature of the GOS sequences. Viruses tell a
dramatic and different story. Of public-100 viral sequences,
89.1% match a Pfam domain, while only 27.5% of GOS-100
viral sequences have a match. This tremendous difference
appears to be due to heavy enrichment of the public data for
minor variants of a few protein families, indicated by the sizes
of the ten most populous Pfams in each kingdom (Figure 5).
Sequences from three Pfam families (envelope glycoprotein
GP120, reverse transcriptase, and retroviral aspartyl pro-
tease) account for a third of all public viral sequences. By
contrast, the most populous three families in the GOS-100
data (bacteriophage T4-like capsid assembly protein [Gp20],
major capsid protein Gp23, and phage tail sheath protein)
account for only about 7% of public-100 sequences. Such a
difference may be due to intentional oversampling of
proteins that come from disease-causing organisms in the
public dataset.
While the total proportion of proteins with a Pfam hit is
fairly similar between public-100 (70%) and GOS-100 (56%)
datasets, there are considerable differences with regard to the
distributions of protein families within these two datasets.
The most highly represented Pfam families in GOS-100
compared to public-100 are shown in Table 7. Notably, we
found that while many known viral families are absent in
GOS-100, viral protein families dominate the list of the
families more highly represented in GOS-100; this is
presumably because of biases in the collection of previously
known viral sequences. Surprisingly few bacterial families
were among the most represented in GOS-100 compared with
public-100. By contrast, we also observed that those families
found more rarely in GOS-100 than public-100 were
frequently bacterial (Table 7). This appears to be a result of
the large number of key bacterial and viral pathogen proteins
in public-100 that are comparatively less abundant in the
oceanic samples and/or less intensively sampled.
GOS-100 Data Suggest That a Number of ‘‘Kingdom-
Specific’’ Pfams Actually Are Represented in Multiple
Kingdoms
Of the 7,868 Pfam models in Pfam 17.0, 4,050 match
proteins from only a single kingdom in public-100. The
additional sequences from GOS-100 reveal that some of these
families actually have representatives in multiple kingdoms.
Table 8 shows 12 families that have a Pfam match to at least
one GOS-100 protein with an E-value  1 3 1010, and which
we confidently assigned to a kingdom different from that of
all the public-100 matches. Because our criteria for a
‘‘confident’’ kingdom assignment are conservative, there are
only one or a few confident assignments for each Pfam
domain to a ‘‘new’’ kingdom. Our ‘‘confident’’ criteria are
especially difficult to meet in the case of kingdom-crossing,
due to the votes contributed by the crossing protein (see
Materials and Methods). Thus, many scaffolds have no
PLoS Biology | www.plosbiology.org | S66
Expanding the Protein Family Universe
confident kingdom assignment. Our examination of each of
the scaffolds responsible for a determination of kingdom-
crossing confirms that each one had both a highly significant
match to the Pfam model in question and an overwhelming
number of votes for the unexpected kingdom. These scaffold
assemblies were also manually inspected. No clear anomalies
were observed. In most instances, the assemblies in question
were composed of a single unitig, and as such are high-
confidence assemblies. Mate pair coverage and consistent
depth of coverage provide further support for the correct-
ness of those assemblies that are built from multiple unitigs.
Examples of kingdom-crossing families include indoleamine
2,3-dioxygenase (IDO), MAM domain, and MYND finger [15],
which have previously only been seen in eukaryotes, but we
find them also to be present in bacteria. These Pfams now
cross kingdoms, due either to their being more ancient than
previously realized or to lateral transfer.
We explored the IDO family further. This family has
representatives in vertebrates, invertebrates, and multiple
fungal lineages [15,61] in public-100. Members of the IDO
family are heme-binding, and mammalian IDOs catalyze the
rate-limiting step in the catabolic breakdown of tryptophan
[62], while family members in mollusks have a myoglobin
function [63]. In mammals, IDO also appears to have a role in
the immune system [62,64–66]. The IDO Pfam has matches to
66 proteins in public-100, all of which are eukaryotic.
However, it also has matches to ten GOS-100 sequences that
we confidently labeled as bacterial proteins and matches to
206 GOS-100 sequences for which a confident kingdom
assignment could not be made (many of these are likely
bacterial sequences due to the GOS sampling bias). To
reconstruct a phylogeny of the IDO family, we searched a
recent version of NCBI-nr (March 5, 2006) for IDO proteins
that were not included in the public-100 dataset. The search
identified two bacterial proteins from the whole genomes of
the marine bacteria Erythrobacter litoralis and Nitrosococcus
oceani, and 24 eukaryotic proteins (see Materials and Methods).
The phylogeny shown in Figure 6 shows 54% bootstrap
support for a separation of the clade containing exclusively
public-100 and NCBI-nr 2006 eukaryotic sequences from a
clade with the GOS-100 sequences as well as the two NCBI-nr
E. litoralis and N. oceani sequences. We confirmed this feature of
the tree topology with multiple other phylogeny reconstruc-
tion methods. Curiously, there is considerable intermixing of
bacterial and eukaryotic sequences in the clade of GOS-100
sequences and the two NCBI-nr bacteria. A manual inspection
of the scaffolds that contain the ten GOS-100 sequences
(containing the IDO domain) that we confidently labeled as
bacterial, overwhelmingly supports the kingdom assignment.
However, a manual inspection of the scaffolds that contain the
ten GOS-100 sequences (containing the IDO domain) that we
confidently labeled as eukaryotes presents a less convincing
picture. These scaffolds are short, with most of them
containing only two voting ORFs. Since the NCBI-nr version
used in the public-100 set has IDO from eukaryotes only, the
ORF with the IDO domain itself would cast four votes for
eukaryotes. Thus, these GOS-100 eukaryotic labelings are not
nearly as confident as the ones labeled bacterial.
Structural Genomics Implications
Knowledge about global protein distributions can be used
to inform priorities in related fields such as structural
0442 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Table 7. Top Pfam Families Represented More Highly or Less Highly in GOS-100 than in Public-100

Category Accession Description Public-100 Hits GOS-100 Hits

Number
Archaea Bacteria Eukaryota Viruses Unknown Total Expected Based Observed Observed/ Chi Square
on Public-100 Expected

Families represented PF07068 Major capsid protein Gp23 0 0 0 41 0 41 8 1,818 23,450% ,1 3 10303
more highly
PF03420 Prohead core protein protease 0 0 0 11 0 11 6 1,223 22,176% ,1 3 10303
PF06841 T4-like virus tail tube protein gp19 0 0 0 13 0 13 6 795 14,036% ,1 3 10303
PF04451 Iridovirus major capsid protein 0 0 1 138 0 139 15 1,692 11,269% ,1 3 10303
Bacteriophage T4-like capsid 0 0 211 0 211 20 1,633 7,992% ,1 3 10303
PF07230 assembly protein (Gp20) 0
PF01818 Bacteriophage translational regulator 0 0 0 10 0 10 5 405 7,444% ,1 3 10303
PF01231 Indoleamine 2,3-dioxygenase 0 0 66 0 0 66 7 226 3,471% ,1 3 10303
PF03322 Gamma-butyrobetaine hydroxylase 0 13 117 0 0 130 60 1,807 3,004% ,1 3 10303
PF04777 Erv1/Alr family 0 0 177 10 0 187 10 309 2,996% ,1 3 10303
PF05367 Phage endonuclease I 0 2 0 10 0 12 13 290 2,152% ,1 3 10303
PF04832 SOUL heme-binding protein 3 8 173 0 1 185 43 714 1,648% ,1 3 10303
PF03159 XRN 59-39 exonuclease N-terminus 0 0 214 2 0 216 11 170 1,584% ,1 3 10303

PLoS Biology | www.plosbiology.org | S67

PF06213 Cobalamin biosynthesis protein CobT 0 33 0 0 0 33 137 2,155 1,569% ,1 3 10303
PF01786 Alternative oxidase 0 5 239 0 0 244 31 479 1,527% ,1 3 10303
PF00274 Fructose-bisphosphate aldolase class-I 0 28 932 0 0 960 143 2,076 1,453% ,1 3 10303
PF03291 mRNA capping enzyme 0 0 149 33 0 182 11 157 1,395% ,1 3 10303
PF04724 Glycosyltransferase family 17 0 1 118 0 0 119 12 155 1,296% ,1 3 10303
PF00940 DNA-dependent RNA polymerase 0 5 208 23 0 236 32 394 1,222% ,1 3 10303
PF03030 Inorganic Hþ pyrophosphatase 11 83 382 0 0 476 355 4,213 1,187% ,1 3 10303
PF02747 Proliferating cell nuclear antigen, 19 0 175 9 0 203 21 243 1,153% ,1 3 10303
C-terminal domain
Families represented PF01617 Surface antigen 0 991 0 0 0 991 3,987 0 0% ,1 3 10303

0443
less highly
PF00516 Envelope glycoprotein GP120 0 0 1 41,115 11 41,127 3,071 0 0% ,1 3 10303
PF00077 Retroviral aspartyl protease 0 0 153 26,747 9 26,909 2,004 0 0% ,1 3 10303
PF04650 YSIRK type signal peptide 0 469 0 0 3 472 1,889 0 0% ,1 3 10303
PF03507 CagA exotoxin 0 333 0 0 0 333 1,343 0 0% 4 3 10294
PF03482 sic protein 0 285 0 0 0 285 1,150 0 0% 4 3 10252
PF01308 Chlamydia major outer membrane protein 0 264 0 0 0 264 1,066 0 0% 8 3 10234
PF02707 Major outer sheath protein N-terminal region 0 264 0 0 0 264 1,066 0 0% 8 3 10234
PF00934 PE family 0 249 0 0 0 249 1,005 0 0% 1 3 10220
PF00820 Borrelia lipoprotein 0 223 0 0 0 223 901 0 0% 6 3 10198
PF02722 Major outer sheath protein C-terminal region 0 223 0 0 0 223 901 0 0% 6 3 10198
PF00921 Borrelia lipoprotein 0 202 0 0 0 202 816 0 0% 1 3 10179
Staphylococcal/streptococcal toxin, 0 197 3 1 2 203 797 0 0% 3 3 10175
PF02876 beta-grasp domain
PF01856 Outer membrane protein 0 176 0 0 0 176 712 0 0% 7 3 10157
Staphylococcal/streptococcal toxin, 0 166 3 1 2 172 672 0 0% 4 3 10148
PF01123 OB-fold domain
PF02474 Nodulation protein A (NodA) 0 157 0 0 0 157 636 0 0% 3 3 10140
PF06458 MucBP domain 0 155 2 0 0 157 628 0 0% 2 3 10138
Bacillus/clostridium GerA spore germination 0 149 0 0 0 149 603 0 0% 3 3 10133
PF03323 protein
PF07548 Chlamydia polymorphic membrane protein 0 146 0 0 0 146 591 0 0% 1 3 10130
middle domain
PF02255 PTS system, lactose/cellobiose-specific IIA subunit 0 141 0 0 0 141 571 0 0% 3 3 10126

Green indicates exclusively bacterial in public-100; blue, exclusively eukaryotic in public-100; red, exclusively viral in public-100. Expected number of matches in GOS-100 to each Pfam model was calculated as described in Materials and Methods. This
calculation is based on the number of matches to each Pfam in public-100 and corrected for the different kingdom proportions in GOS-100 and public-100. For each Pfam model, the percentage representation ratio is the number of observed GOS-100 matches

Special Section from March 2007 | Volume 5 | Issue 3 | e16

Expanding the Protein Family Universe

to that Pfam divided by the number expected, and expressed as a percentage. The top half of the table shows the top 20 most highly represented proteins that have representation ratios . 1,000% and have chi-squared p-value , 1 3 10303. Numbers of
observed matches to these Pfams in public-100 are also indicated according to kingdom. A number of Pfams highly represented in GOS-100 appear to occur exclusively or almost exclusively in a particular kingdom in public-100. For example, Pfams that are
characteristically viral in public-100 (colored in red) dominate the top of this list, and an intriguing protein family (IDO) with a known immune function in higher eukaryotes (blue) also appears. The bottom half of the table shows the 20 Pfam domains not
observed in GOS-100 with the highest expectation based on public-100 (or equivalently, with the most significant chi-squared p-values). Thus, a large number of key bacterial and viral pathogen proteins in public-100 are not observed in the oceanic samples.
doi:10.1371/journal.pbio.0050016.t007
 Expanding the Protein Family Universe

Some Pfam domains observed exclusively in one kingdom in public-100 are found in a different kingdom in GOS-100. The number of sequences in the public dataset that match each Pfam model is listed above the number of sequences in GOS with a
confident kingdom assignment and a highly significant match to the model. The TC bit score is provided for each model, together with the bit score and E-value of the best match to the model in an unexpected kingdom. For this analysis, Pfam
Best E-Value for Match

matches are filtered with an E-value cutoff of 1 3 1010. In every case, the bit score is at least five bits greater than the TC for the model, because of the larger size of the GOS dataset relative to those used for creating the TC thresholds. In addition to
in Novel Kingdom

10100
1071

1011
1016
3.50 3 1013

9.70 3 1060
1017
1014
1022
1012
1.40 3 1014
2.20 3 1072
3
3
3
3

3
3
3
3
2.00
7.00
6.10
4.30

5.40
3.50
2.50
3.40
Best Score for Match
in Novel Kingdom

passing the ‘‘confident’’ criteria (see Materials and Methods), the kingdom assignments are all confirmed by visual inspection of the BLAST kingdom vote distributions for the respective scaffolds.
247.79
342.32

185.71

250.97
35.88
57.91
41.89

60.46
50.82
74.63
40.63
51.44
Pfam TC

107.1

Figure 6. Maximum Likelihood Phylogeny for the IDO Family

17.8
15.1
14.3
36.2

42.1
47.8
38.4
40.7
34.2
45
78

The phylogeny is based on an alignment of 93 sequences from GOS-100

and 51 sequences from public-100 and NCBI-nr from March 2006 that
Unknown

matched the IDO Pfam model and satisfied multiple alignment quality
criteria. The IDO family is eukaryotic specific in public-100. The
0/206
0/165
0/204

0/239

phylogeny shows a clade with all the GOS sequences, predicted to be

0/17

0/13

0/20

0/80
0/92

0/15
0/9

0/2

bacterial (navy blue), eukaryotic (yellow), or unknown (gray), along with

two sequences from the marine bacteria Erythrobacter litoralis and
Matches in Public-100/Matches in GOS-100

Nitrosococcus oceani (lime green) submitted to the sequence database

Viruses

after February 2005, and a public-only clade of only eukaryotic

0/0
0/0
0/0
0/0

0/0

0/0
0/3
0/0
0/0
0/0

0/0
0/0

sequences (orange).
doi:10.1371/journal.pbio.0050016.g006
Eukaryota

66/10
712/1
798/1
197/0

100/1

173/6

genomics. Structural genomics is an international effort to

0/0
0/0
0/0
0/0

0/0
0/0

determine the 3-D shapes of all important biological macro-

molecules, with a primary focus on proteins [67–72]. Previous
Bacteria

108/250

studies have shown that an efficient strategy for covering the

91/42
0/10

protein structure universe is to choose protein targets for

0/6
0/1
0/1

0/1

0/0

0/8
0/3

0/2
0/1

experimental structure characterization from among the

Archaea

largest families with unknown structure [73,74]. If the

structure of one family member is determined, it may be
88/3
27/0

21/0
0/0
0/0
0/0
0/0

0/0

0/1
0/0
0/1

8/0

used to accurately infer the fold of other family members,

even if the sequence similarity between family members is too
Protein of unknown function (DUF1289)

Protein of unknown function (DUF1152)

low to enable accurate structural modeling [75]. Therefore,

large families are a focus of the production phase of the
Eukaryotic DNA topoisomerase I,

Copper resistance protein CopC

Membrane protein of unknown

Protein Structure Initiative (PSI), the National Institutes of

Palmitoyl protein thioesterase
Indoleamine 2,3-dioxygenase

Health–funded structural genomics project that commenced

HTH DNA-binding domain
Coenzyme Q (ubiquinone)
biosynthesis protein Coq4

in October 2005 [76].

DNA binding fragment
Model Description

Ribosomal LX protein

In March 2005, 2,729 (36%) of 7,677 Pfam families had at

function (DUF63)

least one member of known structure; these families could be

MAM domain
MYND finger

used to infer folds for approximately 51% of all pre-GOS

prokaryotic proteins (covering 44% of residues) [74]. The
Pfam5000 strategy is to solve one structure from each of the
Table 8. New Multi-Kingdom Pfams

largest remaining families, until a total of 5,000 families have

at least one member with known structure [73]. As this strategy
Accession
Number

PF01231
PF00629
PF01753
PF02089
PF05019

PF02919
PF06945
PF04234
PF04967
PF01911
PF01889
PF06626

is similar to that being used at PSI centers to choose targets,

doi:10.1371/journal.pbio.0050016.t008
Pfam

projections based on the Pfam5000 should reflect PSI results.

Completion of the Pfam5000, a tractable goal within the
production phase of PSI, would enable accurate fold assign-
Kingdom Specificity

ment for approximately 65% of all pre-GOS prokaryotic

proteins. In the GOS-100 dataset, we observed that 46% of the
in Public-100

proteins might currently be assigned a fold based on Pfam

Database

families of known structure (see Materials and Methods).

A only
B only
E only

Completion of the Pfam5000 would increase this coverage to

55%.

PLoS Biology | www.plosbiology.org | S68 0444 Special Section from March 2007 | Volume 5 | Issue 3 | e16

The GOS sequences will affect Pfam in two ways: some will
be classified in existing protein families, thus increasing the
size of these families; others may eventually be classified into
new GOS-specific families. Both of these will alter the relative
sizes of different families, and thus their prioritization for
structural genomics studies. We calculated the sizes for all
Pfam families based on the number of occurrences of each
family in the public-100 dataset. Proteins in GOS-100 were
then added and the family sizes were recalculated. A total of
190 families that are not in the Pfam5000 based on public-100
are moved into the Pfam5000 after addition of the GOS data.
The 30 largest such families are shown in Table 9. As 20 of the
30 families are annotated as domains of unknown function in
Pfam, structural characterization might be helpful in identi-
fying their cellular or molecular functions. Reshuffling the
Pfam5000 to prioritize these 190 families would improve
structural coverage of GOS sequences after completion of the
Pfam5000 by almost 1% relative to the original Pfam5000
(from 55.4% to 56.1%), with only a small decrease in coverage
of public-100 sequences (from 67.7% to 67.5%).
The Pfam5000 would be further reprioritized by the
classification of clusters of GOS sequences into Pfam.
Assuming each cluster of pooled GOS-100 and public-100
sequences without a current Pfam match would be classified
as a single Pfam family, 885 such families would replace
existing families in the Pfam5000. These 885 clusters contain
a total of 383,019 proteins in GOS-100 and public-100. The
reprioritized Pfam5000 would also retain 1,183 families of
unknown structure from the current Pfam5000; these families
comprise a total of 1,040,330 proteins in GOS-100 and public-
100.
Known Protein Families and Increased Diversity Due to
GOS Data
Several protein families serve as examples to further
highlight the diversity added by the GOS dataset. In this
paper, we examined UV irradiation DNA damage repair
enzymes, phosphatases, proteases, and the metabolic enzymes
glutamine synthetase and RuBisCO (Table 10). The RecA
family (unpublished data) and the kinase family [77] have also
been explored in the context of the GOS data. There are
more than 5,000 RecA and RecA-like sequences in the GOS
dataset (Table 10). An analysis of the RecA phylogeny
including the GOS data reveals several completely new RecA
subfamilies. A detailed study of kinases in the GOS dataset
demonstrated the power of additional sequence diversity in
defining and exploring protein families [77]. The discovery of
16,248 GOS protein kinase–like enzymes enabled the defi-
nition and analysis of 20 distinct kinase-like families. The
diverse sequences allowed the definition of key residues for
each family, revealing novel core motifs within the entire
superfamily, and predicted structural adaptations in individ-
ual families. This data enabled the fusion of choline and
aminoglycoside kinases into a single family, whose sequence
diversity is now seen to be at least as great as the eukaryotic
protein kinases themselves.
Proteins Involved in the Repair of UV-Induced DNA
Damage
Much of the attention in studies of the microbes in the
world’s oceans has justifiably focused on phototrophy, such
as that carried out by the proteorhodopsin proteins.
PLoS Biology | www.plosbiology.org | S69
Expanding the Protein Family Universe
Previously, in the Sargasso Sea study [10] it was shown that
shotgun sequencing reveals a much greater diversity of
proteorhodopsin-like proteins than was previously known
from cloning and PCR studies. However, along with the
potential benefits of phototrophy come many risks, such as
the damage caused to cells by exposure to solar irradiation,
especially the UV wavelengths. Organisms deal with the
potential damage from UV irradiation in several ways,
including protection (e.g., UV absorption), tolerance, and
repair [78]. Our examination of the protein family clusters
reveals that the GOS data provides an order of magnitude
increase in the diversity (in both numbers and types) of
homologs of proteins known to be involved in pathways
specifically for repairing UV damage.
One aspect of the diversity of UV repair genes is seen in the
overrepresentation of photolyase homologs in the GOS data
(see Table 10). Photolyases are enzymes that chemically
reverse the UV-generated inappropriate covalent bonds in
cyclobutane pyrimidine dimers and 6–4 photoproducts [79].
The massive numbers of homologs of these proteins in the
GOS data (11,569 GOS proteins in four clusters; see Table 10)
is likely a reflection of their presence in diverse species and
the existence of novel functions in this family. New repair
functions could include repair of other forms of UV dimers
(e.g., involving altered bases), use of novel wavelengths of light
to provide the energy for repair, repair of RNA, or repair in
different sequence contexts. In addition, some of these
proteins may be involved in regulating circadian rhythms,
as seen for photolyase homologs in various species. Our
findings are consistent with the recent results of a compara-
tive metagenomic survey of microbes from different depths
that found an overabundance of photolyase-like proteins at
the surface [51].
A good deal was known about the functions and diversity of
photolyases prior to this project. However, much less is
known about other UV damage–specific repair enzymes, and
examination of the GOS data reveals a remarkable diversity
of each of these. For example, prior to this project, there were
only some 25 homologs of UV dimer endonucleases (UVDEs)
available [80], and most of these were from the Bacillus
species. There are 420 homologs of UVDE (cluster 6239) in
the GOS data representing many new subfamilies (Figure 7A
and Materials and Methods). A similar pattern is seen for
spore lyases (which repair a UV lesion specific to spores [81])
and the pyrimidine dimer endonuclease (DenV, which was
originally identified in T4 phage [82]). We believe this will also
be true for UV dimer glycosylases [83], but predictions of
function for homologs of these genes are difficult since they
are in a large superfamily of glycosylases.
Our analysis of the kingdom classification assignments
suggests that the diversity of UV-specific repair pathways is
seen for all types of organisms in the GOS samples. This
apparently extends even to the viral world (e.g., 51 of the
UVDE homologs are assigned putatively to viruses), suggest-
ing that UV damage repair may be a critical function that
phages provide for themselves and their hosts in ocean
surface environments. Based on the sheer numbers of genes,
their sequence diversity, and the diversity of types of
organisms in which they are apparently found, we conclude
that many novel UV damage–repair processes remain to be
discovered in organisms from the ocean surface water.
0445 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Table 9. The 30 Largest Structural Genomics Target Families Added to the Pfam5000 Based on Inclusion of GOS Sequences

Accession Number Description Family Size after GOS Family Size before GOS

PF06213.2 Cobalamin biosynthesis protein CobT 2,188 33

PF04244.3 Deoxyribodipyrimidine photolyase-related protein 1,628 51
PF07021.1 Methionine biosynthesis protein MetW 1,305 50
PF03420.3 Prohead core protein protease 1,234 11
PF06347.2 Protein of unknown function (DUF1058) 1,114 40
PF06439.1 Domain of unknown function (DUF1080) 1,021 48
PF06253.1 Trimethylamine methyltransferase (MTTB) 942 38
PF06242.1 Protein of unknown function (DUF1013) 915 36
PF06841.2 T4-like virus tail tube protein gp19 808 13
PF05992.2 SbmA/BacA-like family 746 26
PF04018.3 Domain of unknown function (DUF368) 720 54
PF06230.1 Protein of unknown function (DUF1009) 703 38
PF07583.1 Protein of unknown function (DUF1549) 703 58
PF07864.1 Protein of unknown function (DUF1651) 539 20
PF06539.1 Protein of unknown function (DUF1112) 529 38
PF06684.1 Protein of unknown function (DUF1185) 519 32
PF07586.1 Protein of unknown function (DUF1552) 491 21
PF06844.1 Protein of unknown function (DUF1244) 470 32
PF06938.1 Protein of unknown function (DUF1285) 451 27
PF07075.1 Protein of unknown function (DUF1343) 441 49
PF07587.1 Protein of unknown function (DUF1553) 439 58
PF06041.1 Bacterial protein of unknown function (DUF924) 416 59
PF03209.5 PUCC protein 415 48
PF01996.6 Protein of unknown function (DUF129) 414 53
PF06146.1 Phosphate-starvation-inducible E 393 44
PF07627.1 Protein of unknown function (DUF1588) 372 31
PF05610.1 Protein of unknown function (DUF779) 356 30
PF06245.1 Protein of unknown function (DUF1015) 348 47
PF06175.1 tRNA-(MS[2]IO[6]A)-hydroxylase (MiaE) 342 46
PF01969.7 Protein of unknown function (DUF111) 337 60

The 30 largest families after inclusion of GOS data that were not among the 5000 largest families before inclusion of GOS data are shown here. Family size was calculated as the number of
matches in public-100 (before GOS) and in the combined GOS-100 and public-100 datasets (after GOS).
doi:10.1371/journal.pbio.0050016.t009

Evidence of Reversible Phosphorylation in the Oceans
Reversible phosphorylation of proteins represents a major
mechanism for cellular processes, including signal trans-
duction, development, and cell division [84]. The activity of
protein kinases and phosphatases serve as antagonistic
regulators of the cellular response. Protein phosphatases
are divided into three major groups based on substrate
specificity [85]. The Mg2þ- or Mn2þ-dependent phosphoserine/
phosphothreonine protein phosphatase family, exemplified
by the human protein phosphatase 2C (PP2C), represents the

Table 10. Clustering of Sequences in Families That Are Explored in This and Companion Papers

Protein Family Cluster ID Nonredundant Sequences Total Sequences NCBI-nr PG TGI-EST ENS GOS

RecA 1146 2,897 7,423 1,683 235 288 104 5,113

UVDE 6239 417 484 38 25 1 0 420
Photolyase 411 1,387 2,261 19 9 0 0 2,233
1285 5,907 9,796 302 145 182 15 9,152
3077 319 482 149 2 176 42 113
3454 67 73 1 1 0 0 71
Spore lyase 5283 237 331 39 25 0 0 267
PP2C phosphatase 78 2,917 3,933 762 112 2,295 199 565
3673 62 106 39 0 22 45 0
9118 68 73 0 0 72 1 0
11012181 36 69 34 0 15 20 0
11021747 19 72 13 11 0 0 48
11066319 19 38 14 0 15 9 0
Glutamine synthetase (type I, II, III) 3709 4,284 11,322 1,504 320 489 48 8,961
3072 159 192 46 11 6 0 129
4547 30 32 1 0 1 0 30
RuBisCO (large subunit) 3734 1,979 14,149 13,532 41 148 0 428

doi:10.1371/journal.pbio.0050016.t010

PLoS Biology | www.plosbiology.org | S70 0446 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Figure 7. Phylogenies Illustrating the Diversity Added by GOS Data to Known Families That We Examined
Kingdom assignments of the sequences are indicated by color: yellow, GOS-eukaryotic; navy blue, GOS-bacterial/archaeal; aqua, GOS-viral; orange,
NCBI-nr–eukaryotic; lime green, NCBI-nr–bacterial/archaeal; pink, NCBI-nr–viral; gray, unclassified.
(A) Phylogeny of UVDE homologs.
(B) Phylogeny of PP2C-like sequences.
(C) Phylogeny of type II GS gene family. In addition to the large amount of diversity of bacterial type II GS in the GOS data, a large group of GOS viral
sequences and eukaryotic GS co-occur at the top of the tree with the eukaryotic virus Acanthamoeba polyphaga mimivirus (shown in pink). The red stars
indicate the locations of eight type II GS sequences found in the type I–type II GS gene pairs. They are located in different branches of the phylogenetic
tree. The rest of the type II GS sequences were filtered out by the 98% identity cutoff.
(D) Phylogeny of the homologs of RuBisCO large subunit. A large portion of the RuBisCO sequences from the GOS data forms new branches that are
distinct from the previously known RuBisCO sequences in the NCBI-nr database.
doi:10.1371/journal.pbio.0050016.g007

smallest group in number. An understanding of their
physiological roles has only recently begun to emerge. In
eukaryotes, one of the major roles of PP2C activity is to
reverse stress-induced kinase cascades [86–89].
We identified 613 PP2C-like sequences in the GOS dataset,
and they are grouped into two clusters (Table 10). These
sequences contain at least seven motifs known to be
important for phosphatase structure and function [90,91].
Invariant residues involved in metal binding (aspartate in
motifs I, II, VIII) and phosphate ion binding (arginine in
motif I) are highly conserved among the GOS sequences.
Using the catalytic domain portion of these sequences we

PLoS Biology | www.plosbiology.org | S71 0447 Special Section from March 2007 | Volume 5 | Issue 3 | e16

constructed a phylogeny showing that despite the overall
conserved structure of the PP2C family of proteins, the
known bacterial PP2C-like sequences group together with the
GOS bacterial PP2C-like sequences (Figure 7B, Materials and
Methods). Furthermore, the eukaryotic PP2Cs display a much
greater degree of sequence divergence compared to the
bacterial PP2C sequences.
We also examined the combined dataset of PP2C-like
phosphatases further for potential differences in amino acid
composition between the bacterial and eukaryotic groups.
We observed a striking distinction between the eukaryotic
and bacterial PP2C-like phosphatases in motif II, where a
histidine residue (His62 in human PP2Ca) is conserved in
more than 90% of sequences, but not observed in the
bacterial group. The bacterial PP2C group contains a
methionine (at the corresponding position) in the majority
of the cases (70%). This histidine residue is involved in the
formation of a beta hairpin in the crystal structure of human
PP2C [91]. Furthermore, His62 is proposed to act as a general
acid for PP2C catalysis [92]. Both amino acids lie in the
proximity of the phosphate-binding domain, but at this time
it is unclear how the difference at this position would
contribute to the overall structure and function of the two
PP2C groups. Nonetheless, the large number of diverse PP2C-
like phosphatases in this dataset allowed us to identify a
previously unrecognized key difference between bacterial
and eukaryotic PP2Cs.
Bacterial genes that perform closely related functions can
be organized in close proximity to each other and often in
functional units. Linked Ser/Thr kinase-phosphatase genetic
units have been described in several bacterial species,
including Streptococcus pneumoniae, Bacillus subtilis, and Myco-
bacterium tuberculosis [93–96]. Two major neighboring clusters
are found to be associated with the set of PP2C-like
phosphatases in the GOS bacterial group. We observed that
one of these clusters contained a protein serine/threonine
kinase domain as its most common Pfam domain. An
additional neighboring cluster found to be associated with
the GOS set of bacterial PP2Cs was identified as a set of
sequences containing a PASTA (penicillin-binding protein
and serine/threonine kinase–associated) domain. This domain
is unique to bacterial species, and is believed to play
important roles in regulating cell wall biosynthesis [97].
Our identification of a conserved group of unique PP2C-
like phosphatases in the GOS dataset significantly increases
the number and diversity of this enzyme family. This analysis
of the NCBI-nr, PG ORFs, TGI-EST ORFs, and ENS datasets
along with the sequences obtained from the GOS dataset
significantly increases the overall number of PP2C-like
sequences from that estimated just a year ago [98]. The
presence of genes encoding bacterial serine/threonine kinase
domains located adjacent to PP2Cs in the GOS data supports
the notion that the process of reversible phosphorylation on
Ser/Thr residues controls important physiological processes
in bacteria.
Proteases in GOS Data
Proteases are a group of enzymes that degrades other
proteins and, as such, plays important roles in all organisms
[99]. On the basis of their catalysis mechanism, proteases are
divided into six distinct catalytic types: aspartic, cysteine,
metallo, serine, threonine, and glutamic proteases [99]. They
PLoS Biology | www.plosbiology.org | S72
Expanding the Protein Family Universe
differ from each other by the presence of specific amino acids
in the active site and by their mode of action. The MEROPS
database [100] is a comprehensive source of information for
this large divergent group of sequences and provides a widely
accepted classification of proteases into families, based on the
amino acid sequence comparison, and then into clans based
on the similarity of their 3-D structures.
We identified 222,738 potential proteases in the GOS
dataset based on similarity to sequences in MEROPS (see
Materials and Methods). According to our clustering method,
95% of these sequences are grouped into 190 clusters, with
each cluster on the average containing more than 1,100 GOS
sequences. These sequences were compared to proteases in
NCBI-nr. There are groups of proteases in NCBI-nr that are
highly redundant. For example, there are a large number of
viral proteases from HIV-1 and hepatitis C viruses that
dominate the NCBI-nr protease set. Thus, we computed a
nonredundant set of NCBI-nr proteases and, for the sake of
consistency, a nonredundant set of proteases from the GOS
set using the same parameters. The majority of proteases in
both sets are dominated by cysteine, metallo, and serine
proteases. The GOS dataset is dominated by proteases
belonging to the bacterial kingdom. That is not surprising,
given the filter sizes used to collect the samples. In NCBI-nr
the proteases are more evenly distributed between the
bacterial and the eukaryotic kingdoms.
Our comparison of the protease clan distribution of the
bacterial sequences in the NCBI-nr and GOS sets reveals that
the distribution of clans is very similar for metallo- and serine
proteases. However, the distribution of clans in aspartic and
cysteine proteases is different in the two datasets. Among
aspartic proteases, the most visible difference is the increased
ratio of proteases of the AC clan and the decreased ratio in
the AD clan. Proteases in the former clan are involved in
bacterial cell wall production, while those in the latter clan
are involved in pilin maturation and toxin secretion [99].
Among cysteine proteases, the most apparent is the decrease
in the CA clan and an increase in the number of proteases
from the PB(C) clan. Bacterial members of the CA clan are
mostly involved in degradation of bacterial cell wall compo-
nents and in various aspects of biofilm formation [99]. It is
possible that both activities are less important for marine
bacteria present in surface water. Proteases from the PB(C)
clan are involved in activation (including self-activation) of
enzymes from acetyltransferase family. In fungi this family is
involved in penicillin synthesis, while their function in
bacteria is unknown [99].
We were unable to detect any caspases (members of the CD
clan) in the GOS data. This is consistent with the apoptotic
cell death mechanism being present only in multicellular
eukaryotes, which, based on the filter sizes, are expected to be
very rare in the GOS dataset.
Metabolic Enzymes in the GOS Data
To gain insights into the diversity of metabolism of the
organisms in the sea, we studied the abundance and diversity
of glutamine synthetase (GS) and ribulose 1,5-bisphosphate
carboxylase/oxygenase (RuBisCO), two key enzymes in nitro-
gen and carbon metabolism.
GS is the central player of nitrogen metabolism in all
organisms on earth. It is one of the oldest enzymes in
evolution [101]. It converts ammonia and glutamate into
0448 Special Section from March 2007 | Volume 5 | Issue 3 | e16

glutamine that can be utilized by cells. GS can be classified
into three types based on sequence [101]. Type I has been
found only in bacteria, and it forms a dodecameric structure
[102,103]. Type II has been found mainly in eukaryotes, and in
some bacteria. Type III GS is less well studied, but has been
found in some anaerobic bacteria and cyanobacteria. There
are 18 active site residues in both bacterial and eukaryotic GS
that play important roles in binding substrates and catalyzing
the enzymatic reactions [104].
We found 9,120 GS and GS-like sequences in the GOS data
(Table 10). Using profile HMMs [41,105] constructed from
known GS sequences of different types, we were able to
classify 4,350 sequences as type I GS, 1,021 sequences as type
II GS, and 469 sequences as type III GS (see Materials and
Methods).
The number of type II GS sequences found in the GOS data
is surprisingly high, since previously type II GS were
considered to be mainly eukaryotic and very few eukaryotic
organisms were expected to be included in the GOS
sequencing (Figure 7C and Materials and Methods). We used
gene neighbor analysis to classify the origin of GS genes by
the nature of other proteins found on the same scaffold.
Using this approach, most of the neighboring genes of the
type II GS in the GOS data are identified as bacterial genes.
The neighboring genes of the type II GS include nitrogen
regulatory protein PII, signal transduction histidine kinase,
NH3-dependent NADþ synthetase, A/G-specific adenine gly-
cosylase, coenzyme PQQ synthesis protein c, pyridoxine
biosynthesis enzyme, aerobic-type carbon monoxide dehy-
drogenase, etc. We were able to assign more than 90% of the
type II GS sequences in the GOS data to bacterial scaffolds
based on a BLAST-based kingdom assignment method (see
Materials and Methods). Both neighboring genes and king-
dom assignments suggest that most of the type II GS
sequences in the GOS data come from bacterial organisms.
In comparison, the same type II GS profile HMM detects only
12 putative type II GS sequences from the PG dataset of 222
prokaryotic genomes. Within these, there are only seven
unique type II GS sequences and six unique bacterial species
represented. The reason why bacteria in the ocean have so
many type II GS genes is unclear.
Two hypotheses have been raised to explain the origin of
type II GS in bacterial genomes: lateral gene transfer from
eukaryotic organisms [106] and gene duplication prior to the
divergence of prokaryotes and eukaryotes [101]. The type II
GS sequences in the predominantly bacterial GOS data are
not only abundant, but also diverse and divergent from most
of known eukaryotic GS sequences (Figure 7C). This makes
the hypothesis of lateral gene transfer less favorable. If the GS
gene duplication preceded the prokaryote–eukaryote diver-
gence according to the gene duplication hypothesis, it is
possible that many oceanic organisms retained type II GS
genes during evolution.
Interestingly, we found 19 cases where a type I GS gene is
adjacent to a type II GS gene on the same scaffold. Both GS
genes seem to be functional based on the high degree of
conservation of active site residues. The same gene arrange-
ment was observed previously in Frankia alni CpI1 [107]. The
functional significance of maintaining two types of GS genes
adjacent to one another in the genome remains to be
elucidated. Most of the sequences of these GS genes are
highly similar. We examined the geographic distribution of
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Expanding the Protein Family Universe
these adjacent GS sequences across all the GOS samples. They
are mainly found in the samples taken from two sites. Their
geographic distribution is significantly different from the
distributions of types I and II GS across the samples. The high
sequence similarity among the adjacent GS pairs and their
geographic distribution suggest that these adjacent GS
sequences may come from only a few closely related
organisms. This is consistent with the protein sequence tree
of type II GS, where the type II GS sequences from the GS
gene pairs mainly reside in two distinct branches (Figure 7C).
The active site residues are very well conserved in all GS
sequences in the GOS data, except one residue, Y179, which
coordinates the ammonium-binding pocket. We observed
substitutions of Y179 to phenylalanine in about half of the
type II GS sequences. The activity of type I GS in some
bacteria is regulated by adenylylation at residue Tyr397. In
the GOS data, Tyr397 is relatively conserved in type I GS, with
variations to phenylalanine and tryptophan in about half of
the sequences. This indicates that the activity of some of the
type I GS is not regulated by adenylylation, as shown
previously in some Gram-positive bacteria [108,109].
RuBisCO is the key enzyme in carbon fixation. It is the
most abundant enzyme on earth [110] and plays an important
role in carbon metabolism and CO2 cycle. RuBisCO can be
classified into four forms. Form I has been found in both
plants and bacteria, and has an octameric structure. Form II
has been found in many bacteria, and it forms a dimer in
Rhodospirillum rubrum. Form III is mainly found in archaea,
and forms various oligomers. Form IV, also called the
RuBisCO-like protein (RLP), has been recently discovered
from bacterial genome-sequencing projects [111,112]. RLP
represents a group of proteins that do not have RuBisCO
activity, but resemble RuBisCO in both sequence and
structure [111,113]. The functions of RLPs are largely
unknown and seem to differ from each other.
Contrary to the large number of GS sequences, we
identified only 428 sequences homologous to the RuBisCO
large subunit in the GOS data. The small number of RuBisCO
sequences may partly be due to the fact that larger-sized
bacterial organisms were not included in the sequencing
because of size filtering. However, it could also indicate that
CO2 is not the major carbon source for these sequenced
ocean organisms.
The RuBisCO homologs in the GOS data are more diverse
than the currently known RuBisCOs (Figure 7D, Materials
and Methods). Six of 19 active site residues—N123, K177,
D198, F199, H327, and G404—are not well conserved in all
sequences, suggesting that the proteins with these mutations
may have evolved to have new functions, such as in the case of
RLPs. From the studies of the RLPs from Chlorobium tepidum
and B. subtilis [111,114], it has been shown that the active site
of RuBisCO can accommodate different substrates and is
potentially capable of evolving new catalytic functions
[113,114]. On the other hand, two sequence motifs, helices
aB and a8, that are not involved in substrate binding and
catalytic activity are well conserved in the GOS RuBisCO
sequences. The higher degree of conservation of these
nonactive site residues than that of active site residues
suggests that these motifs are important for their structure,
function, or interaction with other proteins.
We found 47 (31 at 90% identity filtering) GOS sequences
in the branch with known RLP sequences in a phylogenetic
0449 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Figure 8. Distribution of Average HMM Score Difference between GOS
and Public (NCBI-nr, MG, TGI-EST, and ENS)
Only matches to the full length of an HMM are considered, and only
HMMs that have at least 100 matches to each of GOS and public
databases are considered. This results in 1,686 HMMs whose average
scores to GOS and public databases are considered. The mean of the
distribution is 50, showing that GOS sequences tend to score lower
than sequences in public, thereby reflecting diversity compared to
sequences in public.
doi:10.1371/journal.pbio.0050016.g008
tree of RuBisCO (Figure 7D). In this phylogenetic tree, in
addition to the clades for each of the four forms of RuBisCO,
there are also new groups of 65 (58 at 90% identity filtering)
GOS sequences that do not cluster with any known RuBisCO
sequences. This indicates that there could be more than one
type of RuBisCO-like protein existing in organisms. The
novel groups of RuBisCO homologs in the GOS data also
suggest that we have not fully explored the entire RuBisCO
family of proteins (Figure 7D).
GOS Data and Remote Homology Detection
The addition of GOS sequences may help greatly in
defining the range and diversity of many known protein
families, both by addition of many new sequences and by the
increased diversity of GOS sequences. Our comparison of
HMM scores for GOS sequences with those from the other
four datasets shows that GOS sequences consistently tend to
have lower scores, which indicates additional diversity from
that captured in the original HMM (Figure 8). The addition of
GOS data into domain profiles may broaden the profile and
allow it to detect additional remote family members in both
GOS and other datasets. As a trial, we rebuilt the Pfam model
PF01396, which describes a zinc finger domain within
bacterial DNA topoisomerase. The original model finds 821
matches to 481 proteins in NCBI-nr. Our model that includes
GOS sequences reveals 1,497 matches to 722 sequences, an
increase of 50% in sequences and 82% in domains (most
topoisomerases have three such domains, of which one is
divergent and difficult to detect). Of these new matches, 104
are validated by the presence of additional topoisomerase
domains, or they are annotated as topoisomerase, while most
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Expanding the Protein Family Universe
others are unannotated or similar to other DNA-modifying
enzymes not previously thought to have zinc finger domains.
HMM profiles can be further exploited by using matches
beyond the conservative trusted cutoff (TC) used in this study.
For instance, the Pfam for the poxvirus A22 protein family
has no GOS matches above the TC, but 137 matches with E-
values of 1 3 103 to 1 3 1010, containing a short conserved
motif overlap with A22 proteins. Alignment of these matches
shows an additional two short motifs in common with A22,
establishing their homology, and using a profile HMM, we
found a total of 269 family members in GOS and eight family
members in NCBI-nr. Many members of this new family are
surrounded by other novel clusters, or are in putative viral
scaffolds, suggesting that these weak matches are an entry
point into a new clade of viruses.
ORFans with Matches in GOS Data
Further evidence of the diversity added by GOS sequences
is provided by their matches to ORFans. ORFans are
sequences in current protein databases that do not have
any recognizable homologs [117]. ORFan sequences (dis-
counting those that may be spurious gene predictions)
represent genes with organism-specific functions or very
remote homologs of known families. They have the potential
to shed light on how new proteins emerge and how old ones
diversify.
We identified 84,911 ORFans (5,538 archaea, 35,292
bacteria, 37,427 eukaryotic, 5,314 virus, and 1,340 unclassi-
fied) from the NCBI-nr dataset using CD-HIT [116,117] and
BLAST (see Materials and Methods). Of these, 6,044 have
matches to GOS sequences using BLAST (E-value 1 3 106).
Figure 9 shows the distribution of the matched ORFans
grouped by organisms, number of their GOS matches, and
the lowest E-value of the matches. We found matches to GOS
sequences for 13%, 6.3%, 0.89%, and 8.9% of bacterial,
archaeal, eukaryotic, and viral ORFans, respectively. While
most of these ORFans have very few GOS matches, 626 of
them have 20 GOS matches. The similarities between GOS
sequences and eukaryotic ORFans are much weaker than
those between GOS sequences and noneukaryotic ORFans.
The average sequence identity between eukaryotic ORFans
and their closest GOS matches is 38%. This is 6% lower than
the identity between noneukaryotic ORFans and their closest
GOS matches.
The ORFans that match GOS sequences are from approx-
imately 600 organisms. Table 11 lists the 20 most populated
organisms. Out of the 6,044 matched ORFans, approximately
2,000 are from these 20 organisms. For example, Rhodopirellula
baltica SH 1, a marine bacterium, has 7,325 proteins deposited
in NCBI-nr. We identified 1,418 ORFans in this organism, of
which 322 have GOS matches. Another interesting example in
this list is Escherichia coli. Although there are .20 different
strains sequenced, 168 ORFans are identified in strain
CFT073, and 67 of them have GOS matches. The only
eukaryotic organism in this list is Candida albicans SC5314, a
fungal human pathogen, which has 49 ORFans with GOS
matches.
We examined a small but interesting subset of the ORFans
that have 3-D structures deposited in PDB. Out of 65 PDB
ORFans, GOS matches for eight of them are found (see
Supporting Information for their PDB identifiers and names).
0450 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Figure 9. Pie Chart of ORFans That Had GOS Matches

ORFans are grouped by organism (left), number of their GOS matches (middle), and the lowest E-value to their GOS matches in negative logarithm form
(right). For both middle and right charts, inner and outer circles represent noneukaryotic and eukaryotic ORFans, respectively. From the middle chart it
is seen that 626 (¼ 404 þ 180 þ 21 þ 21) ORFans form significant protein families with 20 GOS matches.
doi:10.1371/journal.pbio.0050016.g009

They include four restriction endonucleases, three hypo-
thetical proteins, and a glucosyltransferase.
GOS sequences can play an important role in identifying
the functions of existing ORFans or in confirming protein
predictions. For example, we found that the hypothetical
protein AF1548, which is a PDB ORFan, has matches to 16
GOS sequences. A PSI-BLAST search with AF1548 as the
query against a combined set of GOS and NCBI-nr identified
several significant restriction endonucleases after three
iterations. With the support of 3-D structure and multiple
sequence alignment of AF1548 and its GOS matches, we
predict that AF1548 along with its GOS homologs are
restriction endonucleases (Figure 10). When combined with
an established consensus of active sites of the related
endonucleases families [118], we predicted three catalytic
residues.
Genome Sequencing Projects and Protein Exploration
With respect to protein exploration and novel family
discovery, microbial sequencing offers more promise com-
pared to sequencing more mammalian genomes. This is
illustrated by Figure 11, where the number of clusters that
protein predictions from various finished mammalian ge-
nomes fall into was compared to the number of clusters that
similar-sized random subsets of microbial sequences fall into
(see Materials and Methods). As the figure shows, the rate of
protein family discovery is higher for microbes than for
mammals. Indeed, the rate of new family discovery is
plateauing for mammalian sequences. This is not surprising,

Table 11. Top 20 Organisms with Most ORFans Matched by GOS

Organism Total Proteinsa Total ORFans ORFans Matched

Rhodopirellula baltica SH 1 7,325 1,418 322

Shewanella oneidensis MR-1 4,472 292 206
Cytophaga hutchinsonii 3,686 555 170
Bdellovibrio bacteriovorus HD100 3,587 753 152
Kineococcus radiotolerans SRS30216 4,559 1,070 125
Synechococcus sp. WH 8102 2,517 143 116
Burkholderia cepacia R18194 7,717 198 100
Aeropyrum pernix K1 1,841 1,312 95
Burkholderia cepacia R1808 7,915 292 94
Magnetospirillum magnetotacticum MS-1 10,146 826 92
Microbulbifer degradans 2–40 4,038 386 85
Burkholderia fungorum LB400 7,994 190 84
Desulfitobacterium hafniense DCB-2 4,389 758 75
Escherichia coli CFT073 5,379 168 67
Bradyrhizobium japonicum USDA 110 8,317 580 66
Acanthamoeba polyphaga mimivirus 911 304 57
Caulobacter crescentus CB15 3,737 333 56
Rubrivivax gelatinosus PM1 4,307 287 53
Mesorhizobium loti MAFF303099 7,272 370 53
Candida albicans SC5314 14,107 1,647 49

a
Total number of proteins of this organism deposited at NCBI; may have redundant entries.
doi:10.1371/journal.pbio.0050016.t011

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 Expanding the Protein Family Universe

Figure 10. Structure and GOS Homologs of Hypothetical Protein AF1548

Yellow bars represent b-strands. Highlighted are predicted catalytic residues: 38D, 51E, and 53K.
doi:10.1371/journal.pbio.0050016.g010

as mammalian divergence from a common ancestor is much
more recent than microbial divergence from a common
ancestor, which suggests that mammals will share a larger
core set of less-diverged proteins. Microbial sequencing is
also more cost effective than mammalian sequencing for
acquiring protein sequences because microbial protein
density is typically 80%–90% versus 1%–2% for mammals.
This could be addressed with mammalian mRNA sequencing,
but issues with acquiring rarely expressed mRNAs would need
to be considered. There are, of course, other reasons to
sequence mammalian genomes, such as understanding
mammalian evolution and mammalian gene regulation.
Conclusions
The rate of protein family discovery is approximately
linear in the (current) number of protein sequences. Addi-
tional sequencing, especially of microbial environments, is
expected to reveal many more protein families and sub-
families. The potential for discovering new protein families is
also supported by the GOS diversity seen at the nucleotide
level across the different sampling sites [30]. Averaged over
the sites, 14% of the GOS sequence reads from a site are
unique (at 70% nucleotide identity) to that site [30].
The GOS data provides almost complete coverage of
known prokaryotic protein families. In addition, it adds a
great deal of diversity to many known families and offers new
insights into the evolution of these families. This is illustrated
using several protein families, including UV damage–repair
enzymes, phosphatases, proteases, glutamine synthetase,
RuBisCO, RecA (unpublished data), and kinases [77]. Only a
handful of protein families have been examined thus far, and
many thousands more remain to be explored.
The protein analysis presented indicates that we are far
from exploring the diversity of viruses. This is reflected in
several of the analyses. The GOS-only clusters show an
overrepresentation of sequences of viral origin. In addition,
our domain analysis using HMM profiling shows a lower Pfam
coverage of the GOS sequences in the viral kingdom
compared to the other kingdoms. At least two of the protein
families we explored in detail (UV repair enzymes and
glutamine synthetase) contain abundant new viral additions.
The extraordinary diversity of viruses in a variety of
environmental settings is only now beginning to be under-
stood [57,119–121]. A separate analysis of GOS microbial and
viral sequences (unpublished data) shows that multiple viral
protein clusters contain significant numbers of host-derived

Figure 11. Rate of Cluster Discovery for Mammals Compared to That for Microbes
The x-axis denotes the number of sequences (in thousands), and the y-axis denotes the number of clusters (in thousands). Five mammalian genomes
are considered for the ‘‘Mammalian’’ dataset, and the plot shows the number of clusters that are hit when each additional genome is added. For the
‘‘Mammalian Random’’ dataset, the order of the sequences from the ‘‘Mammalian’’ dataset is randomized. For the NCBI-nr prokaryotic and GOS
datasets, random subsets of size similar to that of the mammalian set are considered.
doi:10.1371/journal.pbio.0050016.g011

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proteins, suggesting that viral acquisition of host genes is
quite widespread in the oceans.
Data generated by this GOS study and similar environ-
mental shotgun sequencing studies present their own analysis
challenges. Methods for various analyses (e.g., sequence
alignment, profile construction, phylogeny inference, etc.)
are generally designed and optimized to work with full
sequences. They have to be tailored to analyze the mostly
fragmentary sequences that are generated by these projects.
Nevertheless, these data are a valuable source of new
discoveries. These data have the potential to refine old
hypotheses and make new observations about proteins and
their evolution. Our preliminary exploration of the GOS data
identified novel protein families and also showed that many
ORFan sequences from current databases have homologs in
these data. The diversity added by GOS data to protein
families also allows for the building of better profile models
and thereby improves remote homology detection. The
discovery of kingdom-crossing protein families that were
previously thought to be kingdom-specific presents evidence
that the GOS project has excavated proteins of more ancient
lineage than that previously known, or that have undergone
lateral gene transfer. This is another example of how
metagenomics studies are changing our understanding of
protein sequences, their evolution, and their distribution
across the various forms of life and environments. Biases in
the currently published databases due to oversampling of
some proteins or organisms are illuminated by environ-
mental surveys that lack such biases. Such knowledge can help
us make better predictions of the real distribution patterns of
proteins in the natural world and indicate where increased
sampling would be likely to uncover new families or family
members of tremendous diversity (such as in the viral
kingdom).
These data have other significant implications for the fields
of protein evolution and protein structure prediction.
Having several hundreds or even tens of thousands of diverse
proteins from a family or examples of a specific protein fold
should provide new approaches for developing protein
structure prediction models. Development of algorithms that
consider the alignments of all these family members/protein
folds and analyze how amino acid sequence can vary without
significantly altering the tertiary structure or function may
provide insights that can be used to develop new ab inito
methods for predicting protein structures. These same
datasets could also be used to begin to understand how a
protein evolves a new function. Finally, this large database of
amino acid sequence data could help to better understand
and predict the molecular interactions between proteins. For
example, they may be used to predict the protein–protein
interactions so critical for the formation of specific func-
tional complexes within cells.
The GOS data also have implications for nearly all
computational methods relying on sequence data. The
increase in the number of known protein sequences presents
challenges to many algorithms due to the increased volume of
sequences. In most cases this increase in sequence data can be
compensated for with additional CPU cycles, but it is also a
foreshadowing of times to come as the pace of large-scale
sequence-collecting accelerates. A related challenge is the
increase in the diversity of protein families, with many new
divergent clades present. With more protein similarity
PLoS Biology | www.plosbiology.org | S77
Expanding the Protein Family Universe
relationships falling into the twilight zone overlapping with
random sequence similarity, the number of false positives for
homology detection methods increases, making the true
relationships more difficult to identify. Nevertheless, a deeper
knowledge of protein sequence and family diversity intro-
duces unprecedented opportunities to mine similarity rela-
tionships for clues on molecular function and molecular
interactions as well as providing much expanded data for all
methods utilizing homologous sequence information data.
The GOS dataset has demonstrated the usefulness of large-
scale environmental shotgun sequencing projects in explor-
ing proteins. These projects offer an unbiased view of
proteins and protein families in an environmental sample.
However, it should be noted that the GOS data reported here
are limited to mostly ocean surface microbes. Even with this
targeted sampling a tremendous amount of diversity is added
to known families, and there is evidence for a large number of
novel families. Additional data from larger filter sizes (that
will sample more eukaryotes) coupled with metagenomic
studies of different environments like soil, air, deep sea, etc.
will help to achieve the ultimate goal of a whole-earth catalog
for proteins.
Materials and Methods
Data description. NCBI-nr [31,32] is the single largest publicly
available protein resource and includes protein sequences submitted
to SWISS-PROT (curated protein database) [122], PDB (a database of
amino acid sequences with solved structures) [123], PIR (Protein
Information Resource) [124], and PRF (Protein Research Founda-
tion). In addition, NCBI-nr also contains protein predictions from
DNA sequences from both finished and unfinished genomes in
GenBank [125], EMBL [126], and DNA Databank of Japan (DDBJ)
[127]. The nonredundancy in NCBI-nr is only to the level of distinct
sequences, and any two sequences of the same length and content are
merged into a single entry. NCBI-nr contains partial protein
sequences and is not a fully curated database. Therefore it also
contains contaminants in the form of sequences that are falsely
predicted to be proteins.
Expressed sequence tag (EST) databases also provide the potential
to add a great deal of information to protein exploration and contain
information that is not well represented in NCBI-nr. To this end,
assemblies of EST sequences from the TIGR Gene Indices [34], an EST
database, were included in this study. To minimize redundancy, only
EST assemblies from those organisms for which the full genome is not
yet known, were included. The protein predictions on metazoan
genomes that are fully sequenced and annotated were obtained by
including the Ensembl database [35,36] in this study.
Both finished and unfinished sequences from prokaryotic genome
projects submitted to NCBI were included. The protein predictions
from the individual sequencing projects are submitted to NCBI-nr.
Nevertheless, these genomes were included in this dataset both for
the purpose of evaluating our approach and also for the purpose of
identifying any proteins that were missed by the annotation process
used in these projects.
Thus, for this study the following publicly available datasets, all
downloaded on February 10, 2005—NCBI-nr, PG, TGI-EST, and
ENS—were used. The organisms in the PG set and the TGI-EST set
are listed in Protocol S1.
Assembly of the GOS dataset. Initial assembly (construction of
‘‘unitigs’’) was performed so that only overlaps of at least 98% DNA
sequence identity and no conflicts with other overlaps were accepted.
False assemblies at this phase of the assembler are extremely rare,
even in the presence of complex datasets [37,128]. Paired-end (also
known as mate-pair) data were then used to order, orient, and merge
unitigs into the final assemblies, but only when two mate pairs or a
single mate pair and an overlap between unitigs implied the same
layout. In one respect, mate pair data was used more aggressively than
is typical in assembly of a single genome in that depth-of-coverage
information was largely ignored [10]. This potentially allows chimeric
assemblies through a repeat within a genome or through an ortholog
between genomes. Thus, a conclusion that relies on the correctness of
a single assembly involving multiple unitigs should be considered
0453 Special Section from March 2007 | Volume 5 | Issue 3 | e16

tentative until the assembly can be confirmed in some way.
Assemblies involved in key results in this paper were subjected to
expert manual review based on thickness of overlaps, presence of
well-placed mate pairs across thin overlaps or across gaps between
contigs, and consistency of depth of coverage.
Data release and availability. All the GOS protein predictions will
be submitted to GenBank. In addition, all the data supporting this
paper, including the clustering and the various analyses, will be made
publicly available via the CAMERA project (Community Cyberinfras-
tructure for Advanced Marine Microbial Ecology Research and
Analysis; http://camera.calit2.net), which is funded by the Gordon
and Betty Moore Foundation.
All-against-all BLASTP search. We used two sets of computer
resources. At the J. Craig Venter Institute, 125 dual 3.06-GHz Xeon
processor systems with 2 Gb of memory per system were used. Each
system had 80 GB local storage and was connected by GBit ethernet
with storage area network (SAN) I/O of ;24 GBit/sec and network
attached storage (NAS) I/O of ;16 GBit/sec. A total of 466,366 CPU
hours was used on this system. In addition, access to the National
Energy Research Scientific Computing Center (NERSC) Seaborg
computer cluster was available, including 380 nodes each with sixteen
375-MHz Power3 processors. The systems had between 16 GB and 64
GB of memory. Only 128 nodes were used at a time. A total of 588,298
CPU hours was used on this system. The dataset of 28.6 million
sequences was searched against itself in a half-matrix using NCBI
BLAST [38] with the following parameters: -F ‘‘m L’’ -U T -p blastp -e
1 3 1010 -z 3 3 109 -b 8000 -v 10. In this paper, similarity of an
alignment is defined to be the fraction of aligned residues with a
positive score according to the BLOSUM62 substitution matrix [129]
used in the BLAST searches.
Identification of nonredundant sequences. Given a set of sequences
S and a threshold T, a nonredundant subset S9 of S was identified by
first partitioning S (using the threshold T) and then picking a
representative from each partition. The set of representatives
constitutes the nonredundant set S9. The process was implemented
using the following graph-theoretic approach. A directed graph G ¼
(V, E) is constructed with vertex set V and edge set E. Each vertex in V
represents a sequence from S. A directed edge (u,v) 2 E if sequence u
is longer than sequence v and their sequence comparison satisfies the
threshold T; for sequences of identical length, the sequence with the
lexicographically larger id is considered the longer of the two. Note
that G does not have any cycles. Source vertices (i.e., vertices with no
in-degree) are sorted in decreasing order of their out-degrees and
(from largest out-degree to smallest) processed in this order. A source
vertex u is processed as follows: mark all vertices that have not been
seen before and are reachable from vertex u as being redundant and
mark vertex u as their representative.
We used two thresholds in this paper, 98% similarity and 100%
identity. The former was used in the first stage of the clustering and
the later was used in the HMM profile analysis. For the 98% similarity
threshold, two sequences satisfy the threshold if the following three
criteria are met: (1) similarity of the match is at least 98%; (2) at least
95% of the shorter sequence is covered by the match; and (3) (match
score)/(self score of shorter sequence)  95%.
For the 100% identity threshold, two sequences satisfy the
threshold if their match identity is 100%.
Description of the clustering algorithm. The starting point for the
clustering was the set of pairwise sequence similarities identified
using the all-against-all BLASTP compute. Because of both the
volume and nature of the data, the clustering was carried out in four
steps: redundancy removal, core set identification, core set merging,
and final recruitment.
A set of nonredundant sequences (at 98% similarity) was identified
using the procedure given in Materials and Methods (Identification of
nonredundant sequences). Only the nonredundant sequences were
considered in further steps of the clustering process.
The aim of the core set identification step was to identify core sets of
highly related sequences. In graph-theoretic terms, this involves
looking for dense subgraphs in a graph where the vertices correspond
to sequences and an edge exists between two sequences if their
sequence match satisfies some reasonable threshold (for instance,
40% similarity match over 80% of at least one sequence and are
clearly homologous based on the BLAST threshold). Dense subgraphs
were identified by using a heuristic. This approach utilizes long edges.
These are edges where the match threshold is computed relative to
the longer sequence. This was done to prevent, as much as possible,
unrelated proteins from being put into the same core set. If all the
sequences were full length, using long edges would have offered a
good solution to keeping unrelated proteins apart. However, the
situation here is complicated by the presence of a large amount of
PLoS Biology | www.plosbiology.org | S78
Expanding the Protein Family Universe
fragmentary sequence data of varying lengths. This was dealt with
somewhat by working with rather stringent match thresholds and a
two-stage process to identify the core sets. We used the concept of
strict long edges and weak long edges. A strict long edge exists between
two vertices (sequences) if their match has the following properties:
(1) 90% of the longer sequence is involved in the match; (2) the match
has 70% similarity; and (3) the score of the match is at least 60% of
the self-score of the longer sequence. A weak long edge exists between
two vertices (sequences) if their match has the following properties:
(1) 80% of the longer sequence is involved in the match; (2) the match
has 40% similarity; and (3) the score of the match is at least 30% of
the self-score of the longer sequence. Core set identification had two
substages: large core initialization and core extension. The large core
initialization step identified sets of sequences where these sets were of
a reasonable size and the sequences in them were very similar to each
other. Furthermore, these sets could be extended in the core
extension step by adding related sequences. In the large core
initialization step, a directed graph G was constructed on the
sequences using strict long edges, with each long edge being directed
from the longer to the shorter sequence. For each vertex v in G, let
S(v) denote the friends set of v consisting of v and all neighbors that v
has an out-going edge to.
Initially all the vertices in G are unmarked. Consider the set of all
friends sets in the decreasing order of their size. For S(v) that is
currently being considered, do the following: (1) initialize seed set A ¼
S(v); (2) while there exists some v9 such that jS(v) \ S(v9)j  k, set A ¼ A
[ S(v9). (Note: k ¼ 10 is chosen); (3) output set A and mark all vertices
in A; and (4) update all friends sets to contain only unmarked vertices.
In the core extension step, we constructed a graph G using weak
long edges. All vertices in seed sets (computed from the large core
initialization step) were marked and the rest of the vertices
unmarked. Each seed set was then greedily extended to be a core
set by adding a currently unmarked vertex that has at least k
neighbors (k ¼ 10 is chosen) in the set; the added vertex was marked.
After this process, a clique-finding heuristic was used to identify
smaller cliques (of size at most k  1) consisting of currently
unmarked vertices; these were also extended to become core sets. A
final step involved merging the computed core sets on the basis of
weak edges connecting them.
In the core set merging step, we constructed an FFAS (Fold and
Function Assignment System) profile [39] for each core set using the
longest sequence in the core set as query. FFAS was then used to carry
out profile–profile comparisons in order to merge the core sets into
larger sets of related sequences. Due to computational constraints
imposed by the number of core sets, profiles were built on only core
sets containing at least 20 sequences.
Final recruitment involved constructing a PSI-BLAST profile [40]
on core sets of size 20 or more (using the longest sequence in the core
set as query) and then using PSI-BLAST (–z 1 3 109, –e 10) to recruit as
yet unclustered sequences or small-sized clusters (size less than 20) to
the larger core sets. For a sequence to be recruited, the sequence–
profile match had to cover at least 60% of the length of the sequence
with an E-value  1 3 107. In a final step, unclustered sequences were
recruited to the clusters using their BLAST search results. A length-
based threshold was used to determine if the sequence is to be
recruited.
Identification of clusters containing shadow ORFs. A well-known
problem in predicting coding intervals for DNA sequences is shadow
ORFs. The key requirement that coding intervals not contain in-
frame stop codons requires that coding intervals be subintervals of
ORFs. Long ORFs are therefore obvious candidates to be coding
intervals. Unfortunately, the constraints on the coding interval to be
an ORF often cause subintervals and overlapping intervals of the
coding interval to also be ORFS in one of the five other reading
frames (two on the same strand and three on the opposite strand).
These coincidental ORFs are called shadow ORFs since they are
found in the shadow of the coding ORF. In rare cases (and more
frequently in certain viruses) coding intervals in different reading
frames can overlap but usually only slightly. Overwhelmingly distinct
coding intervals do not overlap. However, this constraint is not as
strict for ORFs that contain a coding interval, as the exact extent of
the coding interval is not known. Prokaryotes predominate in these
data and are the focus of the ORF predictions. Their 39 end of an
ORF is very likely to be part of the coding interval because a stop
codon is a clear signal for the termination of both the ORF and the
coding interval (this signal could be obscured by frameshift errors in
sequencing). The 59 end is more problematic because the true start
codon is not so easily identified and so the longest ORF with a
reasonable start codon is chosen and this may extend the ORF
beyond the true coding interval. For this reason different criteria
0454 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Table 12. The Number of Sequences in NCBI-nr, PG ORFs, TGI-
EST ORFs, ENS, and GOS ORFs prior to and after the Redundancy
Removal Step of Our Clustering
Data Number of Amino Acid Sequences
Original Set Nonredundant Set
NCBI-nr 2,317,995 1,017,058
PG ORFs 3,049,695 2,424,016
TGI-EST ORFs 5,458,820 5,085,945
ENS 361,668 137,057
GOS ORFs 17,422,766 14,134,842
Total 28,610,944 22,798,918
doi:10.1371/journal.pbio.0050016.t012
were set for when ORFs have a significant overlap depending on the
orientation (or the 59 or 39 ends) of the ORFs involved. Two ORFs on
the same strand are considered overlapping if their intervals overlap
by at least 100 bp. Two ORFs that are on the opposite strands are
considered overlapping either if their intervals overlap by at least 50
bp and their 39 ends are within each others intervals, or if their
intervals overlap by at least 150 bp and the 59 end of one is in the
interval of the other.
ORFs for coding intervals are clustered based on sequence
similarity. In most cases this sequence similarity is due to the ORFs
evolving from a common ancestral sequence. Due to functional
constraints on the protein being coded for by the ORF, some
sequence similarity is retained. There are no known explicit
constraints on the shadow ORFs to constrain drift from the ancestral
sequences. However, the shadow ORFs still tend to cluster together
for some obvious reasons. The drift has not yet obliterated the
similarity. There are implicit constraints due to the functional
constraints on the overlapping coding ORF. There are also other
possible unknown functional constraints beyond the coding ORF. At
first it was surmised that within shadow ORF clusters the diversity
should be higher than for the coding ORF, but this did not prove to
be a reliable signal. The apparent problem is that the shadow ORFs
tend to be fractured into more clusters due to the introduction of
stop codons that are not constrained because the shadow ORFs are
noncoding. What rapidly became apparent is that the most reliable
signal that a cluster was made up of shadow ORFs is that the cluster
was smaller than the coding cluster containing the ORFs overlapping
the shadow ORFs.
The basic rule for labeling a cluster as a shadow ORF cluster is that
the size of the shadow ORF cluster is less than the size of another
cluster that contained a significant proportion of the overlapping
ORFs for the shadow ORF cluster. A specific set of rules was used to
label shadow ORF clusters based on comparison to other clusters that
contained ORFs overlapping ORFS in the shadow ORF cluster (called
the overlapping cluster for this discussion). First, the overlapping
cluster cannot be the same cluster as the shadow ORF cluster (there
are sometimes overlapping ORFs within the same cluster due to
frameshifts). Second, both the redundant and nonredundant sizes of
the shadow ORF cluster must be smaller than the corresponding sizes
of the overlapping cluster. Third, at least one-third of the shadow
ORFs must have overlapping ORFs in the overlapping cluster. Fourth,
less than one-half of the shadow ORFs are allowed to contain their
overlapping ORFs (this test is rarely needed but did eliminate the vast
majority of the very few obvious false positives that were found using
these rules). Finally, the majority of the shadow ORFs that overlapped
must overlap by more than half their length.
When using this rule, 1,274,919 clusters were labeled as shadow
ORF clusters, and 6,570,824 singletons were labeled as shadow ORFs.
The rules need to be somewhat conservative so as not to eliminate
coding clusters. To test these rules, clusters containing at least two
NCBI-nr sequences were examined. Two sequences were used instead
of one because occasional spurious shadow ORFs have been
submitted to NCBI-nr. There were 989 shadow ORF clusters
containing at least two NCBI-nr sequences and with more than
one-tenth as many NCBI-nr sequences as the overlapping cluster.
This was 0.86% of all clusters (114,331 in total) with at least two
NCBI-nr sequences. Of these 989, a few were obvious mistakes, and
the others involved very few NCBI-nr sequences of dubious curation,
PLoS Biology | www.plosbiology.org | S79
Expanding the Protein Family Universe
such as ‘‘hypothetical.’’ Just to be conservative, all of these 989
clusters were rescued and not labeled as shadow ORF clusters.
Ka/Ks test to determine if sequences in a cluster are under selective
pressure. For a cluster containing conserved but noncoding
sequences, it is expected that there is no selection at the codon
level. We checked this by computing the ratio of nonsynonymous to
synonymous substitutions (Ka/Ks test) [130,131] on the DNA
sequences from which the ORFs in the cluster were derived. For
most proteins, Ka/Ks  1, and for proteins that are under strong
positive selection, Ka/Ks  1. A Ka/Ks value close to 1 is an indication
that sequences are under no selective pressure and hence are unlikely
to encode proteins [134,135]. Weakly selected but legitimate coding
sequences can have a Ka/Ks value close to 1. These were identified to
some extent by using a model in which different partitions of the
codons experience different levels of selective pressure. A cluster was
rejected only if no partition was found to be under purifying
selection at the amino acid level.
The Ka/Ks test [130,131] was run only on those clusters (remaining
after the shadow ORF filtering step) that did not contain sequences
with HMM matches or have NCBI-nr sequences in them. Only the
nonredundant sequences in a cluster were considered. Sequences in
each of the clusters were aligned with MUSCLE [134]. For each
cluster, a strongly aligning subset of sequences was selected for the
Ka/Ks analysis. The codeml program from PAML [135,136] was run
using model M0 to calculate an overall (i.e., branch- and position-
independent) Ka/Ks value for the cluster. Clusters with Ka/Ks  0.5,
indicating purifying selection and therefore very likely coding, were
considered as passing the Ka/Ks filter. In addition, the remaining
clusters were examined by running codeml with model M3. This
partitioned the positions of the alignment into three classes that may
be evolving differently (typically, a few positions may be under
positive selection while the remainder of the sequence is conserved).
A likelihood ratio test was applied to select clusters for which M3
explained the data significantly better than M0 [136]. If a cluster was
thus selected, and if one of the resulting partitions had a Ka/Ks  0.5
and comprised at least 10% of the sequence, then that cluster was also
considered as passing the Ka/Ks filter. All other clusters were marked
as containing spurious ORFs.
Statistics for the various stages of the clustering process The
number of sequences that remain after redundancy removal (at 98%
similarity) for each dataset is given in Table 12. Recall that the size of
a cluster is the number of nonredundant sequences in it.
Number of core sets of size two or more totals 1,586,454; number
of nonredundant sequences in core sets of size two or more totals
8,337,256; and total number of sequences in core sets of size two or
more is 12,797,641.
Total number of clusters after profile merging and (PSI-BLAST
and BLAST) recruitment is 1,871,434; number of clusters of size two
or more totals 1,388,287; number of nonredundant sequences in
clusters of size two or more totals 11,494,078; total number of
sequences in clusters of size two or more is 16,565,015.
The final clustering statistics (after shadow ORF detection and Ka/
Ks tests) are as follows: number of clusters of size two or more totals
297,254; number of nonredundant sequences in clusters of size two or
more totals 6,212,610; total number of sequences in clusters of size
two or more is 9,978,637.
In the final BLAST recruitment step, a pattern was seen involving
highly compositionally biased sequences that recruited unrelated
sequences to clusters. This was reflected in the pre- and post-BLAST
recruitment numbers, where the postrecruitment sizes were more
than three to four times the size of the prerecruitment numbers.
There were 75 such clusters, and these were removed.
Searching sequences using profile HMMs. The full set of 7,868
Pfam release 17 models was used, along with additional nonredun-
dant profiles from TIGRFAM (1,720 of 2,443 profiles; version 4.1).
HMM profiling was carried out using a TimeLogic DeCypher system
(Active Motif, Inc., http://www.activemotif.com) and took 327 hours in
total (on an eight-card machine). A sequence was considered as
matching a Pfam (fragment model) if its sequence score was above the
TC score for that Pfam and had an E-value  1 3 103. It was
considered as matching a TIGRFAM if the match had an E-value  1
3 107.
Evaluation of protein prediction via clustering. Our evaluation of
protein prediction via the clustering shows a very favorable
comparison to currently used protein prediction methods for
prokaryotic genomes. We used the PG dataset for this evaluation
(Table 2). Of the 3,049,695 PG ORFs, 575,729 sequences (19%) were
clustered (the clustered set). Of the 614,100 predictions made by the
genome projects, 600,911 sequences could be mapped to the PG ORF
set (the submitted set); 93% of the unmapped sequences were ,60 aa
0455 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Figure 12. Log–Log Plots of Cluster Size Distributions

The x-axis is logarithm of the cluster size X and the y-axis is the logarithm of the number of clusters of size at least X; logarithms are base 10.
(A) Plot comparing the sizes of clusters produced by our clustering approach (red) to those of clusters produced by Pfams (green). The curves track each
other quite well, with both of them having an inflection point around cluster size 2,500 (approximately 3.4 on the x-axis). Each sequence is assigned to
the highest scoring Pfam that it matches. Two sequences that are assigned to the same Pfam can nevertheless be assigned to different clusters by the
full-sequence–based clustering approach if they differ in the remaining portion. This is especially true for commonly occurring domains that are present
in different multidomain proteins. Thus, there tends to be a larger number of big clusters in the Pfam approach as compared to the full-sequence–
based approach. Hence, the green curve is above the red curve at the higher sizes.
(B) Plot of the cluster size distributions for core sets (green) and for final clusters (red). Both curves have an inflection point around cluster size 2,500
(approximately 3.4 on the x-axis). Note that these plots give the cumulative distribution function (cdf), while the power law exponents reported in the
text are for the number of clusters of size X (i.e., the probability density function [pdf]). The relationship between these exponents is bpdf ¼ 1 þ bcdf.
doi:10.1371/journal.pbio.0050016.g012

(recall that the ORF calling procedure only produced ORFs of length
60 aa). The clustered set and submitted set had 493,756 ORFs in
common. Of the 107,155 sequences that were only in the submitted
set, 24,217 sequences (23%) had HMM matches. As with other
unclustered HMM matches, most were weak or partial. These
sequences had an average of only 48% of their lengths covered by
HMMs. Of the remaining 82,938 sequences that did not have an HMM
match, 13,724 (17%) were removed by the filters used, and the rest fell
into clusters with only one nonredundant sequence (and thus were
not labeled as predicted proteins by the clustering analysis). Based on
NCBI-nr sequences in them, these clusters were mostly labeled as
‘‘hypothetical,’’ ‘‘unnamed,’’ or ‘‘unknown.’’ Our clustering method
identified 81,973 ORFs not predicted by the genome projects, of
which 16,042 (20%) were validated by HMM matches (with average
HMM coverage of 69% of sequence length) and an additional 27,120
(33%) had significant BLAST matches (E-value  1 3 1010) to
sequences in NCBI-nr. Thus, if the submitted set is considered as
truth, then protein prediction via clustering produces 493,756 true
positives (TP), 81,973 false positives (FP), and 107,155 false negatives
(FN), thereby having a sensitivity (TP/[TP þ FN]) of 83% and
specificity (TP/[TP þ FP]) of 86%. However, if truth is considered as
those sequences that are common to both the clustered and
submitted sets in addition to those sequences with HMM matches,
then our protein prediction method via clustering has 95% sensitivity
and 89% specificity, while protein prediction by the prokaryotic
genome projects has 97% sensitivity and 86% specificity.
Evaluation of protein clustering. We used Pfams to evaluate the
clustering method in two ways. For both evaluations the clustering
was restricted to only those sequences with Pfam matches. It should
be kept in mind that there are redundancies among Pfams in that
there can be more than one Pfam for a homologous domain family
(for instance, the kinase domain Pfams—PF00069 protein kinase
domain and PF07714 protein tyrosine kinase), and these redundan-
cies can affect the evaluation statistics reported below.
For the first evaluation, each sequence was represented by the set
of Pfams that match it. This is referred to as the domain architecture for
a sequence. While Pfams provide a domain-centric view of proteins,
the domain architecture attempts to approximate the full sequence-
based approach used here, and thus could be used to shed light on the
general performance of the clustering. We measured how often
unrelated sequences were present in a given cluster. Two sequences
were defined to be unrelated if their domain architectures each had
at least one Pfam that was not present in the other’s domain
architecture. Note that this measure did not penalize the case when
the domain architecture of one sequence was a proper subset of the
domain architecture of the other sequence. This was done to allow
fragmentary sequences in clusters to be included in the evaluation as
well (and also because it is not always easy to determine whether an
amino acid sequence is fragmentary or not). For each cluster, we
computed the percentage of sequence pairs that are unrelated under
this measure. A total of 92% of the clusters had at most 2% unrelated
pairs. Then we carried out an assessment of how many instances of a
given domain architecture appear in a single cluster. A total of 58%
of the domain architectures were confined to single clusters (i.e.,
100% of their occurence is in one cluster), and 88% of the domain
architectures was such that .50% of their occurences is in one
cluster.
For the second evaluation, we selected all sequences with Pfam
matches, and each sequence was assigned to the Pfam that matches it
with the highest score. With this assignment, the Pfams induce a
partition on the sequences. The distribution of the number of
sequences in clusters induced by the Pfams was compared to those of
clusters from the clustering method. Figure 12A shows comparison as
a log–log plot of the number of sequences versus the number of
clusters with at least that many sequences for the two cases. The plot
shows that cluster size distributions are quite similar, with both the
methods having an inflection point around 2,500. The difference
between the two curves is that there are more big clusters (and also
fewer small clusters) induced by the Pfams as compared to the
clustering method. This can be explained by noting that two
sequences that are in the same Pfam cluster can nevertheless be put
into different clusters by the clustering method if they differ in their
remaining portions.
Our clustering also shows a good correspondence with HMM
profiling on the phylogenetic markers that we looked at. The
clustering identifies 7,423, 12,553, and 13,657 sequences, respectively,
for RecA (cluster ID 1146), Hsp70 (cluster ID 197), and RpoB (cluster
ID 1187). HMM profiling identifies 5,292, 12,298, and 12,165
sequences, respectively, for these families. For each of these families,
there are at least 94% of sequences (relative to the smaller set) in
common between clustering and HMM profiling.
Difference in ratio of predicted proteins to total ORFs for the PG
set and the GOS set. The ratio of clustered ORFs to total ORFs is
significantly higher for the GOS ORFs (0.3471) compared to the PG
ORFs (0.1888). This can be explained by the fragmentary nature of
the GOS data. For the large majority of the GOS data, the average
sequence length is 920 bp compared to full-length genomes for the
PG data. For the PG data, clustered ORFs have a mean length of 325
aa and a median length of 280 aa. Unclustered ORFs have a mean
length of 119 aa and a median length of 87 aa. Assuming that the
genomic GOS data has a similar underlying ORF structure to PG data,
the effect that GOS fragmentation had on ORF lengths is estimated.
Each reading frame will have a mixture of clustered and unclustered
ORFs, but on average there will be 2 ORFs per reading frame per 920-
bp GOS fragment, and both ORFs will be truncated. Assuming the
truncation point for the ORF is uniformly distributed across the
ORF, the truncated ORF will drop below the 60-aa threshold to be
considered as an ORF with a probability of 60/(length of the ORF).
Using the median length, the percentage of clustered ORFs dropping
below the threshold due to truncation is 21%; for unclustered ORFs,
it is 69%. Accounting for this truncation, the expected ratio of
clustered ORFs to total ORFs for the GOS ORFs based on the PG
ORFs would be 0.3708, which is very close to the observed value.
Kingdom assignment strategy and its evaluation. We used several

PLoS Biology | www.plosbiology.org | S80 0456 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Table 13. BLAST-Based Classification Rate per Kingdom Table 14. The Values for Cd(n), the Number of Clusters of Size
d, as a Function of the Power Law Exponent b and Constant a
Kingdom Total Number Correct Classification Percent Correct
b a Cd(n)
Eukaryota 440,951 422,173 95.7
Bacteria 465,692 430,014 92.3 b,1 nb1 1
Archae 36,894 25,527 69.2 b¼1 1 ln n
Viruses 36,346 32,381 89.0 1,b,2 nb1 ðn= d Þb1
b¼2 ð n= ln nÞ ð n=d b1
ln nÞ
b.2 n n d
doi:10.1371/journal.pbio.0050016.t013

approaches to assign kingdoms for GOS sequences. They are all
fundamentally based upon a strategy that takes into account top
BLAST matches of a GOS sequence to sequences in NCBI-nr, and
then voting on a majority.
We evaluated a simple strict-majority voting scheme (of the top
four BLAST matches) using the NCBI-nr set. First, the redundancy in
NCBI-nr was removed using a two-staged process. A nonredundant
set of NCBI-nr sequences was computed involving matches with 98%
similarity over 95% of the length of the shorter sequence (using the
procedure discussed in Materials and Methods [Identification of
nonredundant sequences]). This set was made further nonredundant
by considering matches involving 90% similarity over 95% of the
length of the shorter sequence. The nonredundant sequences that
remained after this step constituted the evaluation dataset S. For each
sequence in S, its top four BLAST matches to other sequences in S
(ignoring self-matches) were used to assign a kingdom for it (based on
a strict majority rule). This predicted kingdom assignment for the
sequence was compared to its actual kingdom. A correct classification
is obtained for 93% of the sequences. The correct classification rate
per kingdom is given in Table 13.
While this evaluation shows that the BLAST-based voting scheme
provides a reasonable handle on the kingdom assignment problem,
there are caveats associated with it. The kingdom assignment for a set
of query sequences is greatly influenced by the taxonomic groups
from each kingdom that are represented in the reference dataset
against which these queries are being compared. If certain taxa are
only sparsely represented in the reference set, then, depending on
their position in the tree of life, queries from these taxa can be
misclassified (using a nearest-neighbor type approach based on
BLAST matches). This explains why the archaeal classification rate is
quite low compared to the others. Thus, the true classification rate
for the GOS dataset based on this approach will also depend on the
differences in taxonomic biases in the GOS dataset (query) and the
NCBI-nr set (reference).
The kingdom proportion for the GOS dataset reported in Figure 1
is based on a kingdom assignment of scaffolds. Those GOS ORFs with
BLAST matches to NCBI-nr were considered, and the top-four
majority rule was used to assign a kingdom to each of them. Using the
ORF coordinates on the scaffold, the fraction (of bp) of a scafffold
assigned to each kingdom was computed. The scaffold was labeled as
belonging to a kingdom if the fraction of the scaffold assigned to that
kingdom was .50%. All ORFs on this scaffold were then assigned to
the same kingdom.
Cluster size distribution, the power law, and the rate of protein
family discovery. Earlier studies of protein family sizes in single
organisms [137–139] have suggested that P(d), the frequency of
protein families of size d, satisfies a power law: that is, P(d) ’ d  b
with exponent b reported between 2.68 and 4.02. Power laws have
been used to model various biological systems, including protein–
protein interaction networks and gene regulatory networks [42,43].
Figure 12B illustrates the distribution of the cluster sizes from our
data on a log–log scale, a scale for which a power law distribution
gives a line. In contrast to family size distributions reported in single
organisms, the cluster sizes from our data are not well described by a
single power law. Rather, there appear to be different power laws:
one governs the size distribution of very large clusters, and another
describes the rest. This behavior is observed both in the distribution
of the core set sizes and also in the distribution of the final cluster
sizes. We identified an inflection point for both the core set
distribution and the final clusters at around size 2,500, and estimated
the power law exponent b via linear regression separately in each size
regime. For the core set distribution, the exponent b ¼ 1.99 (R2 ¼
0.994) for clusters of size  2,500, and b ¼ 3.34 (R2 ¼ 0.996) for
clusters of size . 2,500. For the final cluster sizes, the exponent b ¼
doi:10.1371/journal.pbio.0050016.t014
1.72 (R2 ¼ 0.995) for clusters of size  2,500, and b ¼ 2.72 (R2 ¼ 0.995)
for clusters of size . 2,500. The estimates for b are different for the
core clusters compared to the final clusters, reflecting a larger
number of medium and large clusters in the final clustering as a
result of the cluster-merging and additional recruitment steps. A
similar dichotomy between the size distributions of large and small
protein families was observed in a study [140] of protein families
contained in the ProDom, Protomap, and COG databases, where the
exponent b reported was in the range of 1.83 to 1.98 for the 50
smallest clusters and 2.54 to 3.27 for the 500 largest clusters in these
databases.
Our clustering method was run separately on the following seven
datasets: set 1 consisted of only NCBI-nr sequences; set 2 consisted of
all sequences in NCBI-nr, ENS, TGI-EST, and PG; sets 3 through 6
consisted of set 2 in combination with a random subset of 20%, 40%,
60%, and 80% of the GOS sequences, respectively; set 7 consisted of
set 2 in combination with all the GOS sequences. On each of the
seven datasets, the redundancy removal (using the 98% similarity
filter) was run, followed by the core set detection steps. Figure 2
shows the number of core sets of varying sizes (3, 5, 10, and 20)
as a function of the number of nonredundant sequences for each
dataset.
The observed linear growth in number of families with increase in
sample size n is related to the power law distribution in the following
way. We model protein families as a graph where each vertex
corresponds to a protein sequence and an edge between two vertices
indicates sequence similarity between the corresponding proteins.
Consider a clustering (partitioning) of the vertices of a graph with n
vertices such that the cluster sizes obey a power law distribution. Let
Cd(n) [respectively, Cd(n)] denote the number of clusters of size d
(respectively, d). Since the distribution of cluster sizes follows a
power law, there exist constants a, b such that for all x  n, Cx(n) ¼
axb.
As every vertex of the graph is a member of exactly one cluster,
8  
Xn Xn < n2b  1
n¼ xCx ðnÞ ¼ ax 1b
’ a b 6¼ 2 ð1Þ
: 2b
x¼1 x¼1 alnn b¼2
The number of clusters of size at least d is
8  
Xn < n1b  d1b
Cd ðnÞ ¼ Cx ðnÞ ’ a b 6¼ 1 ð2Þ
: 1b
x¼d alnn b¼1
Combining the two equations, we obtain values (up to a multiplicative
constant) for Cd(n) as shown in Table 14. In all cases with b . 1, the
number of clusters Cd(n) increases as n increases, and as d decreases.
Specifically, for b . 2, the growth is linear in n for all d, with slope
decreasing as d increases. For 1 , b , 2, the growth is sublinear in n
for all d.
Note that while the observed distribution of protein family sizes is
fit by two different power laws, one for clusters of size less than 2,500
with b ¼ 1.99 and another for clusters of size greater than 2,500 with b
¼ 3.34 for the current number of (nonredundant) sequences, the
contribution of large families to the rate of growth is negligible
compared to the small families.
The above formulas for Cd(n) also suggest the dependence of the
rate of growth of clusters on the cluster size d. For example, in the
case when b is very close to 2,
n
Cd ðnÞ ¼ m b1 ð3Þ
d

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Figure 13. Log–Log plot of Slopes m(d) of Linear Regression Fit to the
Rate of Growth in Figure 2 for Different Values of Cluster Size d
According to the equation derived in the text, m(d) ¼ md1b for some
constant m. The best linear fit to log [m(d)] gives a line with slope 0.91
(R2 ¼ 0.98) that is close to the predicted value 1  b ¼ 0.99.
doi:10.1371/journal.pbio.0050016.g013
for some constant m. Thus, the rate of growth of cluster sizes is linear,
and the slope m(d) of rate of growth is given by m(d) ¼ md1b. Figure 13
shows how well the observed rates of growth match the values
predicted by this equation. A fit to a sublinear function (not shown)
also gives similar results as in Figure 13.
GOS versus known prokaryotic versus known nonprokaryotic.
Examples of top five clusters in the various categories (except GOS-
only) are given below. The cluster identifiers are in parentheses.
Known prokaryotic only: (Cluster ID 1319) outer surface protein in
Anaplasma ovis, Wolbachia, Ehrlichia canis; (Cluster ID 10911) nitrite
reductase in uncultured bacterium; (Cluster ID 1266) outer mem-
brane lipoprotein in Borrelia; (Cluster ID 8595) methyl-coenzyme M
reductase subunit A in uncultured archaeon; (Cluster ID 2959) outer
membrane protein in Helicobacter. Known nonprokaryotic only:
(Cluster ID 2226) Pol polyprotein HIV sequences; (Cluster ID 4023)
maturase K; (Cluster ID 6257) NADH dehydrogenase subunit 2;
(Cluster ID 8644) HIV protease; (Cluster ID 12196) MHC class I and II
antigens. GOS and known prokaryotic only: (Cluster ID 3369)
carbamoyl transferase; (Cluster ID 688) apolipoprotein N-acyltrans-
ferase; (Cluster ID 3726) potassium uptake proteins; (Cluster ID 300)
primosomal protein N9; (Cluster ID 4605) DNA polymerase III delta
subunit. GOS and known nonprokaryotic only: (Cluster ID 186) seven
transmembrane helix receptors; (Cluster ID 2069) zinc finger
proteins; (Cluster ID 3092) MAP kinase; (Cluster ID 1413) potential
mitochondrial carrier proteins; (Cluster ID 233) pentatricopeptide
(PPR) repeat-containing protein. Known prokaryotic and known
nonprokaryotic only: (Cluster ID 3510) immunoglobulin (and
immunoglobulin-binding) proteins; (Cluster ID 600) expansin; (Clus-
ter ID 50) pectin methylesterase; (Cluster ID 6492) lectin; (Cluster ID
986) BURP domain-containing protein. GOS and known prokaryotic
and known nonprokaryotic: (Cluster ID 2568) ABC transporters;
(Cluster ID 49) short-chain dehydrogenases; (Cluster ID 4294)
epimerases; (Cluster ID 1239) AMP-binding enzyme; (Cluster ID
2630) envelope glycoprotein.
Neighbor functional linkage methods. For the sequences in each
GOS-only cluster, we determined if neighboring ORFs occurring on
the same strand had a similar biological process in the GO [49]. If this
shared biological process of the neighbors occurred statistically more
often than expected by chance, that inferred a potential operon
linkage and a biological process term for the GOS-only cluster. This
approach weighted ORFs by sequence similarity to reduce the
skewing effect of sequences from highly related organisms.
For definition of linked ORFs, we collected pairs of same-strand
ORF protein predictions with intergenic distances less than 500 bp.
Negative distances were possible if the 59 end of the downstream ORF
in the pair occurred 59 to the 39 end of the upstream ORF. We used a
probability function to estimate the probability that two putative
genes belong to the same operon given their intergenic distance [47].
Because sequences come from a variety of unknown organisms, the
probability distribution was created by averaging properties of 33
randomly chosen divergent genomes. The exact choice of genomes
did not greatly affect the ability of the distribution to separate
experimentally determined same-operon gene pairs from adjacent,
same-strand gene pairs in different known operons annotated in a
version of RegulonDB downloaded on March 29, 2005 [141].
We measured the functional linkage between two protein clusters
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Expanding the Protein Family Universe
Figure 14. Receiver Operating Characteristic Curve Used to Evaluate
Various Methods of Scoring Pairs of Clusters for Functional Similarity
Pairs of clusters with 1 example of neighboring ORFs and assigned GO
terms were divided into a set of functionally related (true positive) and
functionally unrelated (true negative) cluster pairs based on the similarity
of their GO terms. The scoring methods evaluated are described in the
text.
doi:10.1371/journal.pbio.0050016.g014
by searching for all occurrences of nearby pairs of ORFs belonging to
the two clusters of interest. Sufficiently close pairs were more likely to
be encoded in the same operon. We devised a scoring mechanism to
reward those pairs of clusters for which many divergent examples of
likely operon pairs existed in the set of ORF pairs. For each pair of
clusters, a weight was applied to the contribution of each pair of
ORFs, and this was proportional to how similar the pair of ORFs was
to other example pairs. Thus, many near-identical pairs of ORFs,
likely from the same or similar species, are not overrepresented in the
final cluster pair score, while conserved examples of neighboring
position from more divergent sequences contribute an increased
weight. The score for each cluster pair is calculated as:
i¼n
SðC1 C2 Þ ¼ 1  P ½1  PrðOgi1 gi2 jdistÞ  wi1  wi2  ð4Þ
i¼1
where S(C1C2) is the linkage score of clusters C1 and C2. The
probability PrðOgi1 gi2 jdistÞ that any two genes gi1 from C1 and gi2 from
C2 are in an operon is dependent on the distance between them as
calculated by [47], and is weighted according to the sequence weights
wi1 and wi2 described below, for all example pairs i.
We calculated sequence weights in a manner similar to that used in
progressive multiple sequence alignment [142]. Briefly, neighbor-
joining trees were built for all clusters using the QuickJoin [143] and
QuickTree programs [144] based on a distance matrix constructed
from all-against-all BLAST scores within a cluster, normalized to self-
scores. For those few clusters with more than 30,000 members, trees
were not built. Instead, equal sequence weights for all members were
assigned because of computational limitations. The root of each tree
was placed at the midpoint of the tree by using the retree package in
PHYLIP [145]. The individual sequence weights were then computed
by summing the distance from each leaf to the root after dividing
each branch’s weight by the number of nodes in the subtree below it.
Weights were normalized so that the sum of weights in any given tree
was equal to 1.0. This weighting scheme is superior to one in which
weights are normalized to the largest weight in the tree, one that does
not weight sequences according to divergence, and one that only
considers the number of example pairs seen (Figure 14). To compare
the different scoring methods, pairs of clusters annotated with GO
terms that contained adjacent ORFs in the data were gathered. These
pairs were divided into into functionally related and unrelated
clusters based on a measure of GO term similarity (p-value  0.01)
[146]. We evaluated scoring methods for the ability to recover
0458 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Figure 15. Novel GOS-Only Clusters Are More Interconnected Than a
Size-Matched Sample of Clusters
Red line, novel clusters; green line, size-matched sample; blue line (right
axis), log2 ratio of fraction novel clusters recovered divided by fraction
sample clusters recovered.
doi:10.1371/journal.pbio.0050016.g015
functionally similar pairs. In all analyses, linkages between clusters
were ignored if there were fewer than five examples of cluster
member ORFs adjacent to each other on a scaffold.
Function for novel families was inferred as follows. (1) Assignment
of GO terms to clusters. We downloaded the GO [49] database on
September 21, 2005, from http://www.geneontology.org, along with the
files gene_association.goa_uniprot and pfam2go.txt dated July 12,
2005. Only the biological process component of the ontology was
considered. If a cluster had at least 10% of its redundant sequences
annotated by the most abundant Pfam domain for that cluster, and
that Pfam domain had a GO biological process term provided by the
pfam2go mapping, then we assigned a cluster the GO term of its most
abundant Pfam annotation. In addition, if a cluster contained at least
20% of its Uniprot GO annotations the same, it was assigned that GO
term. For each cluster, redundant GO terms found on the same path
to the root were removed. (2) Identification of neighbors to GOS-only
clusters. Neighbors of GOS-only clusters were defined as those
clusters that had a cluster linkage score above a predetermined
threshold (1 3 106) and had at least five examples of cluster members
adjacent to each other in the data. These neighbors were then
screened for those that had been annotated with a GO term by the
process described above. (3) Overrepresentation of neighbor GO
terms. We attempted to define GO terms for a set of GOS-only
neighbors that were statistically overrepresented. Because of the
highly dependent nature of the terms in the GO, a simulation-based
approach was chosen to determine which terms might be over-
represented. Annotated neighbors to a cluster of unknown function
were identified as described above. For each annotated neighbor,
counts for the associated GO term and all terms on the path to the
root of the ontology were incremented. A total of 100,000 simulated
neighbor lists of the same size as the true neighbor list were computed
by selecting without replacement from those clusters with annotated
GO terms, and an identical counting scheme was performed for each
simulation. Overrepresentation of neighbor terms was calculated for
each term on the ontology by asking how many times out of the
100,000 simulations the count for each GO term in the ontology met
or exceeded the observed count for the actual neighbors. This
fraction of simulations was interpreted as a p-value. If a term is
unusually prevalent in the true observed neighbors, it should be
relatively infrequent in the simulated data. For the purpose of the
metric used here, ‘‘is-a’’ and ‘‘part-of’’ relationships were treated
equally. In cases where a cluster had more than one GO term assigned
to it, any redundant terms occurring on each other’s path to the root
were first removed. For any remaining clusters with nonredundant,
multiple GO annotations, all possible lists of functions for each list of
neighbor clusters were enumerated, and one function from each
cluster was chosen. Each node in the ontology was assigned the
maximum count observed from the enumerated function lists. We
consistently applied this rule for the observed and simulated data.
The following descriptive measures of the novel GOS-only cluster
set were obtained. Transmembrane helix prediction was carried out
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Expanding the Protein Family Universe
with the programs TMHMM [147] and SPLIT4 [148]. GC content was
calculated as (G þ C)/(G þ C þ A þ T) bases for each ORF in a cluster,
and averaged for each cluster within a set. The GC content, reported
as the mean and standard deviation of the cluster averages, is as
follows for each cluster set: Group I, 36.7% 6 8.0%; Group II, 35.9%
6 7.9%. Group I size-matched sample, 48.8% 6 11.1%; Group II size-
matched sample, 49.5% 6 11.2%; Group I viral fraction, 37.8% 6
5.1%; Group II viral fraction, 37.3% 6 4.6%. To address the
interconnectivity of the novel clusters within the context of all
operon linkages, we constructed a graph with clusters as nodes and
inferred operon linkages (with score  1 3 106) as edges. We then
asked for every node in the set of novel clusters what was the
cumulative fraction of novel nodes that could be reached within a
varying edge distance from the starting node. The expectation of this
fraction was calculated at each distance, and the procedure was
repeated for the set of size-matched clusters (Figure 15).
We tried three different BLAST-based approaches for kingdom
assignment of ORFs. The first method, used in the analysis, required a
majority of the four top BLAST matches to vote for the same
kingdom (archaea, bacteria, eukaryota, or viruses; see Materials and
Methods [Kingdom assignment strategy and its evaluation]). The
second method required all eight top BLAST matches to vote for the
same kingdom. The last method we used was the scaffold-based
kingdom assignment described in Materials and Methods (Kingdom
assignment strategy and its evaluation). Figure 16 shows the results of
using these assignments to infer the kingdom of GOS-only clusters
(Figure 16D–16F) and their neighboring ORFs (Figure 16A–16C).
GOS-only clusters were assigned a kingdom only if .50% of their
neighboring ORFs were assigned the same kingdom. The general
trends observed are the same for each method, though the coverage
decreases slightly for the more stringent methods.
Characteristics and kingdom distribution of known protein
domains. For these analyses we used the predicted proteins from
the public (NCBI-nr, PG, TGI-EST, and ENS) and GOS datasets. The
public dataset contains multiple identical copies of some sequences
due to overlaps between the source datasets. For example, many
sequences in PG are also found in NCBI-nr. We filtered the public set
at 100% identity to avoid overcounting these sequences. Because this
filtering was necessary for the public dataset, the GOS dataset was
also filtered at 100% identity. If two or more sequences were 100%
identical at the residue level, but were of different lengths, only the
longest sequence was kept. The resulting datasets of nonredundant
proteins are referred to as public-100 and GOS-100.
We assigned each protein in public-100 to a kingdom based on the
species annotations provided in the source datasets (NCBI-nr,
Ensembl, TIGR, and PG). The NCBI taxonomy tree was used to
determine the kingdom of each species. Of 3,167,979 protein
sequences in public-100, 3,158,907 can be annotated by kingdom.
The remaining 9,072 sequences are largely synthetic.
Determining the kingdom of origin of an environmental sequence
can be difficult; while an unambiguous assignment can be made for
some sequences, others can be assigned only tentatively or not at all.
Therefore, we took a probabilistic approach (kingdom-weighting
method), calculating ‘‘weights’’ or probabilities that each protein
sequence originated from a given kingdom.
The top four BLAST matches (E-value , 1 3 1010) of GOS ORFs to
NCBI-nr were considered. The kingdom of origin for each match was
determined. We pooled these ‘‘kingdom votes’’ for each scaffold,
since (presuming accurate assembly) each scaffold must come from a
single species and hence from a single kingdom. Each ORF on a
scaffold contributed up to four votes. If an ORF had fewer than four
BLAST matches with an E-value , 1 3 1010, then it contributed
fewer votes. ORFs with no BLAST matches contributed no votes.
In many cases, the votes were not unanimous, indicating that some
uncertainty must be associated with any kingdom assignment. An
additional source of uncertainty is the finite number of votes. We
accounted for these statistical issues by applying the following
procedure to each scaffold. First, two pseudocounts were added to
the votes for the ‘‘unknown’’ kingdom to represent the uncertainty
that remains even when votes are unanimous (especially when there
are few votes). The frequency of votes for each kingdom was
calculated. The vote frequency for a kingdom provides the maximum
likelihood estimate of the kingdom probability (i.e., the vote
frequency that would have been observed on a scaffold of similar
composition but with infinitely many voting ORFs). However, that
estimate may not be accurate or precise. Therefore, the multinomial
standard deviation was calculated for each vote frequency p as SQRT
[p 3 (1  p)/(n  1)], where n is the number of votes. A distance of two
standard deviations from the mean corresponds roughly to a 95%
confidence interval. Thus, two standard deviations were subtracted
0459 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Figure 16. GOS-Only Clusters Are Enriched for Sequences of Viral Origin
Independently of the Kingdom Assignment Method Employed
For each panel, clusters are as in Figure 4. For (A–C), a kingdom is
assigned to each neighboring ORF within each cluster set; the
percentage of all neighboring ORFs with a given kingdom assignment
is plotted. For (D–F), a kingdom is assigned to each cluster if more than
50% of all that cluster’s neighbors with a kingdom assignment share the
same assignment; the percentage of clusters in each set with a given
assignment is plotted. In (A) and (D), a kingdom is assigned to a
neighboring ORF by a majority vote of the top four BLAST matches to a
protein in NCBI-nr (Materials and Methods). In (B) and (E), a kingdom is
assigned if all eight highest-scoring BLAST matches agree in kingdom. In
(C) and (F), all ORFs on a scaffold are assigned the same kingdom by
voting among all ORFs with BLAST matches to NCBI-nr on that scaffold
(Materials and Methods). In all graphs, only clusters with at least one
assignable neighbor are considered. When compared to the size-
matched controls, in all cases the GOS-only clusters show enrichment
for viral sequences.
doi:10.1371/journal.pbio.0050016.g016
from each vote frequency, and called the result (or zero, if the result
was negative) the ‘‘kingdom weight.’’ This ‘‘kingdom weight’’ is a
conservative estimate. There is 95% chance that the actual kingdom
probability is greater.
The kingdom weights do not sum to one because of the standard
deviation penalty. The difference between the sum of the kingdom
weights and unity is a measure of the total uncertainty about the
kingdom assignment. This is called the ‘‘unknown weight.’’
Finally, we assigned each ORF the kingdom weights calculated for
the scaffold as a whole. This procedure assigned kingdom weights to
many ORFs with no BLAST matches. Overall, 4,745,649 (84%) of the
5,654,638 proteins in GOS-100 receive nonzero kingdom weights.
The kingdom weights calculated in this way provide a basis for
estimating the proportion of sequences originating from each
kingdom, pGOS(K). The weights over all sequences in GOS-100 were
summed for each of the known kingdoms, and divided by the sum of
the weights for all kingdoms (excluding the unknown weight). This
procedure suggested that 96% of the sequences are bacterial, a
somewhat higher proportion than is estimated by the method
described in Materials and Methods (Kingdom assignment strategy
and its evaluation). Similarly, kingdom proportions, pGOS–Pfam(K),
were calculated for the subset of GOS-100 sequences that have a
significant Pfam hit, and 97% are found to be bacterial.
We used the kingdom weights directly in the analyses where
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Expanding the Protein Family Universe
possible (e.g., to calculate the expected kingdom distribution of a
given set of proteins by summing the weights). However, it was
necessary in some cases to use discrete assignments of a single
kingdom to each ORF. A tentative assignment can be made for a
given scaffold by choosing the kingdom with the highest weight. The
possibility remains, in this case, that a fraction of the ‘‘unknown’’
weight should rightfully belong to a different kingdom. However, if a
kingdom weight is greater than 0.5, then this danger is averted, and a
‘‘confident’’ assignment of the scaffold and its constituent ORFs to
that kingdom can be made.
Given the uncertainty penalty above, achieving a kingdom weight
greater than 0.5 generally requires overwhelming support for one
kingdom over the others. In particular, on a given scaffold, at least
eight unanimous votes for a kingdom are needed (i.e., two ORFs
contributing four votes each) to make a confident assignment to that
kingdom. Any disagreement between the votes increases the required
number rapidly: for instance, 15 votes for a single kingdom are
required to override four votes for other kingdoms.
‘‘Confident’’ kingdom assignments were made for 2,626,178 (46%)
of the 5,654,638 proteins in GOS-100.
In the analysis that identified new multi-kingdom Pfams, we used
the subset of confidently kingdom-annotated proteins. Here, a Pfam
model was designated as ‘‘kingdom-specific’’ in public-100 if there
were only matches to proteins in one particular kingdom, and no
‘‘unknown’’ matches. A Pfam model that was kingdom specific in
public-100 was further designated as newly ‘‘multi-kingdom’’ if it had
matches to one or more GOS-100 proteins that were confidently
labeled as belonging to a kingdom different from that found in the
public-100 matches. Also, we filtered Pfam matches with an E-value
cutoff of 1 3 1010. In every case, the bit score is at least five bits
greater than the trusted cutoff for the model. In addition to passing
the ‘‘confident’’ criteria, the kingdom assignments were all confirmed
by visual inspection of the BLAST kingdom vote distributions for the
respective scaffolds. Because the criteria for a ‘‘confident’’ kingdom
assignment were conservative, there were only one or a few confident
assignments for each domain to a ‘‘new’’ kingdom. The ‘‘confident’’
criteria are especially difficult to meet in the case of kingdom-
crossing due to the votes contributed by the crossing protein. For
instance, because the IDO domain itself always contributes four votes
for ‘‘Eukaryota,’’ at least 15 votes for ‘‘Bacteria’’ were required to call
a scaffold ‘‘bacterial.’’ Thus, many scaffolds have no confident
kingdom assignment.
We compared the relative diversities of protein families between
GOS-100 and public-100 as represented by Pfam sequence models. In
order to do this, the number of matches expected to be found for
each Pfam model in the GOS-100 data was computed, assuming that
the matches were distributed among the models in the same
proportions that they were in the public-100 data. These ‘‘expected’’
match counts were compared with the observed counts to identify
domains that are more diverse in GOS-100 than in public-100 and
vice versa.
Because kingdoms differ in their protein usage, Pfam models
match sequences from different kingdoms with different frequencies,
and some models match sequences exclusively from one kingdom.
Thus, to calculate the expected number of matches to a given Pfam in
GOS-100 based on the number of matches observed in public-100, we
corrected for the radically different kingdom composition of the two
datasets.
The expected proportion of all Pfam matches in GOS-100 that are
to a given model M was calculated as follows. First, we made a
simplifying assumption that sequences from different kingdoms were
equally likely to have a Pfam hit, and thus that the Pfam matches in
GOS-100 would be distributed among the kingdoms according to the
kingdom proportions calculated using the weighted method above
(for instance, it is assumed that 97% of the matches would be to
bacterial sequences). Probability that a Pfam hit in GOS-100 is from K
’ pGOS-Pfam(K) (for sequences in GOS-100 with at least one Pfam hit)
for kingdoms K in fArchae, Bacteria, Eukaryotes, Virusesg.
Second, we assumed that Pfam models match with the same
relative rates within each kingdom in GOS-100 as they do in public-
100. For instance, since twice as many SH3 domains as SH2 domains
are found in public-100 eukaryotic sequences, the same ratio is
expected to be found in GOS-100 eukaryotic sequences. Using the
public-100 data, we calculated the frequency of matches for each
Pfam model M within each kingdom, relative to the total number of
Pfam matches to that kingdom. Pseudocounts of one were added to
both the ‘‘match’’ and ‘‘no match’’ counts (i.e., using a uniform
Dirichlet prior), to allow proper statistical treatment of families with
few or no matches in the public databases for some kingdom. In
Equation 5 below, Obspublic(M,K) is the observed number of public-
0460 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Figure 17. Content of Protease Types in NCBI-nr and GOS, and Kingdom
Distribution of All Proteases
Due to the highly redundant nature of some NCBI-nr protease groups,
nonredundant sets for both NCBI-nr and GOS are computed; these
nonredundant sets are referred to as NCBI-nr60 and GOS60.
doi:10.1371/journal.pbio.0050016.g017
100 hits to M in K, and Obspublic(K) is the observed number of public-
100 hits to all models in K.
Obspublic ðM; KÞ þ 1
pGOSPfam ðMjKÞ ’ ppubPfam ðMjKÞ ¼ ð5Þ
Obspublic ðKÞ þ 2
By multiplying the conditional probability of each model given a
kingdom by the respective kingdom probability (pGOS-Pfam(K),
calculated as described above in ‘‘Kingdom annotation of GOS-100
proteins: kingdom weighting method’’), the proportions of Pfam
matches in GOS-100 due to each combination of kingdom and Pfam
model were then predicted. Finally, these predictions were summed
across kingdoms to obtain the expected proportion of matches to
each model.
pGOSPfam ðMÞ ¼ SUMðK ¼ fA; B; E; VgÞ½pGOSPfam ðMjKÞpGOSPfam ðKÞ
ð6Þ
Relatively fewer GOS-100 sequences than public-100 sequences
have a Pfam hit (likely because Pfam is based on sequences in the
public databases). To avoid systematically overestimating the number
of GOS-100 hits for each Pfam model due to this global effect, the
predicted counts were based on the observed total number of Pfam
matches to all models in GOS-100, and an attempt was made to predict
only how these matches are distributed among models. Thus, the
expected number of Pfam hits to a given model in GOS-100 is equal to
the expected proportion of hits to that model, as calculated above,
multiplied by the total number of Pfam hits. In the equation below,
ObsGOS is the total number of Pfam hits to all models in GOS-100.
Expected count of hits to M in GOS100 ¼ pGOSPfam ðMÞ 3 ObsGOS
ð7Þ
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Expanding the Protein Family Universe
In summary, calculation of the expected number of Pfam hits to a
model M in GOS-100 for all kingdoms can be expressed in one
equation as follows:
ðSUMðK 2 fA; B; E; VgÞ½ððObspublic ðM; KÞ þ 1Þ=ðObspublic ðKÞ þ 2ÞÞ ð8Þ
3 pGOSPfam ðKÞÞ 3 ObsGOS
where Obspublic(M,K) is the observed number of public-100 hits to
model M in K, Obspublic(K) is the observed number of public-100 hits
to all models in K, pGOS-Pfam(K) is the proportion of GOS-100
sequences that have at least one Pfam hit in K, and ObsGOS is the total
number of Pfam hits to all models in GOS-100.
The ratio of the observed to the predicted number of hits for each
Pfam model is a measure of the relative diversity of that Pfam family
in GOS-100 compared to public-100, corrected for the differing
kingdom proportions in the two datasets. We computed the
significance of this ratio using the CHITEST function in Excel, which
implements the standard Pearson’s Chi-square test with one degree of
freedom and expresses the result as a probability. For many protein
families, the difference in diversity between the two datasets was so
pronounced that Excel reports a probability of zero due to numerical
underflow, indicating a p-value less than 1 3 10303.
IDO analysis. The GOS-100 and public-100 sequences selected for
the IDO family alignment matched the PF01231 Pfam fs model with a
score above the trusted bit-score cutoff at the sequence level. In
addition, the sequences were required to have the width of their
matching region spanning over 50% of the Pfam IDO HMM model
length. Next, all sequence matches to the Pfam IDO model from the
NCBI-nr database downloaded on March 6, 2006, were added (these
also satisfied the trusted score cutoff and model alignment span
criteria). An additional 26 IDO sequences were found in the new
sequence database relative to the GOS public sequence data freeze
after filtering for identical and 1 aa different sequences and presence
of first and last residues in the final trimmed alignment. Jevtrace
(version 3.14) [149] was used to assess alignment quality, to remove
sequences problematic for alignment, to remove sequence redun-
dancy (at the 0-aa and 1-aa difference levels) while allowing for
redundant nonoverlapping sequences, to trim the alignment to a
block of aligned columns, to delete columns with more than 50%
gaps, and to remove sequences with missing first or last residues. One
sequence (GenBank ID 72038700) was likely a multidomain protein
problematic for alignment and was removed manually. This set of
procedures produced a block sequence alignment of 144 sequences
and 231 characters. We aligned sequences with MUSCLE (version
3.52) [134] using default parameters. The final alignment was used to
reconstruct phylogenies with a series of phylogeny reconstruction
methods: PHYML [150], Tree-Puzzle [151], Weighbor [152], and the
protpars program from the PHYLIP package (version 3.6a3) [145].
Bootstrapping was performed with the protpars program using 1,000
bootstrap replicates, each with 100 jumbles; the majority consensus
tree was produced by the consense program in the PHYLIP package.
Structural genomics implications. The Pfam5000 families used in
this study were chosen from among the manually curated (Pfam-A)
families in from Pfam version 17. We added 2,932 families with a
structurally characterized representative as of October 27, 2005, to
the Pfam5000 in descending order by family size, followed by 2,068
additional families without a structurally characterized representa-
tive, in descending order by family size. Pre-GOS family size was
calculated as the number of sequences in public-100 that had a match
to the Pfam family. Post-GOS family size was calculated as the
number of sequences in public-100 and GOS-100 that matched each
family. We used the results of the HMM profiling effort (using Pfams)
used for this analysis.
Coverage of GOS-100 and public-100 sequences by both versions
of the Pfam5000 was measured using the subset of families in Pfam 17
that were also in Pfam 16. This was done in order to enable direct
comparison of coverage results with a previous study of coverage of
fully sequenced bacterial and eukaryotic genomes [73]. The versions
of Pfam are similar in size (Pfam 16 contains 7,677 families, and Pfam
17 contains 7,868 families).
Phylogeny construction for various families. For the UVDE family,
sequences were aligned using MUSCLE [134] and a tree was built
using QuickTree [144].
For the PP2C family, the catalytic domain portions of the
sequences were identified and aligned using the PP2C Pfam model.
Sequences that contained 70% nongaps in this alignment were used
to generate a phylogenetic tree of all the PP2C-like sequences. The
phylogeny was inferred using the protdist and neighbor-joining
programs in PHYLIP [145]. We used 1,941 total PP2C-like sequences
for the phylogenetic analysis. The breakdown was as follows: public
0461 Special Section from March 2007 | Volume 5 | Issue 3 | e16
 Expanding the Protein Family Universe

Figure 18. Content of Bacterial Protease Clans

doi:10.1371/journal.pbio.0050016.g018

PLoS Biology | www.plosbiology.org | S86 0462 Special Section from March 2007 | Volume 5 | Issue 3 | e16

Table 15. Clustering Information for Ensembl Sequences for H.
sapiens, M. musculus, R. norvegicus, C. familiaris, and P.
troglodytes
Genome Number of Number of Number of
Sequences Sequences Clusters
from Ensembl in Clusters
H. sapiens 33,860 31,268 10,536
M. musculus 32,442 30,025 9,734
R. norvegicus 28,545 27,486 9,485
C. familiaris 30,308 29,041 9,397
P. troglodytes 38,822 34,697 9,978
doi:10.1371/journal.pbio.0050016.t015
eukaryotic sequences, 73%; public bacterial sequences, 14%; GOS-
eukaryotic sequences, 2%; GOS-bacterial sequences, 10%; and GOS-
viral and GOS-unknown sequences, less than 1% combined.
For the type II GS family, sequences in GOS and NCBI-nr were
searched with a type II GS HMM constructed from 17 previously
known bacterial and eukaryotic type II GS sequences. Matching
sequences from NCBI-nr and GOS were filtered separately for
redundancy at 98% identity; the combined set of sequences was
aligned and a neighbor-joining tree was constructed.
For the RuBisCO family, matching RuBisCO sequences from GOS
and NCBI-nr were filtered separately for redundancy at 90% identity,
resulting in 724 sequences in total. The 724 RuBisCO sequences were
then aligned and a neighbor-joining tree was constructed.
Identification of proteases. We clustered sequences in the MEROPS
Peptidase Database [100] using CD-HIT [116,117] at 40% similarity
level. This resulted in 7,081 sequences, which were then divided into
groups based on catalytic type and Clan identifier. These sequences
were used as queries to search against a clustered version of NCBI-nr
(clustered at 60% similarity threshold) using BLASTP (E-value  1 3
1010). A similar search was carried out against GOS (clustered at 60%
similarity threshold). Figure 17 shows the content of protease types in
NCBI-nr and GOS together with the kingdom distributions. Figure 18
shows the content of bacterial protease clans.
Metabolic enzymes in GOS. Hmmsearch from the HMMER
package [105] was used to search the GOS sequences for different
GS types. The GlnA TIGRFAM model was used for finding GSI
sequences. The HMMs built from known examples of 17 GSII and 18
GSIII sequences from NCBI-nr were used to search the GOS
sequences.
Identification of ORFans in NCBI-nr. ORFans are proteins that do
not have any recognizable homologs in known protein databases. A
straightforward way to identify ORFans is through all-against-all
sequence comparison using relaxed match parameters. However, this
is not computationally practical. An effective approach is to first
remove the non-ORFans that can be easily found, and then to identify
ORFans from the remaining sequences.
We identified non-ORFans by clustering the NCBI-nr with CD-HIT
[116,117], an ultrafast sequence clustering program. A multistep
iterated clustering was performed with a series of decreasing
similarity thresholds. NCBI-nr was first clustered to NCBI-nr90,
where sequences with .90% similarities were grouped. NCBI-nr90
was then clustered to NCBI-nr80/70/60/50 and finally NCBI-nr30.
After each clustering stage, the total number of clusters of NCBI-nr
was decreased and non-ORFans were identified. A one-step clustering
from NCBI-nr directly to NCBI-nr30 can be performed. However, the
multistep clustering is computationally more efficient.
At the 30% similarity level, all the NCBI-nr proteins were grouped
into 391,833 clusters, including 259,571 singleton clusters. The
proteins in nonsingleton clusters are by definition non-ORFans.
However, proteins that remain as singletons are not necessarily
ORFans, because their similarity to other proteins may not be
reported for two reasons: (1) significant sequence similarity can be
,30%; and (2) in order to prevent a cluster from being too diverse,
CD-HIT, like all other clustering algorithms, may not add a sequence
to that cluster even if the similarity between this sequence and a
sequence in that cluster meet the similarity threshold.
The 259,571 singletons were compared to NCBI-nr with BLASTP
[38] to identify real ORFans from them. The default low-complexity
PLoS Biology | www.plosbiology.org | S87
Expanding the Protein Family Universe
filter was enabled in the BLAST comparisons, and similarity thresh-
old in the form of an E-value was set to 1 3 106. In the end, 84,911
proteins with at least 100 aa are identified as ORFans. About 100,000
short ORFans less than 100 aa were removed from this study, because
they may not be real proteins.
Genome sequencing projects and rate of discovery. We used
Ensembl sequences for Homo sapiens, Mus musculus, Rattus norvegicus,
Canis familiaris, and Pan troglodytes. Their clustering information is
shown in Table 15. When we considered the datasets in the order HS,
HS þ MM, HS þ MM þ RN, HS þ MM þ RN þ CF, and HS þ MM þ RN þ
CF þ PT, the numbers of distinct clusters were 10,536, 12,731, 13,605,
14,606, and 14,993, respectively. These numbers were compared
against a random subset of NCBI-nr bacterial sequences (of a similar
size) and also against a random subset of GOS sequences. We also
randomized the order of the mammalian sequences to produce a
dataset that was independent of the genome order being considered.
Supporting Information
Protocol S1. Supplementary Information
Found at doi:10.1371/journal.pbio.0050016.sd001 (25 KB DOC).
Accession Numbers
All NCBI-nr sequences from February 10, 2005 were used in our
analysis. Protocol S1 lists the GenBank (http://www.ncbi.nlm.nih.gov/
Genbank) accession numbers of (1) the genomic sequences used in
the PG set, (2) the sequences used in building GS profiles, and (3) the
NCBI-nr sequences used in building the IDO phylogeny. The other
GenBank sequences discussed in this paper are Bacillus sp. NRRL B-
14911 (89089741), Janibacter sp. HTCC2649 (84385106), Erythrobacter
litoralis (84785911), and Nitrosococcus oceani (76881875). The Pfam
(http://pfam.cgb.ki.se) structures discussed in this paper are envelope
glycoprotein GP120 (PF00516), reverse transcriptase (PF00078),
retroviral aspartyl protease (PF00077), bacteriophage T4-like capsid
assembly protein (Gp20) (PF07230), major capsid protein Gp23
(PF07068), phage tail sheath protein (PF04984), IDO (PF01231),
poxvirus A22 protein family (PF04848), and PP2C (PF00481). The
glutamine synthetase TIGRFAM (http://www.tigr.org/TIGRFAMs) used
in the paper is GlnA: glutamine synthetase, type I (TIGR00653). The
PDB (http://www.rcsb.org/pdb) identifiers and the names of the eight
PDB ORFans with GOS matches are: restriction endonuclease MunI
(1D02), restriction endonuclease BglI (1DMU), restriction endonu-
clease BstYI (1SDO), restriction endonuclease HincII (1TX3); alpha-
glucosyltransferase (1Y8Z), hypothetical protein PA1492 (1T1J),
putative protein (1T6T), and hypothetical protein AF1548 (1Y88).
Acknowledgments
We are indebted to a large group of individuals and groups for
facilitating our sampling and analysis. We thank the governments of
Canada, Mexico, Honduras, Costa Rica, Panama, and Ecuador and
French Polynesia/France for facilitating sampling activities. All
sequencing data collected from waters of the above-named countries
remain part of the genetic patrimony of the country from which they
were obtained. We also acknowledge TimeLogic (Active Motif, Inc.)
and in particular Chris Hoover and Joe Salvatore for helping make
the DeCypher system available to us; the Department of Energy for
use of their NERSC Seaborg compute cluster; Marty Stout, Randy
Doering, Tyler Osgood, Scott Collins, and Marshall Peterson (J. Craig
Venter Institute) for help with the compute resources; Peter Davies
and Saul Kravitz (J. Craig Venter Institute) for help with data
accessibility issues; Kelvin Li and Nelson Axelrod (J. Craig Venter
Institute) for discussions on data formats; K. Eric Wommack
(University of Delaware, Newark) and the captain and crew of the
R/V Cape Henlopen for their assistance in field collection of
Chesapeake Bay virioplankton samples; John Glass (J. Craig Venter
Institute) for assistance with the collection and processing of the
virioplankton samples; Beth Hoyle and Laura Sheahan (J. Craig
Venter Institute) for help with paper editing; and Matthew LaPointe
and Jasmine Pollard (J. Craig Venter Institute) for help with figure
formatting. STM, MPJ, CvB, DAS, and SEB acknowledge Kasper
Hansen for statistical advice. We also acknowledge the reviewers for
their valuable comments.
Author contributions. SY contributed to the design and imple-
mentation of the clustering process, and the subsequent analyses of
the clusters; he also contributed to and coordinated all of the analyses
in the paper, and wrote a large portion of the paper. GS contributed
0463 Special Section from March 2007 | Volume 5 | Issue 3 | e16

to the design and analysis of the clustering process, contributed ideas,
analysis, and also wrote parts of the paper. DBR identified ORFs from
the assemblies, performed the all-against-all BLAST searches, con-
tributed to GOS kingdom assignment, and contributed analysis tools
and ideas. ALH performed the assembly of GOS sequences, and
contributed analysis tools and ideas. SW contributed to the analysis
of viral sequences. KR contributed to project planning and paper
writing. JAE performed the analysis of UV damage repair enzymes,
and also contributed to paper writing. KBH, RF, and RLS contributed
to project planning. GM performed the profile HMM searches,
carried out the domain analysis, and contributed to paper writing.
WL and AG carried out the ORFan analysis and contributed to paper
writing. LJ contributed to the profile-profile search process. PC and
AG carried out the analysis of proteases and contributed to paper
writing. CSM, HL, and DE carried out the analysis of novel clusters,
the analysis of metabolic enzymes and contributed to paper writing.
YZ contributed to the profile HMM searches and domain analysis.
STM, MPJ, CvB, DAS, and SEB carried out the analysis of Pfam
domain distributions in GOS and current proteins, analysis of IDO,
contributed to GOS kingdom assignment, and also contributed to
paper writing. DAS and SEB also contributed to the Ka/Ks test. JMC
and SEB carried out the analysis on the implications for structural
genomics and contributed to paper writing. SL, KN, SST, and JED
carried out the phosphatase analysis and contributed to paper
writing. SST and JED also contributed to project planning. BJR and
VB contributed to the analysis of cluster size distribution, family
discovery rate, and contributed to paper writing. MF contributed to
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Funding. The authors acknowledge the Department of Energy
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the Gordon and Betty Moore Foundation, the Discovery Channel and
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 PLoS BIOLOGY

Structural and Functional Diversity

of the Microbial Kinome
Natarajan Kannan1,2, Susan S. Taylor1,2, Yufeng Zhai3, J. Craig Venter4, Gerard Manning3*
1 Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America, 2 Howard Hughes Medical Institute, University
of California San Diego, La Jolla, California, United States of America, 3 Razavi-Newman Center for Bioinformatics, Salk Institute for Biological Studies, La Jolla, California,
United States of America, 4 J. Craig Venter Institute, Rockville, Maryland, United States of America

The eukaryotic protein kinase (ePK) domain mediates the majority of signaling and coordination of complex events in
eukaryotes. By contrast, most bacterial signaling is thought to occur through structurally unrelated histidine kinases,
though some ePK-like kinases (ELKs) and small molecule kinases are known in bacteria. Our analysis of the Global
Ocean Sampling (GOS) dataset reveals that ELKs are as prevalent as histidine kinases and may play an equally
important role in prokaryotic behavior. By combining GOS and public databases, we show that the ePK is just one
subset of a diverse superfamily of enzymes built on a common protein kinase–like (PKL) fold. We explored this huge
phylogenetic and functional space to cast light on the ancient evolution of this superfamily, its mechanistic core, and
the structural basis for its observed diversity. We cataloged 27,677 ePKs and 18,699 ELKs, and classified them into 20
highly distinct families whose known members suggest regulatory functions. GOS data more than tripled the count of
ELK sequences and enabled the discovery of novel families and classification and analysis of all ELKs. Comparison
between and within families revealed ten key residues that are highly conserved across families. However, all but one
of the ten residues has been eliminated in one family or another, indicating great functional plasticity. We show that
loss of a catalytic lysine in two families is compensated by distinct mechanisms both involving other key motifs. This
diverse superfamily serves as a model for further structural and functional analysis of enzyme evolution.
Citation: Kannan N, Taylor SS, Zhai Y, Venter JC, Manning G (2007) Structural and functional diversity of the microbial kinome. PLoS Biol 5(3): e17. doi:10.1371/journal.pbio.
0050017

sequence methods, but retain structural and mechanistic

Introduction
The eukaryotic protein kinase (ePK) domain is the most
abundant catalytic domain in eukaryotic genomes and medi-
ates the control of most cellular processes, by phosphorylation
of a significant fraction of cellular proteins [1–3]. Most
prokaryotic protein phosphorylation and signaling is thought
to occur through structurally distinct histidine-aspartate
kinases [4]. However, there is growing evidence for the existence
and importance of different families of ePK-like kinases (ELKs)
in prokaryotes [5–10]. ePKs and ELKs share the protein kinase–
like (PKL) fold [11] and similar catalytic mechanisms, but ELKs
generally display very low sequence identity (7%–17%) to ePKs
and to each other. Crystal structures of ELKs such as amino-
glycoside, choline, and Rio kinases reveal striking similarity to
ePKs [12–14], and other ELKs have been defined by remote
homology methods [6,15] and motif conservation [16]. Another
set of even more divergent PKL kinases are undetectable by
conservation with ePKs. These include the phosphatidyl
inositol kinases (PI3K) and related protein kinases, alpha
kinases, the slime mold actin fragmin kinases, and the
phosphatidyl inositol 59 kinases [17–20].
These studies demonstrate that PKL kinases conserve both
fold and catalytic mechanisms in the presence of tremendous
sequence variation, which allows for an equivalent diversity in
substrate binding and function. This makes the PKL fold a
model system to investigate how sequence variation maps to
functional specialization. Previous studies along these lines
include the study of ePK-specific regulatory mechanisms,
through ePK–ELK comparison [16], and the sequence
determinants of functional specificity within one group
(CMGC [CDK, MAPK, GSK3, and CLK kinases]) of ePKs [21].
Previous studies have been hampered by poor annotation
and classification of ELK families and their low representa-
tion in sequence databases relative to ePKs. Recent large-
Academic Editor: Tony Pawson, Samuel Lunenfeld Research Institute, Canada
Received May 18, 2006; Accepted September 20, 2006; Published March 13, 2007
Copyright: Ó 2007 Kannan et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: aa, amino acid; CAK, choline and aminoglycoside kinase; ChoK,
choline kinase; ELK, ePK-like kinase; ePK, eukaryotic protein kinase; GOS, Global
Ocean Sampling; HMM, hidden Markov model; NCBI, National Center of
Biotechnology Information; PKL, protein kinase–like
* To whom correspondence should be addressed. E-mail: manning@salk.edu
This article is part of Global Ocean Sampling collection in PLoS Biology. The full
collection is available online at http://collections.plos.org/plosbiology/gos-2007.
php.

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 Microbial Kinome

Author Summary

The huge growth in sequence databases allows the characterization of

every protein sequence by comparison with its relatives. Sequence
comparisons can reveal both the key conserved functional motifs that
define protein families and the variations specific to individual
subfamilies, thus decorating any protein sequence with its evolu-
tionary context. Inspired by the massive sequence trove from the
Global Ocean Survey project, the authors looked in depth at the
protein kinase–like (PKL) superfamily. Eukaryotic protein kinases
(ePKs) are the pre-eminent controllers of eukaryotic cell biology and
among the best studied of enzymes. By contrast, their prokaryotic
relatives are much more poorly known. The authors hoped to both
characterize and better understand these prokaryotic enzymes, and
also, by contrast, provide insight into the core mechanisms of the
eukaryotic protein kinases. The authors used remote homology
methods, and bootstrapped on their discoveries to detect more than
45,000 PKL sequences. These clustered into 20 major families, of which
the ePKs were just one. Ten residues are conserved between these
families: 6 were known to be important in catalysis, but four more—
including three highly conserved in ePKs—are still poorly understood,
despite their ancient conservation. Extensive family-specific features
were found, including the surprising loss of all but one of the ten key
residues in one family or another. The authors explored some of these
losses and found several cases in which changes in one key motif
substitute for changes in another, demonstrating the plasticity of
these sequences. Similar approaches can be used to better under-
stand any other family of protein sequences.

scale microbial genomic sequencing, coupled with Global

Ocean Sampling (GOS) metagenomic data, now allow a much
more comprehensive analysis of these families. In particular,
the GOS data provides more than 6 million new peptide
sequences, mostly from marine bacteria [22,23], and more
than triples the number of ELK sequences. Here, for the first
time, we define the extent of 20 known and novel PKL
families, define a set of ten key conserved residues within the
catalytic domain, and explore specific elaborations that
mediate the unique functions of distinct families. These
highlight both underappreciated aspects of the catalytic core
as well as unique family specific features, which in several
cases reveal correlated changes that map to concerted
variations in structure and mechanism.
Results
Discovery and Classification of PKL Kinase Families
Kinase sequences were detected using hidden Markov
model (HMM) profiles of known PKLs as well as with a motif
model focused on key conserved PKL motifs [16,24]. Results
of each approach were used to iteratively build, search, and
refine new sets of HMMs, using both public and GOS data.
Weak but significant sequence matches were used as seeds to
define and elaborate novel families. The final result was
16,248 GOS sequences (Dataset S1) classified into 20 HMM-
defined PKL families (Table 1; Figure 1; Dataset S2). A similar
analysis of the National Center of Biotechnology Information
nonredundant public database (NCBI-nr) revealed 24,924
ePK and 5,151 ELK sequences (Dataset S1). More than 1,400
of the NCBI ELK sequences were annotated as hypothetical
or unknown, and several hundred more are misannotated or
have no functional annotation. GOS data at least doubles the
size of most families, and permits an in-depth analysis of
Figure 1. Sequence and Structure Based Clustering of PKL Families
Despite minimal sequence similarity, relationships between families can
be estimated by profile–profile matching and alignments restricted to
conserved motifs. Three main clusters of families are seen (shaded ovals):
CAK, ePK, and KdoK. Four more families (towards bottom) are distantly
related to these clusters, while three more (PI3K, AlphaK, IDHK, at
bottom) have no sequence similarity outside a subset of key motifs. The
area of each sphere represents the family size within GOS data.
doi:10.1371/journal.pbio.0050017.g001
family structure and conservation. Two families that are more
than 10-fold enriched in GOS (CapK and HSK2) are found
largely in a proteobacteria, which are also highly enriched in
GOS. Both CapK and HRK contain viral-specific subfamilies
that are also greatly GOS enriched, indicating that differ-
ences in kinase distribution between databases are largely due
to taxonomic biases. As expected, eukaryotic-specific families,
(ePK, Bub1, PI3K, AlphaK) are underrepresented in GOS.
Functional Diversity of PKL Families
These 20 PKL families display great functional and
sequence diversity, though common sequence motifs and
functional themes recur. Some families are entirely unchar-
acterized, and few have been well studied, though most have
some characterized members, many with known kinase
activity. Their substrates include proteins and small mole-
cules such as lipids, sugars, and amino acids, and they
generally appear to have regulative functions (Table 1). This
is in contrast to the diversity of several other structurally
unrelated, small-molecule kinase families that play largely
metabolic roles [25]. Profile–profile alignments show clear but
distant relationships between several families, which are
enclosed by ovals in Figure 1. The ePK cluster includes pknB,
which is highly similar to but distinct from ePK and is

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 Microbial Kinome

Table 1. The 20 PKL Families, Their Gene Counts in GOS and NCBI-nr, and Functional Notes

Family GOS Count/NR Count Description

ePK 2753/24924 Eukaryotic protein kinase, almost exclusively eukaryotic.

pknB 1525/1047 Bacterial-specific, enriched in several phyla, closely related to ePK.
BLRK 21/28 Bacterial leucine-rich kinase, an uncharacterized ePK-like family containing leucine-rich repeats. Mostly restricted to
proteobacteria.
GLK 38/17 Glycosylase-linked kinase. Previously unannotated. Sequences from bacterial phyla fused to or neighbors of a DNA
glycosylase domain. Archaeal members are neighbors of tRNA-associated genes.
HRK 259/78 Haspin-related kinase. Consists of eukaryotic haspin protein kinases [55] and two distinct sets of largely viral kinases,
one of which lacks the GxGxxG and VAIK motifs, suggesting that they may be catalytically inactive and/or interfere with
host kinase signaling.
Bub1 9/112 Pan-eukaryotic protein kinase, functions in mitotic spindle assembly.
Bud32 139/123 Universal/single copy gene in eukaryotes and archaeae. In vertebrates it phosphorylates p53, while in S. cerevisiae it is
involved in bud site selection, both unconserved processes [56]. Recently implicated in telomere regulation [57].
Rio 133/249 Universal eukaryotic/archaeal protein kinase, implicated in control of translation, a function that is highly conserved
between eukaryotes and archaeae [58]. Also found in some bacteria, particularly proteobacteria.
KdoK 389/199 Small family of bacterial kinases known to phosphorylate sugar moieties of LPS [59]. Reportedly autophosphorylates on
tyrosine [60]. High sequence variation, even at key motifs, suggests diverse functions.
CAK 3997/1427 Choline and aminoglycoside kinases. Includes many novel subfamilies. Bacterial choline kinase (ChoK, licA), modifies
LPS, enabling mucosal binding for several human-commensal and pathogenic bacteria [61]. Expression is controlled by
phase variation. Metazoan choline kinases are involved in the production of phosphatidyl choline and acetylcholine,
and metazoans also have related ethanolamine kinases. Aminoglycoside kinases (Aminoglycoside phosphotransferases,
APH) phosphorylate and inactivate aminoglycosides, antibiotics that target the bacterial ribosome [62]. They are pro-
duced as antidotes by aminoglycoside-producing bacteria, and by many of their targets.
HSK2 1649/93 One of several structurally distinct homoserine kinases, involved in threonine biosynthesis. Found mostly in a-proteo-
bacteria, mirroring the distribution of HSK1 in c-proteobacteria. Unlike HSK1, it does not chromosomally cluster with
other threonine biosynthesis genes, but is usually linked to the lytB gene involved in isoprenoid biosynthesis, to RNAse
H1 and to clusters of novel genes (NK, GM, unpublished data). These suggest additional functions for this family.
FruK 390/136 Fructosamine kinase. Initiates repair of aging proteins by phosphorylating residues damaged by glycosylation, leading
to their repair [63]. Found in most eukaryotes and many bacteria, and may also have sugar kinase activities [64].
MTRK 144/38 MethylThioRibose kinase. Involved in a sulphur salvage pathway of methionine synthesis. Expression is controlled by
methionine levels in K. pneumonia [65] and by starvation in B. subtilis [66]. Present in select bacteria and plants, but
not in higher eukaryotes.
UbiB 4110/623 UbiB (ABC1 in eukaryotes). Regulates the ubiquinone (co-enzyme Q) biosynthesis pathway in both prokaryotes and
yeast [67,68]. It is speculated to activate an unknown mono-oxygenase in the ubiquinone biosynthesis pathway, possi-
bly in response to aerobic induction. Ubiquitous in eukaryotes and widespread in bacteria.
MalK 29/80 Maltose kinase. Contains two members shown biochemically to be maltose kinases [69]. Most public members are
annotated as trehalose synthases, based on transitive annotation from a member that is fused to trehalose synthase.
RevK 116/77 Reverse kinase. Novel family that lacks the N-terminal ATP-binding GxGxxG loop, but on the C-terminus is usually fused
a P-type ATPase domain, including an ATP-binding GxxGxG motif. No functional annotation.
CapK 308/21 Capsule kinase. Uncharacterized family. Chromosomal neighbors in two bacterial phyla involved in capsule synthesis. A
viral subset lacks obvious motifs upstream of the H164xD motif, reminiscent of the viral HRK subfamily.
PI3K 79/702 Eukaryotic PI39 and PI49 lipid kinases and associated PIKK protein kinases.
AlphaK 13/100 Eukaryotic protein kinases with diverse functions.
IDHK 111/58 Isocitrate dehydrogenase kinase (AceK). Small, highly conserved family. Activates the glyoxylate bypass in E. coli, used
for survival on acetate or fatty acids, by phosphorylating and inhibiting isocitrate dehydrogenase [70].

doi:10.1371/journal.pbio.0050017.t001

distinguished by its exclusive bacterial specificity, as opposed
to the mostly eukaryotic ePK family. The other major cluster
is centered on the large and divergent CAK (choline and
aminoglycoside kinase) family, and includes three other
families of small-molecule kinases. CAK itself is particularly
diverse, containing subfamilies that are specific for choline/
ethanolamine and aminoglycosides, as well as many novel
subfamilies, some of which are specific to eukaryotic
sublineages. A looser cluster is formed between the Rio and
Bud32 families, which are universal among both eukaryotes
and archaeae, and the bacterial lipopolysaccharide kinase
family KdoK. An additional four families (UbiB, revK, MalK,
CapK) are distantly related to all three clusters, and are
distinct from another set—PI3K, AlphaK, and IDHK—which
have even less similarity to any other kinase; for PI3K and
AlphaK, the relationship to kinases was determined by
structural comparisons [11], while IDHK displays only
conservation of the key residues and motifs found in all
PKL kinases.
Sequence similarity between these 20 families varies from
very low (;20%) to almost undetectable. Sequence-profile
methods are generally required to align families within the
oval clusters of Figure 1, while alignments between clusters
require profile–profile methods. The diversity of this collec-
tion is demonstrated by comparison with the automated
sequence- and profile-based clustering of the overall GOS
analysis [22], which assigns 93% of these sequences into 32
clusters, each of which is largely specific to one of our 20
families.
Key Conserved Residues Unify Diverse Kinase Families
Comparison between all families reveals a set of ten key
residues that not only account for one-third of the residues
conserved within each family, but also are consistently

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Figure 2. The Conserved Core and Variable Regions of the Catalytic Domain
The conserved core in three distinct families, namely ePK (PKA [52]), Rio (A. fulgidis Rio2 [14]), and CAK (APH(39)-IIIa [12]). The conserved regions are
shown in ribbon representation and the variable regions in surface representation. The illustrations were created in PyMOL (http://www.pymol.org).
Some highly conserved residues (see Figure 3) and their associated interactions are shown.
doi:10.1371/journal.pbio.0050017.g002
conserved between families, constituting a core pattern of
conservation that helps define this superfamily (Table 2,
Figure 2, Figure 3). These residues are conserved across the
major divisions of life, which diverged one to two billion years
ago, and across diverse families, which presumably diverged
even earlier. Thus, they are likely to mediate core functions of
the catalytic domain rather than merely maintaining their
structures. Six of these residues are known to be involved in
ATP and substrate binding and catalysis (G52, K72, E91, D166,
N171 D184; residues numbered based on PKA structure 1ATP
except where otherwise noted; see Table 3). The full functions
of the other four remain unclear, though three of them
(H158, H164, and D220) are part of a hydrogen-bonding
network that links the catalytically important DFG motif with
substrate binding regions (Figure 2). The conservation of this
network across diverse PKL structures suggested a role for
this network in coupling DFG motif-associated conforma-
tional changes with substrate binding and release [16].
Despite this ancient conservation, different families of ePKs
have lost individual members of this triad without destroying
structure or catalytic function: H164 is changed to a tyrosine
in PKA and many other AGC families; H158 is lost in most
tyrosine kinases; and D220 is lost in the Pim family. The Pim1
structure retains an ePK-like structure, perhaps in part due
to stabilization of the catalytic loop by the activation loop, a
function normally performed by D220 [26], suggesting a novel
mode of coupling ATP and substrate binding in this family.
The individual loss of each member of this triad suggests that
they have independent functions yet to be understood.
Sequence and Structural Diversity
Family-specific functions are mediated by features that are
highly conserved within families, but that are divergent
between families (Figure 4). Many family-selective residues
map to the motifs surrounding the ten key residues, or to the
divergent C-terminal substrate-binding region (Tables 2 and
S1). The proximity of these residues to the active site suggests
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that they are key in selecting substrates or tuning mechanism
of action. For instance, the 4–amino acid (aa) stretch between
the HxD166 and N171 residues is highly conserved but distinct
between families (Figure 4), and provides a discriminative
signature that defines each family. Within ePKs, tyrosine and
serine/threonine-specific kinases display distinct patterns of
conservation within this 4-aa stretch [27]. Serine/threonine
kinases conserve a [LI]KPx motif within this stretch, while
tyrosine kinases conserve a [LI]AAR motif. These variations
alter the surface electrostatics of the substrate-binding
pocket, thereby contributing to substrate specificity [27].
The C-terminal region of ;100 aa following the DFG motif
is highly divergent between families, apart from the con-
served D220 at the beginning of the F-helix (Figure 2; Dataset
S3). Secondary structure is generally predicted to be helical,
but the poor sequence conservation and known structures
[11] suggest that the overall orientation of the helices may be
different between families. Notably, in the crystal structures
of APH bound to its substrate, kanamycin [28], the relative
positioning of the substrate-binding helices (aH–aI) is
distinct from that of ePKs (Figure 2). The presence of unique
patterns of conservation in each family (Table 2) also suggests
that this region is involved in family-specific functions.
Several families contain sizeable (;30–100 aa) insert
segments between core subdomains that are specific to
clusters of families. Most CAK members have an insert
segment between subdomains VIa and VIb. There is very little
sequence similarity within this segment across CAK members,
but structures of APH and ChoK indicate some structural
similarity and highlight its role in substrate binding [28,29].
An equivalent insert is seen in the other CAK cluster families,
FruK, HSK2, and MTRK. Similarly, KdoK and Rio contain an
insert between subdomains II and III, which shows some
sequence similarity between these families. In the Rio2
structure, this insert is disordered, but the presence of a
conserved threonine suggests a possible regulatory role [14].
This region also contains an insert in the distinct UbiB family.
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 Microbial Kinome

Figure 3. Conservation of Secondary Structure, Key Motifs, and Residues between Families
The ePK secondary structure is shown with standard annotations of subdomains [53] and structural elements. Subdomains I–IX are generally conserved
in all PKLs. Key residues are bolded and numbered; dashed lines point to positions within secondary structure elements. The table below shows the
conservation (% identity) of the ten key residues, showing their broad conservation across families, but the successful replacement of almost all of them
in at least one family. Parentheses indicate changes to another conserved residue and dashes indicate unconserved positions. Key residues are
numbered based on their position in PKA: G52, K72, E91, P104 (VPKA), H158, H164 (YPKA), D166, N171, D184, and D220. More detailed figures are shown
in Dataset S3.
doi:10.1371/journal.pbio.0050017.g003

Finally, the ePK, pknB, and HRK families contain an
extended activation loop between subdomains VIII and IX.
These kinases are generally activated by phosphorylation of
this loop, the negative charge of which helps to coordinate
key structural elements during the activation process,
including a family-selective HRD arginine in the catalytic
loop [30,31].
Mechanistic Diversity of the Catalytic Core
A surprising finding was that while ten key residues are
conserved both within and between families, all but one of
them was dispensable in one family or another (Figure 3),
indicating that even catalytic residues are malleable in the
appropriate context. Here we explore the effect of loss of the
‘‘catalytic lysine’’ K72, which typically positions the a and b
phosphates of ATP (Figure 5A). Mutation of this lysine in ePKs
is a common method to make inactive kinases [32]. Yet this
residue is conserved as an arginine (R111ChoK) in most CAK
subfamilies, as a methionine in the CAK-chloro subfamily, and
as a threonine in the related HSK2 family (Figure 4).
In the two major CAK subfamilies with a conserved R72
(FadE and choline kinase [ChoK]), we see correlated changes in
the glycine-rich and DFG motifs (Figure 4). Specifically, the Phe
and Gly within the GxGxFG motif (F54 and G55) are changed to
Ser/Thr and Asn, respectively (S86ChoK, N87ChoK), and G186
within the DFG motif is changed to E. Both the GxGxFG and
DFG motifs are spatially proximal to K72 (Figure 5A). Thus,
correlated changes in these two motifs could structurally
account for the K-to-R change. Indeed, in the ChoK crystal
structure [13], N55 protrudes into the ATP binding pocket, and
hydrogen bonds to R72. In addition, the conserved E91 in helix
C, which typically forms a salt bridge with K72, is hydrogen
bonded (via a water molecule) to the covarying E186, thus
linking these three correlated changes and stabilizing R72 in a
unique conformation (Figure 5B). By contrast, the two solved
APH structures (1ND4 and 2BKK) retain the ‘‘ancestral’’
sequence state with K72 and G186, and lack N55.
Mutation of R72 or E186 to alanine in ChoK reduces the
catalytic rate by several fold [33]. To test the possible role of
these residues in the ChoK catalytic mechanism, we modeled
an ATP in the active site of ChoK (based on the nucleotide-
bound structures of APH and PKA). This revealed that R72
partially occludes the ATP binding site and is likely to move
upon ATP binding. Notably, a K72-to-R mutation in Erk2 [34]
also exhibits a conformational change in R72 upon nucleotide

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 Microbial Kinome

Table 2. Distribution of Residues That Are .90% Identical within Each Family

Family Key Residues Motif-Associated C-Term Unique Semiconserved Other Total

ePK 8 4 1 4 0 17
pknB 9 3 0 8 0 20
BLRK 8 12 4 4 18 46
HRK 5 2 0 1 0 8
Bub1 8 7 3 5 4 27
GLK 7 1 2 2 0 12
Bud32 10 6 3 2 2 23
Rio 9 4 0 1 0 14
KdoK 2 0 0 1 0 3
CAK 5 0 0 0 0 5
HSK2 9 9 11 7 4 40
FruK 9 6 5 5 2 27
MTRK 9 10 4 2 8 33
UbiB 8 6 0 1 4 19
MalK 8 13 15 0 10 46
revK 5 1 2 0 5 13
CapK 1 0 0 0 0 1
PI3K 4 3 1 1 2 11
AlphaK 5 4 6 1 7 23
IDHK 9 17 10 1 23 60
Total 138 (31%) 108 (24%) 67 (15%) 46 (10%) 89 (20%) 448 (100%)

Across the ;250-aa domain, almost one-third of .90% conserved residues map to the ten key residues that are also conserved between families, and more than half map to these key
residues or their surrounding motifs (GxGxxGxxxx, vaiK, E, vP, LxxLH, xxHxDxxxNxx, xxDxGxx, DLA; boldfacing indicates the ten key residues). An additional 15% are found in the largely
unalignable region C-terminal of DxG, strongly suggesting family-specific functions, and 10% are semi-conserved, being found in some, but not most families. See Dataset S3 for details.
doi:10.1371/journal.pbio.0050017.t002

binding (Figure 5C). A similar conformational change in ChoK
upon ATP binding could result in formation of a R72–E91 salt
bridge similar to the activation of ePKs (Figure 5A). In this
conformation, R72 could potentially hydrogen bond to both
E91 as well as to the covarying E186 in ChoKs, which might
explain the covariation of R72 and E186 in these families.
Variation on a Theme
Other CAK members display distinct coordinated changes
at the G55, K72, and G186 positions. The chloro subfamily of
CAK loses the positive charge at position 72 altogether,
replacing it with methionine, and has concurrent changes to
R55 and Q186 (Figure 4). This may reflect a shift of the
positive charge from position 72 to 55, an event that also
happened in Wnk kinases, the only functional ePK family that
lacks K72. The conserved K55 of Wnks is required for
catalysis and has been shown to interact with ATP similarly to
K72 of PKA [35] (Figure 5D). Hence, two evolutionary
inventions may have converted the same core motif residue
from one function to another. In CAK-chloro, the unpaired
E91 position loses its charge to become a conserved Phe. The
function of this Phe is unknown, but is likely to be important
since it is also conserved in HSK2, a related family, and the
only other kinase family to conserve a Phe at the E91 position
(Figure 4).
Evolution of Conformational Flexibility and Regulation in
ePKs
The ePK catalytic domain is highly flexible and undergoes
extensive conformational changes upon ATP binding [36]. In
contrast, crystal structures of APH, solved in both ATP-
bound and -unbound forms, revealed modest structural
changes in the ATP-binding pocket [37]. This difference in
conformational flexibility is reflected in the patterns of
conservation at key positions within the ATP-binding glycine-
rich loop (Figure 4). Specifically, two conserved glycines (G50
and G55), which contribute to the conformational flexibility
of this loop in ePKs, are replaced by non-glycines in APH.
These two glycines are absent in several PKL families (Figure
4) while G52, which is involved in catalysis, is present in most,
suggesting that the conformational flexibility of the nucleo-
tide-binding loop is a feature of selected PKL families such as
ePKs. Since conformational flexibility allows for regulation, it
is likely that modest structural changes associated with
nucleotide binding gradually evolved into quite dramatic
structural rearrangements required to ensure that key players
in various signaling pathways act only at the right place and at
the right time. The conserved glycine (G186) within the
catalytically important DFG motif may likewise have evolved
for regulatory functions in ePKs [38]. This glycine is highly
conserved in the ePK cluster but is absent from most other

Figure 4. Sequence Logos Depicting Conservation of Core Motifs and Neighboring Sequences across Most Kinase Families and Selected CAK
Subfamilies
Motifs are GxGxxGxxxx, VAIK, E, LxxLH, xxHxDxxxxNxx, xxDFGxx, and Dxx. The size of the letters corresponds to their information content [54]. Families
with less than 100 members (BLRK, GLK) are omitted. The diverse CAK family is represented by four distinct subfamilies: APH contains many
aminoglycoside resistance kinases and ChoK includes most ChoKs, while FadE and chloro are less well described. For the HRK family, the first two motif
logos omit the viral subfamily that lacks these motifs.
doi:10.1371/journal.pbio.0050017.g004

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 Microbial Kinome

Table 3. Structural/Functional Role of Highly Conserved Residues

Residue (PKA Number) Structural Location Structural/Functional Role

G52 Glycine-rich loop The backbone of this glycine coordinates the c-phosphate of ATP and facilitates
phosphoryl transfer [71].
K72 b3 strand Hydrogen bonds to the a-oxygen and b phosphate of ATP [72].
E91 C-helix Forms a salt bridge interaction with K72 and functions as a regulatory switch in
ePKs [73].
P104 aC-b4 loop Unknown function. Absent from ePKs.
H158 C-terminus of E helix Hydrogen bonds to D220 in the F-helix and is part of a hydrogen bond network
that couples ATP and substrate-binding regions [16].
H(Y164) Catalytic loop aE-b6 Hydrogen bonds to the backbone of the residue before the DFG-Asp and inte-
grates substrate and ATP binding regions [16].
D166 Catalytic loop Catalytic base [72].
N171 Catalytic loop Coordinates the second Mg2þ ion and involved in phosphoryl transfer.
D184 N-terminus of activation loop Coordinates the first Mg2þ ion.
D220 N-terminus of F helix Hydrogen bonds to the backbone of the catalytic loop and positions this loop re-
lative to substrate-binding regions [16].

doi:10.1371/journal.pbio.0050017.t003

Figure 5. Mechanistic Diversity of the ATP-Binding Pocket.

(A) PKA showing structural interactions associated with K72 in active ATP-bound state. The salt bridge interaction between K72 and E91 is shown by
dotted lines.
(B) Structural interactions associated with Arg111ChoK in ChoK.
(C) Conformational changes associated with Arg52Erk2 in the Erk2 mutant structure. Here, the arginine does not form a salt bridge interaction with
Glu69Erk2 (E91), but moves closer towards Glu69Erk2 upon ATP binding.
(D) Inactive state of Wnk1: K72 is shifted over to the G-loop (K233Wnk1) and E91 (Glu268Wnk1) hydrogen bonds to a conserved Arg (R348Wnk1 within the
HRD motif) in the catalytic loop.
(A–D) Residues conserved across all the major families are colored in magenta, while family-specific residues are colored in gold. Hydrogen bonds are
indicated in dotted lines.
doi:10.1371/journal.pbio.0050017.g005

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Figure 6. ePK-Specific Motifs and Interactions in the Substrate-Binding Region
(A) The ePK-specific activation loop and G-helix are shown in PKA (PKA [52]). The corresponding regions are shown in Rio (A. fulgidis Rio2 [14]). The
activation loop and G-helix are colored in red, and the core-conserved residues are shown in stick representation.
(B) The three ePK-specific motifs in the C-terminal substrate-binding lobe and their structural interactions are shown. Hydrogen bonds are indicated by
dotted lines. The conserved buried water is shown in CPK representation.
doi:10.1371/journal.pbio.0050017.g006
PKL families. However, within the small subfamily of
magnesium-dependent Mnk ePK kinases, G185 is changed
to aspartate (DFD). In the Mnk2 crystal structure, this DFD
motif adopts an ‘‘out’’ conformation in which F185 protrudes
into the ATP-binding site. This is in contrast to the ‘‘in’’
conformation, where it packs up below the C-helix [39].
Mutation of the Mnk2 D186 ‘‘back’’ to glycine results in both
in and out conformations of the DFG motif, supporting the
role of G186 in DFG-associated conformational changes.
Such conformational transitions may facilitate regulation of
activity since the conformation of the catalytic aspartate is
also changed during this transition [38]. This may also explain
why the ePK-specific extended activation loop, which is
phosphorylated and undergoes dramatic conformational
changes, is directly attached to the DFG motif (Figure 6A).
In addition to the flexible catalytic core, the substrate-
binding regions appear to have evolved for tight regulation of
ePK activity. In particular, the conserved G helix, which was
recently shown to undergo a conformational changes upon
substrate binding [40], is uniquely oriented in ePK/pknB
(Figure 6A). Several ePK-conserved residues and motifs are at
the interface between the G helix and the catalytic core
(Figure 6B). These include the APE motif, located at the C-
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Microbial Kinome
terminal end of the activation loop, a W-[SA]-X-[G] motif in
the F-helix, and an arginine (R280), at the beginning of the I
helix (Figure 6B). These three motifs structurally interact with
each other and form a network that couples the substrate-
and ATP-binding regions (Figure 6B). This network also
involves conserved buried water molecules, which are known
to contribute to the conformational flexibility of proteins
[41]. Thus, this ePK/pknB-conserved network may also
facilitate regulation by increasing the conformational flexi-
bility of the substrate-binding regions [16].
Discussion
Data from the GOS voyage provides a huge increase in
available sequences for most prokaryotic gene families,
enabling new studies in discovery, classification, and evolu-
tionary and structural analysis of a wide array of gene
families. Even for a eukaryotic family such as ePK kinases,
GOS provides insights by greatly increasing understanding of
related PKL families. GOS increases the number of known
ELK sequences more than 3-fold, and has enabled both the
discovery of novel families of kinases as well as a detailed
analysis of conservation patterns and subfamilies within
known families. We believe that the GOS data, coupled with
Special Section from March 2007 | Volume 5 | Issue 3 | e17

the recent strong growth in whole-genome sequencing,
provide the opportunity for similar insights into virtually
every gene family with prokaryotic relatives.
PKL kinases are largely involved in regulatory functions, as
opposed to the metabolic activities of other kinases with
different folds [25]. The characteristics of this fold that lead
to the explosion of diverse regulatory functions of eukaryotic
ePKs have also been exploited for many different functions
within prokaryotes. While these kinases reflect only ;0.25%
of genes in both GOS and microbial genomes (ePKs represent
;2% of eukaryotic genes [42]), indicating a simpler prokary-
otic lifestyle, they now outnumber the count of ;12,000
histidine kinases that we observe in GOS [22], suggesting that
ELKs may be at least as important in bacterial cellular
regulation as the ‘‘canonical’’ histidine kinases.
PKL kinases cross huge phylogenetic and functional spaces
while still retaining a common fold and biochemical function
of ATP-dependent phosphorylation. The presence of Rio and
Bud32 genes in all eukaryotic and archaeal genomes suggests
that at least this cluster dates back to the common ancestor of
these domains of life. Similarly, the presence of UbiB in all
eukaryotes and most bacterial groups, the close similarity of
pknB/ePK families, and the widespread bacterial/eukaryotic
distribution of FruK suggest their origins before the
emergence of eukaryotes, or from an early horizontal
transfer. Their ancient divergence leaves little or no trace
of their shared structure within their protein sequence other
than at functional motifs, which include a set of ten key
residues that are highly conserved across all PKLs.
Despite the huge attention paid to ePKs, four key residues
(P104, H158, H164, D220), three of which are highly conserved in
ePKs, are still functionally obscure and worthy of greater
attention, both in ELKs and ePKs. Conversely, it appears that
nine of the ten key residues have been eliminated or
transformed in individual families while maintaining fold
and function, showing that almost anything is malleable in
evolution given the right context. That right context is
frequently a set of additional changes in the family-specific
motifs surrounding these key residues, and we see that in the
case of K72, a substitution to arginine triggers a cascade of
other core substitutions that serve to retain basic function,
while a substitution to methionine involves a shift of the
positive charge normally provided by K72 to another
conserved residue, in both CAK-chloro and Wnk kinases.
Other core changes are also seen independently in very
distinct families, such as the G55-to-A change in UbiB and the
chloro subfamily of CAK, or the E91-to-F change in both
chloro and HSK2, suggesting that these kinases are sampling a
limited space of functional replacements.
These families vary greatly in diversity. While the ePK
family has expanded to scores of deeply conserved functions
[42], other families, including Bud32, Rio, Bub1, and UbiB,
usually have just one or a handful of members per genome,
suggesting critical function but an inability to innovate. The
largely prokaryotic CAK family is also functionally and
structurally diverse, containing several known functions and
many distinct subfamilies likely to have novel functions. The
diversity of both CAK and KdoK sequences may be related to
their involvement in antibiotic resistance and immune
evasion, likely to be evolutionarily accelerated processes.
Comparison of CAK to the related and more functionally
constrained HSK2, FruK, and MTRK families may reveal
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Microbial Kinome
adaptive changes such as the ePK-specific flexibility changes
that may assist in its diversity of functions.
GOS data are rich in highly divergent viral sequences, and
accordingly we find a number of new subfamilies of viral kinases,
including two of the three subfamilies of HRK and a subfamily
of CapK. In both cases we see loss of N-terminal–conserved
elements, suggesting that these kinases may have alternative
functions or even act as inactive competitors to host kinases.
These patterns of sequence conservation and diversity raise
many questions that can only be fully addressed by structural
methods. The combination of structural and phylogenetic
insights for ChoK enabled insights that were not clear from
the structure alone, and enabled us to reject other inferences
from the crystal structure that were not conserved within this
family, highlighting the value of combining these approaches.
The relative ease of crystallization of PKL domains, the
emergence of high-throughput structural genomics, and our
understanding of the diversity of these families make them
attractive targets for structure determination of selected
members, and position this family as a model for analysis of
deep structural and functional evolution.
Materials and Methods
Discovery and classification of kinase genes. Sequences used
consisted of 17,422,766 open reading frames from GOS, 3,049,695
predicted open reading frames from prokaryotic genomes, and
2,317,995 protein sequences from NCBI-nr of February 10, 2005, as
described [22]. Profile HMM searches were performed with a Time
Logic Decypher system (Active Motif, http://timelogic.com) using in-
house profiles for ePK, Haspin, Bub1, Bud32, Rio, ABC1 (UbiB), PI3K,
and AlphaK domains, as well as Pfam profiles [43] for ChoK, APH,
KdoK, and FruK, and TIGRFAM profiles [44] for HSK2 (thrB_alt),
UbiB, and MTRK. A number (69) of additional ePK-annotated models
from Superfamily 1.67 [45] were used to capture initial hits but not for
further classification. Initial hits were clustered and re-run against all
models, and each model was rebuilt and rerun three to seven times
using ClustalW [46], MUSCLE [47], and hmmalign (http://hmmer.
janelia.org) to align, followed by manual adjustment of alignments
using Clustal and Pfaat [48] and model building with hmmbuild. Low-
scoring members of each family (e . 1 3 105) were used as seeds to
build new putative families, and profile–profile and sequence–profile
alignments were used to merge families into a minimal set (Dataset S2).
A motif-based Markov chain Monte Carlo multiple alignment model
[49] based on the conserved motifs of Figure 3 was run independently
and used to verify HMM hits and seed new potential families for blast-
based clustering, model building, and examination for conserved
residues. Final family assignment was by scoring against the set of HMM
models, with manual examination of sequences with borderline scores
(e . 1 3 105 or difference in e-values between best two models ..01).
Family annotations. Annotations of chromosomal neighbors used
SMART [50] and a custom analysis of GOS neighbors ([22]; C. Miller,
H. Li, D. Eisenberg, unpublished data). Annotation analysis was based
on GenBank annotations and PubMed references. Taxonomic
analysis used a mapping of GOS scaffolds to taxonomic groupings
[22] and NCBI taxonomy tools.
Family alignments and logos. Residue conservation (Dataset S3)
was counted from the final alignment using a custom script that
omitted gap counts. These counts were then used to construct family
logos using WebLogo (http://weblogo.berkeley.edu; [51]).
Family comparisons. Relatedness between families was estimated
using several methods. HMM–HMM alignments and scores were
computed using PRC (http://supfam.org/PRC), and sequence–profile
alignments using hmmalign were analyzed using custom scripts and
by inspection. Both full-length and motif multiple alignments were
also created and used for the family comparisons.
Supporting Information
Dataset S1. FastA-Formatted Sequence Files for Each of the 20 Kinase
Families, Including Both GOS and Public Sequences
Found at doi:10.1371/journal.pbio.0050017.sd001 (10 MB BZ2).
0476 Special Section from March 2007 | Volume 5 | Issue 3 | e17

Dataset S2. HMM Profiles for the 20 Kinase Families in HMMer
Format
Found at doi:10.1371/journal.pbio.0050017.sd002 (2.4 MB HMM).
Dataset S3. Domain Profiles for 20 PKL Families
These 20 spreadsheets show the conservation profile at each residue
of the kinase domain for each family, including annotations and
classifications of individual residues. Each worksheet details the
alignment of one kinase family to its HMM. Every row corresponds to
a position within the alignment, listing the four most common amino
acids (aa) in that row along with their fractional popularity. The
number of aa’s and number of gaps at that position within the
alignment is also listed. The ‘‘Notes’’ column annotates conservation
status of selected residues and other notes, while the ‘‘.90%
Conserved’’ annotates those corresponding residues as to their class
(Core, Motif, Motif-Associated, Semi-Conserved, C-terminal, Unique,
or external to the kinase domain). A number of color highlights are
used. (1) Positions with few aa’s in the alignment (typically inserts
within the domain that are not of great interest) are shaded gray:
typically dark gray for 20 aa at that position, and light gray for .20
but still low (the range varies depending on the depth of the
alignment). Rows highlighted in gray have no highlights in any other
columns and are assumed not to be part of the core domain. (2) Core
motifs are highlighted in bold and blue. (3) The fractional count for
the most popular aa is labeled green if 1, dark yellow if .0.9, and light
yellow if .0.8 and ,0.9.
Found at doi:10.1371/journal.pbio.0050017.sd003 (1.6 MB XLS).
Accession Numbers
The Protein Databank (http://www.pdb.org) accession numbers for
the structures discussed in this paper are PKA (1ATP), A. fulgidis Rio2
(1TQP), C. elegans choline kinase (INW1), Erk2 (1GOL), Wnk1 (1T4H),
and APH(39)-IIIa (1J7L). The Pfam (http://pfam.cgb.ki.se) accession
numbers for the structures discussed in this paper are ChoK
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(PF01633.8), APH (PF01636.9), KdoK (PF06293.3), and FruK
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Acknowledgments
We thank the Governments of Bermuda, Canada, Mexico, Honduras,
Costa Rica, Panama, Ecuador, and French Polynesia for facilitating
sampling activities. All sequencing data collected from waters of the
above-named countries remain part of the genetic patrimony of the
country from which they were obtained. We thank Chris Miller,
Huiying Li, and David Eisenberg for access to analyses of chromo-
somal neighbors for function prediction, and to Doug Rusch, Shibu
Yooseph, and other members of the Venter Institute GOS team for
taxonomic predictions, geographic analysis, and other data and tools.
We thank Eric Scheeff and Tony Hunter for critical comments and
structural insights, and Nina Haste for help with PyMOL.
Author contributions. JCV proposed and enabled collaboration.
SST conceived of collaboration and provided structural insights and
critical evaluation. NK and GM conceived and designed the experi-
ments. NK, YZ, and GM performed the experiments. NK and GM
analyzed the data. YZ and JCV contributed reagents/materials/analysis
tools. NK and GM wrote the paper.
Funding. This work was supported by funding from the Razavi-
Newman Center for Bioinformatics to GM and National Institutes of
Health grant IP01DK54441 to SST. We gratefully acknowledge the US
Department of Energy (DOE) Genomics: GTL Program, Office of
Science (DE-FG02-02ER63453), the Gordon and Betty Moore Foun-
dation, and the J. Craig Venter Science Foundation for funding of the
GOS expedition.
Competing interests. The authors have declared that no competing
interests exist.
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