Microbial

Ecology

Metaproteomics: A New Approach for Studying Functional

Microbial Ecology
Pierre-Alain Maron, Lionel Ranjard, Christophe Mougel and Philippe Lemanceau
UMR Microbiologie et Géochimie des Sols, INRA/Université de Bourgogne, CMSE, BP 86510, 17 rue de Sully, 21065, Dijon Cedex, France

Received: 17 November 2006 / Accepted: 26 November 2006 / Online publication: 13 March 2007

Abstract be an integrative science with strong interconnections

between systematics, genetics, biochemistry, molecular
In the postgenomic era, there is a clear recognition of the
biology, physiology, modeling, paleobiology, soil science,
limitations of nucleic acid-based methods for getting
parasitology, epidemiology, etc., with important food,
information on functions expressed by microbial com-
public health, and environmental implications. Microbial
munities in situ. In this context, the large-scale study of
ecology can be considered apart from Bclassical^ ecology
proteins expressed by indigenous microbial communities
by the specifics of the organisms involved. The small size
(metaproteome) should provide information to gain
of microorganisms, the difficulty defining bacterial
insights into the functioning of the microbial component
species and the huge genetic/metabolic diversity among
in ecosystems. Characterization of the metaproteome is
them in the various environments they colonize [50] led
expected to provide data linking genetic and functional
to the development of specific concepts and methodo-
diversity of microbial communities. Studies on the
logical approaches for elucidating the role of microbes in
metaproteome together with those on the metagenome
ecosystem functioning. Analysis of historical and recent
and the metatranscriptome will contribute to progress in
advances in microbial ecology shows a Bstep-by-step^
our knowledge of microbial communities and their
evolution managed by methodological developments
contribution in ecosystem functioning. Effectiveness of
([8], Fig. 1).
the metaproteomic approach will be improved as
In the 1960s, the most comprehensive studies
increasing metagenomic information is made available
focused on monoxenic cultures lacking interactions
thanks to the environmental sequencing projects cur-
between microorganisms and between microorganisms
rently running. More specifically, analysis of metapro-
and their habitat. In the 1980s, one of the first advances
teome in contrasted environmental situations should
was to take into consideration not only single organisms
allow (1) tracking new functional genes and metabolic
but density, diversity, and activity of microbial popula-
pathways and (2) identifying proteins preferentially
tions isolated from natural environments. This was
associated with specific stresses. These proteins consid-
initiated by Brock (1987) [5] who stated that the
ered as functional bioindicators should contribute, in the
properties of an organism cultivated in the laboratory
future, to help policy makers in defining strategies for
may not necessary reflect its activity and physiology in
sustainable management of our environment.
the environment where factors such as resource com-
petition, environmental heterogeneity, predation, and
other interactions are prevalent. In the 1990s, many
Past, Present, and Future Prospects studies were dedicated to this type of approach and
provided the basis for understanding the microbial
Microbial ecology is a scientific domain derived (40 years
world and its role in ecosystem functioning. However,
ago) from medicine and agronomy by the need to
the statements made by Bakken (1985) [3] and Amann
elucidate relationships between microbes and their
et al. (1995) [1] that more than 90% of the micro-
natural habitats (soil, water, sediments, rhizosphere,
organisms in the environment are not cultivable
alimentary canal...). Soil microbial ecology is known to
highlighted the limit of culture-dependent approaches
to describe natural diversity and that most of the
Correspondence to: Philippe Lemanceau; E-mail: lemanceau@dijon.inra.fr microbial diversity remained unexplored. Faced with

486 DOI: 10.1007/s00248-006-9196-8 & Volume 53, 486–493 (2007) & * Springer Science + Business Media, LLC 2007
P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY 487

First symposium of First manual of First scientific journals

microbial ecology microbial ecology Appl Environ Microbiol.
Brock and
Microbiol. Ecol. ‘omic era’

1st ISME
time
1957 1966 1970 1974-76 1980 1990 1998 nowadays

Integration level of studies in microbial ecology

Individual level
(monoxenic culture)

Population level
Community level
Link between genetic and functional diversity

Methodological developments
Development of culture media for isolating microbial organisms

2-D gel Mass Bio

electrophoresis PCR Spectrometry informatic Genomic
Development of molecular
biology and biochemistry DNA-SIP
Metagenomic
Development of methodologies to extract, amplify,
clone and sequence DNA from microbial communities
Metatranscriptomic
Development of methodologies to extract, amplify
and sequence RNA from microbial communities
Metaproteomic
Development of methodologies to extract and
characterize proteins from microbial communities
Figure 1. Historical and step-by-step evolution of microbial ecology.

this major limitation, Pace et al. (1985) [34] introduced
a cultivation-independent approach based on the ex-
traction, amplification, cloning, and characterization of
rDNA genes directly from natural environments.
Beginning with these early works, many efforts have
been dedicated to developing molecular methods to
characterize microbial information contained in the nucleic
acids extracted from environmental samples [1, 20]. These
developments enabled the characterization of variations of
the microbial community structure and diversity in mul-
tiple situations allowing the identification of populations
preferentially associated with environmental perturbations
(for review, see [44]). Further methodological progress
allowed the cloning and sequencing of large genome frag-
ments (about 40 kb) from microbial communities of a
planktonic marine archaeon [49]. This work provided the
first glimpse into content and diversity of marine archae
but was also the first example of the feasibility of meta-
genome characterization [collective genome from all
(micro-) organisms present in an ecosystem] [46]. Recent
advances in high-throughput screening and sequencing
facilitate this type of study and have provided the majority
of DNA sequences now found in databases. Metagenomic
approaches provide new insights into genetic diversity and
evolution of uncultured microorganisms. However, indi-
cations of genetic potential does not contribute to the
elucidation of the functionality (level of expression of the
genetic potential) (Fig. 2) of microbial communities in
ecosystems [8, 45, 50, 52].
Different methods have been developed to discrimi-
nate active populations from quiescent ones in natural
habitats by incorporation of labeled markers in microbial
biomass such as 13C (DNA-Stable isotope probing meth-
ods [41]), or BrdU [4]. However, these approaches only
provide limited information on the populations associated
with a specific process rather than a complete description
of their functional role within a Bcommunity^. Bioinfor-
matic data acquired over the last 20 years on putative
functional genes allows the design of primers and probes
to target specific functional communities in complex
environments. The design of such primers enables
(1) the quantification of gene copy number [real time
polymerase chain reaction (PCR)] and (2) the character-
ization of polymorphism(s) of functional gene(s). These
studies performed at the DNA level can be further linked
to measurement of the corresponding activities [27].
However, this integrated approach is restricted to a
limited number of functions (denitrification, nitrification,
488 P.-A. MARON ET AL.:
Figure 2. Schematic representation of the FMeta_ levels in the ecology of microbial communities.
and methane oxidation) for which genes involved in each
step of the metabolic pathway are known and sufficiently
conserved to allow the design of consensus primers.
Furthermore, the presence of a gene within populations
of a given Bfunctional community^ does not necessarily
mean that it is expressed in the habitat.
In the postgenomic era, a major challenge is to
elucidate the functional role of the metagenome by
linking genetic structure and diversity of microbial
communities with their functions. To fulfill this chal-
lenge, advances in the understanding of the evolution of
microorganisms must be achieved by developing new
approaches (Fig. 1). Databases also include putative func-
tional gene sequences from microbial groups which have
yet to be cultivated in isolation in the laboratory. Until
we manage to grow them, progress in our knowledge of
their functions in complex environments can only be
achieved by untargeted culture-independent character-
ization of their functionality. As shown in Fig. 2,
microbial functionality can be characterized either by
the analysis of transcripts (metranscriptome: collective
RNA from all microorganisms present in an ecosystem)
and/or proteins (metaproteome: collective proteins from
all microorganisms present in an ecosystem [45]). So far,
major limitations related to the short half-life of RNA,
difficulty in eliminating humic acids during the extrac-
tion process, differential transcription kinetics of similar
genes in different populations, low correlation between
RNA levels and synthesis of the corresponding proteins
have hampered the study of the metatranscriptome of
indigenous microbial communities [17, 58].
These limitations, together with progress in protein
analysis (see below), have stimulated interest in meta-
proteome characterization. The term Bmetaproteomics^
was first proposed by Wilmes and Bond (2004) [55] as
Bthe large-scale characterization of the entire protein
complement of environmental microbiota at a given
point in time^. As proteins, and more precisely enzymes,
are involved in biotransformation processes, metapro-
teome analysis constitutes a suitable way to characterize
the dynamics of microbial function in a holistic way,
METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY
which represents the last and crucial step toward our
understanding of metabolome regulation (collective
metabolites from all microorganisms present in an
ecosystem [12]) (Fig. 2). The aim of this position paper
is to stress the relevance of metaproteome analysis in
(1) describing new functional genes and (2) relating
genetic and taxonomic diversity to the functionality of
microbial communities in complex environments.
On the Track of Metaproteomics?
Proteomics, Bthe large-scale study of proteins expressed
by an organism^ [54], truly emerged in the middle of the
1970s, when scientists started to map protein expression
using the newly developed two-dimensional (2-D) gel
electrophoresis [29]. By applying the 2-D technique, it
became possible to separate proteins from complex
mixtures of cellular extracts into individual polypeptides,
thus, allowing the analysis of bacterial response to
various growth conditions [38]. However, protein iden-
tification was time consuming and tedious due to the
lack of sensitive and fast sequencing technologies for
protein analysis. From the 1990s, proteomics has been
made advanced, thanks to the development of high-
efficiency peptide ionization methods in mass spectrom-
etry (MS), allowing rapid and highly sensitive protein
identification [36, 56]. In parallel, progress was made in
bioinformatic tools and in their adaptation to the
analysis of information from 2-D gels and MS, making
possible (1) the identification of proteins by database
searching with MS information [35], (2) the character-
ization of the corresponding genes by reverse genetics
[22], and (3) the determination of protein posttranslational
modifications [2]. Over the last decade, the advances in
proteomic technologies, together with the sequencing of
an increasing number of complete genomes of different
microorganisms, provide the opportunity to link phylog-
eny with the function of microorganisms. In this context,
the proteomic characterization of model organisms
currently being sequenced further facilitates the descrip-
P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH
tion of those physiological pathways involved in various
functions and interactions within and between organisms
such as symbiosis [14], pathogenicity [28], antibiotic
resistance [6], and adaptation to stresses [16, 51].
These studies stress the relevance of proteome
analysis for investigating global modifications in genome
expression of prokaryotic organisms and to progress in
our knowledge of those processes that regulate gene
expression. However, these investigations have largely
been performed under laboratory conditions, at the
organism level, without taking into account the biotic
(among microorganisms) and abiotic (microorganisms
with their natural habitat) interactions governing the
ecology of microbial communities in situ. Therefore, new
approaches are needed for in situ characterization of the
global protein expression at the population, or more
widely, at the community level. The main challenge of
environmental proteomics is to map proteins (proteo-
typing) extracted from indigenous microbial communi-
ties and identify in an untargeted way new physiological
pathways and their associated coding genes.
How to Do It?
Metaproteome analysis of soil microbial communities
implies the development of different technical steps,
from the extraction of microbial proteins from the
Environmental
samples
statistical analysis
of encoded profile
Link to Modifications of
genetic structure functional structure
Functional
communities
Figure 3. Experimental strategy and expected outcomes of the metaproteome characterization.
FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY 489
environmental matrix to the resolution of their diversity
and their identification (Fig. 3).
As with in situ nucleic acid-based studies, the most
crucial step in the metaproteome analysis is ensuring that
the quality and quantity of the proteins extracted are
representative of the sample. This is particularly true for
environmental proteomic studies because of the com-
plexity of indigenous microbial communities, the het-
erogeneity of natural environments, especially soil, and
the presence of interfering compounds (phenolic com-
pounds, humic acids...) making difficult the extraction of
a suitable protein fraction for analysis. The extraction
strategy varies according to the targeted protein fraction
(i.e., procaryota/eucaryota, extracellular/cell associated)
and by the subsequent methods of protein analysis
(i.e., 2-D comparative protein maps or measurement/
detection of specific polypeptides or enzymatic activities).
Recently, Schulze et al. (2004) [47] characterized extra-
cellular proteins isolated from dissolved organic matter
in different environments and showed that the relative
proportion of the proteins originating from bacteria
varied from 78% in lake water to less than 50% in a
forest soil solution.
For an exhaustive recovery of environmental pro-
teins (cellular + extracellular), organisms may be lyzed
directly in the environmental matrix before purification,
quantification, and analysis [30–32, 48, 55]. This strate-
gy, based on direct lysis, was applied by several authors
Extraction of microbial proteic pool Protein separation
2D-gel electrophoresis 1D-gel electrophoresis
Protein
profile
=
Fingerpinting of
functional structure
In situ spot digestion
reverse
Identification Protein identification by
genetic
of new functional Mass spectrometry
genes
Functional Physiological
bioindicator response
490 P.-A. MARON ET AL.:
to characterize the metaproteome in different environ-
ments such as a natural microbial biofilm [42], water
[18, 30–32], sediment [30], soil [30, 48], and activated
sludge [55]. From these studies, the protein complement
of the metagenome appears to be very complex and vary
according to the target environment [30] and with the
surrounding local conditions [31, 48]. Thus, in situ lysis
allows an exhaustive protein recovery from indigenous
bacteria, fungi, protozoa, and multicellular organisms;
this mixture being likely to introduce difficulties in the
taxonomic delineation of the detected proteins. Further-
more, a direct lysis strategy is technically difficult to
apply to natural environments as protein extracts are
widely contaminated with interfering compounds, mak-
ing protein characterization difficult with the biochem-
ical methods available.
Another option is to follow an indirect lysis strategy
in which proteins are extracted, purified, and separated
from organisms that have been previously extracted from
the environmental matrix [9, 23, 24]. The relevance of
this indirect approach for genetic diversity analysis
based on nucleic acid characterization was recently
shown [7]. This strategy allows the precise targeting of
the bacterial fraction and to obtain a cellular fraction
only slightly contaminated by soil compounds. Howev-
er, the efficiency of bacterial extraction is dependent on
the physicochemical characteristics of the environmen-
tal matrix, which may introduce further bias in the
quantitative and qualitative recovery of environmental
bacterial proteins [25]. By using this approach, Ehlers
and Clote (1999) [9] demonstrated the similarity of the
functional structure of 21 different activated sludge
systems which differed in design and phosphorous
removal; similarly, we have shown that water pollution
with mercury and cadmium leads to a modification of
the metaproteome of freshwater when compared to the
unpolluted control [26].
Once the protein samples are obtained, different
biochemical methods can be applied for metaproteome
analysis according to the type of information and the
level of resolution required. To get a Bproteofingerprint^
of the bacterial community in an untargeted way,
environmental proteins can be separated by one-dimensional
or two-dimensional gel electrophoresis before being stained
(Fig. 3). 2-D gel electrophoresis is preferred to obtain better
protein separation, facilitating the further identification of
polypeptides by database searches with MS information.
However, this technique remains limited by major problems
such as the impossibility of reliably monitoring low
abundant [15], very hydrophobic, very acidic or very basic
proteins [19]. For these reasons, 2-D gel electrophoresis has
also been designated as Bthe Achilles_ heel of proteomics^
[11]. Others consider that proteomics is still Btechnology-
driven and technology-limited discovery science^ [19]. In
this context, there is a need for alternative separation
METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY
methods. Considerable efforts have already been dedicated
in this direction with the development of various technol-
ogies such as: chromatographic/capillary electrophoresis
separation [57] and protein microarray developments [10,
43]. This last methodology should enable high throughput
analysis of complex protein mixtures and increase the
possible applications of proteomics in environmental
studies.
Despite the technical limitations of 2-D gel electro-
phoresis, this technique still provides a vast amount of
new data and remains the traditional technique used to
separate proteins for proteomic studies. Recently, Wilmes
and Bond (2004) [55] analyzed the diversity of the
protein pool of microbial communities in activated
sludges by combining 2-D polyacrylamide gel electro-
phoresis with MS and demonstrated that it is possible to
isolate and identify microbial proteins from a complex
environment. Schulze et al. (2004) [47] also characterized
protein pools, from complex environments such as lake
water, soil solutions, and soil particles by electrophoresis
coupled with MS.
Within a protein pool, proteins synthesized in
response to a stress can be detected by isotopic labeling
[13, 31] and visualized by autoradiography after separa-
tion on acrylamide gels [31]. Specific proteins can also be
detected and quantified in complex environments such
as soil using immunological methods [23, 24]. Their
quantification gives access to functionalities (i.e., level of
expression of the genetic potential at a given point in
time) of the microbial community. However, the
application of such strategy is limited by the specificity
of the antibodies targeting a class of functional proteins
(such as the dissimilative nitrate reductase) being
produced by a wide diversity of populations belonging
to different species [24, 39]. The strategy is also limited
by the small number of enzymes catalyzing biological
functions that have been identified (i.e., enzymes in-
volved in nitrogen cycle). Alternatively, specific polypep-
tides can be detected on the basis of their metabolic
activities by blotting of environmental protein samples
and applying staining techniques [21, 32, 33]. This
strategy requires the preservation of the catabolic
potential of the extracted enzymes implying the use of
gentle and nondenaturing protein extraction procedures
that may preclude the exhaustive recovery and accurate
separation of proteins from environmental samples.
The step after the identification of proteins associat-
ed with specific environments or those produced after
imposed stress is to relate these proteins to the cor-
responding gene sequences (Fig. 3). One of the goals of
the metaproteomic approach is to link biological func-
tions to gene sequences. Technologies (extraction and
separation) for characterizing the protein complement of
genome are still limited compared to those used for
nucleic acids and need to be optimized. However, to
P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH
meet the major scientific challenge of this postgenomic
area, proteomic analysis is presently one of the fastest
developing areas of biological research [47].
Expected Outcomes
Development of Functional Bioindicators. Nowadays,
specific attention is given to the impact of anthropogenic
activities, and more generally, of global change on the
quality of the environment. In this context, there is a
strong demand for bioindicators that will enable
characterization of the dynamics and sustainability of
environmental quality. The metaproteomic approach
appears to be a relevant option for the identification of
functional bioindicators. This was demonstrated by a
global approach based on the quantification of the total
soil proteins by Singleton et al. (2003) [48] who showed
that contamination of a soil with cadmium leads to a
significant decrease in its protein content. It can be
assumed that the qualitative analysis of the metaproteome
will provide more sensitive and specific bioindicators of
stress.
As indicated above, the total protein pool of
indigenous microbial communities can be resolved by
gel electrophoresis, providing a proteofingerprint (Fig. 3).
Possible shifts of the proteofingerprint in relation to
environmental stresses would be related to changes in the
functional structure of the microbial communities. This
type of relation can be compared to that established
between variations of DNA fingerprints of microbial
communities and shifts in their genetic structure after
being submitted to stresses. Specific proteins identified as
being induced or repressed by a given perturbation may
be considered as functional bioindicators, after validation
of their sensitivity, specificity (of the perturbation), and
ubiquity in different environments. These proteins could
be applied to design easy, fast, and sensitive tests that
allow the evaluation of the impact of different stresses on
ecosystem function by immunological approaches and/or
protein chips technologies that still need development
(for review, see [37]).
Tracking New Functional Genes and Complex
Metabolic Pathways. Metaproteomics is expected to
allow the identification of new functions involved in
complex biological pathways. Most of the functional
genes identified, so far, code for enzymes catalyzing
simple reactions such as those involved in nitrogen cycle.
These genes are used as genetic markers to characterize
the structure of specific functional communities.
However, this strategy, based on a limited number of
functional genes, does not allow one to study complex
biochemical pathways involving numerous and diverse
genes. This is especially true for complex biogeochem-
FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY 491
ical cycles such as that of carbon. Indeed, little infor-
mation is available on the dynamics of biochemical
transformations involved in the degradation of complex
carbon molecules from plant residues or exudates.
Furthermore, the role of the dynamic succession of
populations, belonging to fungi, bacteria and fauna,
expressing complementary functions is poorly doc-
umented. In that context, metaproteomics presents the
advantage of allowing the identification of proteins
involved in a biochemical process in an untargeted way.
Sequences of the corresponding coding genes can then be
deduced from amino acid sequences by reverse genetics
and ultimately used to develop tool probes for DNA/RNA
analysis (see Fig. 3). Combination of molecular and
biochemical tools, targeting functional genes, and
corresponding proteins allows the dynamics of the
functions and the associated communities in complex
environments to be followed.
Revisiting Microbial Ecology Concepts with a
Functional Point of View. The stability of ecosystems
depends on (1) their resistance (the magnitude of change
caused by a disturbance), (2) their resilience (the speed
with which they return to their predisturbance level) [40],
and (3) their functional redundancy (same function
achieved by different populations) [53]. These
parameters, well identified for higher organisms, have so
far been poorly addressed in microbial ecology. This
limitation is related to the huge taxonomic and functional
diversity within microbial communities, together with the
difficulty of studying them in their natural habitats, which
hamper the characterization of population dynamics and
definition of functional groups. Up to now, the three
parameters (resistance, resilience, and functional redun-
dancy) are only documented by diversity data based on
DNA analysis that addresses taxonomic and genetic diver-
sity. Metaproteomics should provide complementary
functional insights supporting the application of these
parameters to microbial ecology. Resistance and
resilience of ecosystems could be evaluated by the
magnitude of modifications occurring in the functional
structure of microbial communities similar to the
assessment currently made for the genetic structure
using DNA fingerprinting. Specific functional markers
deduced from these modifications would then be used to
define functional groups involved in the community
response to perturbations. Further diversity analysis of
these reactive functional groups would give information
on their level of functional redundancy and on the
contribution of this redundancy to ecosystem stability.
Functional insights provided by metaproteomics
should help the scientific community to address major
questions in microbial ecology related to (1) the link
between genetic and functional diversity in microbial
communities and (2) the relative contribution of
492 P.-A. MARON ET AL.:
taxonomic diversity and functional diversity on the
stability of ecosystems.
Acknowledgements
The authors are grateful to K. Klein for helpful com-
ments and correcting the English text.
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