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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 evolution of uncultured microorganisms. However, indi-
a cultivation-independent approach based on the ex- cations of genetic potential does not contribute to the
traction, amplification, cloning, and characterization of elucidation of the functionality (level of expression of the
rDNA genes directly from natural environments. genetic potential) (Fig. 2) of microbial communities in
Beginning with these early works, many efforts have ecosystems [8, 45, 50, 52].
been dedicated to developing molecular methods to Different methods have been developed to discrimi-
characterize microbial information contained in the nucleic nate active populations from quiescent ones in natural
acids extracted from environmental samples [1, 20]. These habitats by incorporation of labeled markers in microbial
developments enabled the characterization of variations of biomass such as 13C (DNA-Stable isotope probing meth-
the microbial community structure and diversity in mul- ods [41]), or BrdU [4]. However, these approaches only
tiple situations allowing the identification of populations provide limited information on the populations associated
preferentially associated with environmental perturbations with a specific process rather than a complete description
(for review, see [44]). Further methodological progress of their functional role within a Bcommunity^. Bioinfor-
allowed the cloning and sequencing of large genome frag- matic data acquired over the last 20 years on putative
ments (about 40 kb) from microbial communities of a functional genes allows the design of primers and probes
planktonic marine archaeon [49]. This work provided the to target specific functional communities in complex
first glimpse into content and diversity of marine archae environments. The design of such primers enables
but was also the first example of the feasibility of meta- (1) the quantification of gene copy number [real time
genome characterization [collective genome from all polymerase chain reaction (PCR)] and (2) the character-
(micro-) organisms present in an ecosystem] [46]. Recent ization of polymorphism(s) of functional gene(s). These
advances in high-throughput screening and sequencing studies performed at the DNA level can be further linked
facilitate this type of study and have provided the majority to measurement of the corresponding activities [27].
of DNA sequences now found in databases. Metagenomic However, this integrated approach is restricted to a
approaches provide new insights into genetic diversity and limited number of functions (denitrification, nitrification,
488 P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY

Figure 2. Schematic representation of the FMeta_ levels in the ecology of microbial communities.

and methane oxidation) for which genes involved in each which represents the last and crucial step toward our
step of the metabolic pathway are known and sufficiently understanding of metabolome regulation (collective
conserved to allow the design of consensus primers. metabolites from all microorganisms present in an
Furthermore, the presence of a gene within populations ecosystem [12]) (Fig. 2). The aim of this position paper
of a given Bfunctional community^ does not necessarily is to stress the relevance of metaproteome analysis in
mean that it is expressed in the habitat. (1) describing new functional genes and (2) relating
In the postgenomic era, a major challenge is to genetic and taxonomic diversity to the functionality of
elucidate the functional role of the metagenome by microbial communities in complex environments.
linking genetic structure and diversity of microbial
communities with their functions. To fulfill this chal-
lenge, advances in the understanding of the evolution of
On the Track of Metaproteomics?
microorganisms must be achieved by developing new
approaches (Fig. 1). Databases also include putative func- Proteomics, Bthe large-scale study of proteins expressed
tional gene sequences from microbial groups which have by an organism^ [54], truly emerged in the middle of the
yet to be cultivated in isolation in the laboratory. Until 1970s, when scientists started to map protein expression
we manage to grow them, progress in our knowledge of using the newly developed two-dimensional (2-D) gel
their functions in complex environments can only be electrophoresis [29]. By applying the 2-D technique, it
achieved by untargeted culture-independent character- became possible to separate proteins from complex
ization of their functionality. As shown in Fig. 2, mixtures of cellular extracts into individual polypeptides,
microbial functionality can be characterized either by thus, allowing the analysis of bacterial response to
the analysis of transcripts (metranscriptome: collective various growth conditions [38]. However, protein iden-
RNA from all microorganisms present in an ecosystem) tification was time consuming and tedious due to the
and/or proteins (metaproteome: collective proteins from lack of sensitive and fast sequencing technologies for
all microorganisms present in an ecosystem [45]). So far, protein analysis. From the 1990s, proteomics has been
major limitations related to the short half-life of RNA, made advanced, thanks to the development of high-
difficulty in eliminating humic acids during the extrac- efficiency peptide ionization methods in mass spectrom-
tion process, differential transcription kinetics of similar etry (MS), allowing rapid and highly sensitive protein
genes in different populations, low correlation between identification [36, 56]. In parallel, progress was made in
RNA levels and synthesis of the corresponding proteins bioinformatic tools and in their adaptation to the
have hampered the study of the metatranscriptome of analysis of information from 2-D gels and MS, making
indigenous microbial communities [17, 58]. possible (1) the identification of proteins by database
These limitations, together with progress in protein searching with MS information [35], (2) the character-
analysis (see below), have stimulated interest in meta- ization of the corresponding genes by reverse genetics
proteome characterization. The term Bmetaproteomics^ [22], and (3) the determination of protein posttranslational
was first proposed by Wilmes and Bond (2004) [55] as modifications [2]. Over the last decade, the advances in
Bthe large-scale characterization of the entire protein proteomic technologies, together with the sequencing of
complement of environmental microbiota at a given an increasing number of complete genomes of different
point in time^. As proteins, and more precisely enzymes, microorganisms, provide the opportunity to link phylog-
are involved in biotransformation processes, metapro- eny with the function of microorganisms. In this context,
teome analysis constitutes a suitable way to characterize the proteomic characterization of model organisms
the dynamics of microbial function in a holistic way, currently being sequenced further facilitates the descrip-
P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY 489

tion of those physiological pathways involved in various environmental matrix to the resolution of their diversity
functions and interactions within and between organisms and their identification (Fig. 3).
such as symbiosis [14], pathogenicity [28], antibiotic As with in situ nucleic acid-based studies, the most
resistance [6], and adaptation to stresses [16, 51]. crucial step in the metaproteome analysis is ensuring that
These studies stress the relevance of proteome the quality and quantity of the proteins extracted are
analysis for investigating global modifications in genome representative of the sample. This is particularly true for
expression of prokaryotic organisms and to progress in environmental proteomic studies because of the com-
our knowledge of those processes that regulate gene plexity of indigenous microbial communities, the het-
expression. However, these investigations have largely erogeneity of natural environments, especially soil, and
been performed under laboratory conditions, at the the presence of interfering compounds (phenolic com-
organism level, without taking into account the biotic pounds, humic acids...) making difficult the extraction of
(among microorganisms) and abiotic (microorganisms a suitable protein fraction for analysis. The extraction
with their natural habitat) interactions governing the strategy varies according to the targeted protein fraction
ecology of microbial communities in situ. Therefore, new (i.e., procaryota/eucaryota, extracellular/cell associated)
approaches are needed for in situ characterization of the and by the subsequent methods of protein analysis
global protein expression at the population, or more (i.e., 2-D comparative protein maps or measurement/
widely, at the community level. The main challenge of detection of specific polypeptides or enzymatic activities).
environmental proteomics is to map proteins (proteo- Recently, Schulze et al. (2004) [47] characterized extra-
typing) extracted from indigenous microbial communi- cellular proteins isolated from dissolved organic matter
ties and identify in an untargeted way new physiological in different environments and showed that the relative
pathways and their associated coding genes. 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-
How to Do It? teins (cellular + extracellular), organisms may be lyzed
Metaproteome analysis of soil microbial communities directly in the environmental matrix before purification,
implies the development of different technical steps, quantification, and analysis [30–32, 48, 55]. This strate-
from the extraction of microbial proteins from the gy, based on direct lysis, was applied by several authors

Environmental
Extraction of microbial proteic pool Protein separation
samples

2D-gel electrophoresis 1D-gel electrophoresis

Protein
statistical analysis profile
of encoded profile
Link to Modifications of =
genetic structure functional structure Fingerpinting of
functional structure

In situ spot digestion

reverse
Identification Protein identification by
Functional genetic
of new functional Mass spectrometry
communities
genes

Functional Physiological
bioindicator response
Figure 3. Experimental strategy and expected outcomes of the metaproteome characterization.
490 P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY

to characterize the metaproteome in different environ- methods. Considerable efforts have already been dedicated
ments such as a natural microbial biofilm [42], water in this direction with the development of various technol-
[18, 30–32], sediment [30], soil [30, 48], and activated ogies such as: chromatographic/capillary electrophoresis
sludge [55]. From these studies, the protein complement separation [57] and protein microarray developments [10,
of the metagenome appears to be very complex and vary 43]. This last methodology should enable high throughput
according to the target environment [30] and with the analysis of complex protein mixtures and increase the
surrounding local conditions [31, 48]. Thus, in situ lysis possible applications of proteomics in environmental
allows an exhaustive protein recovery from indigenous studies.
bacteria, fungi, protozoa, and multicellular organisms; Despite the technical limitations of 2-D gel electro-
this mixture being likely to introduce difficulties in the phoresis, this technique still provides a vast amount of
taxonomic delineation of the detected proteins. Further- new data and remains the traditional technique used to
more, a direct lysis strategy is technically difficult to separate proteins for proteomic studies. Recently, Wilmes
apply to natural environments as protein extracts are and Bond (2004) [55] analyzed the diversity of the
widely contaminated with interfering compounds, mak- protein pool of microbial communities in activated
ing protein characterization difficult with the biochem- sludges by combining 2-D polyacrylamide gel electro-
ical methods available. phoresis with MS and demonstrated that it is possible to
Another option is to follow an indirect lysis strategy isolate and identify microbial proteins from a complex
in which proteins are extracted, purified, and separated environment. Schulze et al. (2004) [47] also characterized
from organisms that have been previously extracted from protein pools, from complex environments such as lake
the environmental matrix [9, 23, 24]. The relevance of water, soil solutions, and soil particles by electrophoresis
this indirect approach for genetic diversity analysis coupled with MS.
based on nucleic acid characterization was recently Within a protein pool, proteins synthesized in
shown [7]. This strategy allows the precise targeting of response to a stress can be detected by isotopic labeling
the bacterial fraction and to obtain a cellular fraction [13, 31] and visualized by autoradiography after separa-
only slightly contaminated by soil compounds. Howev- tion on acrylamide gels [31]. Specific proteins can also be
er, the efficiency of bacterial extraction is dependent on detected and quantified in complex environments such
the physicochemical characteristics of the environmen- as soil using immunological methods [23, 24]. Their
tal matrix, which may introduce further bias in the quantification gives access to functionalities (i.e., level of
quantitative and qualitative recovery of environmental expression of the genetic potential at a given point in
bacterial proteins [25]. By using this approach, Ehlers time) of the microbial community. However, the
and Clote (1999) [9] demonstrated the similarity of the application of such strategy is limited by the specificity
functional structure of 21 different activated sludge of the antibodies targeting a class of functional proteins
systems which differed in design and phosphorous (such as the dissimilative nitrate reductase) being
removal; similarly, we have shown that water pollution produced by a wide diversity of populations belonging
with mercury and cadmium leads to a modification of to different species [24, 39]. The strategy is also limited
the metaproteome of freshwater when compared to the by the small number of enzymes catalyzing biological
unpolluted control [26]. functions that have been identified (i.e., enzymes in-
Once the protein samples are obtained, different volved in nitrogen cycle). Alternatively, specific polypep-
biochemical methods can be applied for metaproteome tides can be detected on the basis of their metabolic
analysis according to the type of information and the activities by blotting of environmental protein samples
level of resolution required. To get a Bproteofingerprint^ and applying staining techniques [21, 32, 33]. This
of the bacterial community in an untargeted way, strategy requires the preservation of the catabolic
environmental proteins can be separated by one-dimensional potential of the extracted enzymes implying the use of
or two-dimensional gel electrophoresis before being stained gentle and nondenaturing protein extraction procedures
(Fig. 3). 2-D gel electrophoresis is preferred to obtain better that may preclude the exhaustive recovery and accurate
protein separation, facilitating the further identification of separation of proteins from environmental samples.
polypeptides by database searches with MS information. The step after the identification of proteins associat-
However, this technique remains limited by major problems ed with specific environments or those produced after
such as the impossibility of reliably monitoring low imposed stress is to relate these proteins to the cor-
abundant [15], very hydrophobic, very acidic or very basic responding gene sequences (Fig. 3). One of the goals of
proteins [19]. For these reasons, 2-D gel electrophoresis has the metaproteomic approach is to link biological func-
also been designated as Bthe Achilles_ heel of proteomics^ tions to gene sequences. Technologies (extraction and
[11]. Others consider that proteomics is still Btechnology- separation) for characterizing the protein complement of
driven and technology-limited discovery science^ [19]. In genome are still limited compared to those used for
this context, there is a need for alternative separation nucleic acids and need to be optimized. However, to
P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY 491

meet the major scientific challenge of this postgenomic ical cycles such as that of carbon. Indeed, little infor-
area, proteomic analysis is presently one of the fastest mation is available on the dynamics of biochemical
developing areas of biological research [47]. 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,
Expected Outcomes
expressing complementary functions is poorly doc-
Development of Functional Bioindicators. Nowadays, umented. In that context, metaproteomics presents the
specific attention is given to the impact of anthropogenic advantage of allowing the identification of proteins
activities, and more generally, of global change on the involved in a biochemical process in an untargeted way.
quality of the environment. In this context, there is a Sequences of the corresponding coding genes can then be
strong demand for bioindicators that will enable deduced from amino acid sequences by reverse genetics
characterization of the dynamics and sustainability of and ultimately used to develop tool probes for DNA/RNA
environmental quality. The metaproteomic approach analysis (see Fig. 3). Combination of molecular and
appears to be a relevant option for the identification of biochemical tools, targeting functional genes, and
functional bioindicators. This was demonstrated by a corresponding proteins allows the dynamics of the
global approach based on the quantification of the total functions and the associated communities in complex
soil proteins by Singleton et al. (2003) [48] who showed environments to be followed.
that contamination of a soil with cadmium leads to a
significant decrease in its protein content. It can be Revisiting Microbial Ecology Concepts with a
assumed that the qualitative analysis of the metaproteome Functional Point of View. The stability of ecosystems
will provide more sensitive and specific bioindicators of depends on (1) their resistance (the magnitude of change
stress. caused by a disturbance), (2) their resilience (the speed
As indicated above, the total protein pool of with which they return to their predisturbance level) [40],
indigenous microbial communities can be resolved by and (3) their functional redundancy (same function
gel electrophoresis, providing a proteofingerprint (Fig. 3). achieved by different populations) [53]. These
Possible shifts of the proteofingerprint in relation to parameters, well identified for higher organisms, have so
environmental stresses would be related to changes in the far been poorly addressed in microbial ecology. This
functional structure of the microbial communities. This limitation is related to the huge taxonomic and functional
type of relation can be compared to that established diversity within microbial communities, together with the
between variations of DNA fingerprints of microbial difficulty of studying them in their natural habitats, which
communities and shifts in their genetic structure after hamper the characterization of population dynamics and
being submitted to stresses. Specific proteins identified as definition of functional groups. Up to now, the three
being induced or repressed by a given perturbation may parameters (resistance, resilience, and functional redun-
be considered as functional bioindicators, after validation dancy) are only documented by diversity data based on
of their sensitivity, specificity (of the perturbation), and DNA analysis that addresses taxonomic and genetic diver-
ubiquity in different environments. These proteins could sity. Metaproteomics should provide complementary
be applied to design easy, fast, and sensitive tests that functional insights supporting the application of these
allow the evaluation of the impact of different stresses on parameters to microbial ecology. Resistance and
ecosystem function by immunological approaches and/or resilience of ecosystems could be evaluated by the
protein chips technologies that still need development magnitude of modifications occurring in the functional
(for review, see [37]). structure of microbial communities similar to the
assessment currently made for the genetic structure
Tracking New Functional Genes and Complex using DNA fingerprinting. Specific functional markers
Metabolic Pathways. Metaproteomics is expected to deduced from these modifications would then be used to
allow the identification of new functions involved in define functional groups involved in the community
complex biological pathways. Most of the functional response to perturbations. Further diversity analysis of
genes identified, so far, code for enzymes catalyzing these reactive functional groups would give information
simple reactions such as those involved in nitrogen cycle. on their level of functional redundancy and on the
These genes are used as genetic markers to characterize contribution of this redundancy to ecosystem stability.
the structure of specific functional communities. Functional insights provided by metaproteomics
However, this strategy, based on a limited number of should help the scientific community to address major
functional genes, does not allow one to study complex questions in microbial ecology related to (1) the link
biochemical pathways involving numerous and diverse between genetic and functional diversity in microbial
genes. This is especially true for complex biogeochem- communities and (2) the relative contribution of
492 P.-A. MARON ET AL.: METAPROTEOMICS: A NEW APPROACH FOR STUDYING FUNCTIONAL MICROBIAL ECOLOGY

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