Environmental proteomics - Briefings in Functional Genomics


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Environmental proteomics:applications
of proteome profiling in environmental
microbiology and biotechnology
Carla M.R.Lacerda and Kenneth F.Reardon
In this review,we present the use of proteomics to advance knowledge in the field of environmental biotechnology,
including studies of bacterial physiology,metabolism and ecology.Bacteria are widely applied in environmental
biotechnology for their ability to catalyze dehalogenation,methanogenesis,denitrification and sulfate reduction,
among others.Their tolerance to radiation and toxic compounds is also of importance.Proteomics has an impor-
tant role in helping uncover the pathways behind these cellular processes.Environmental samples are often highly
complex,which makes proteome studies in this field especially challenging.Some of these challenges are the lack of
genome sequences for the vast majority of environmental bacteria,difficulties in isolating bacteria and proteins from
certain environments,and the presence of complex microbial communities.Despite these challenges,proteomics
offers a unique dynamic view into cellular function.We present examples of environmental proteomics of model
organisms,and then discuss metaproteomics (microbial community proteomics),which has the potential to provide
insights into the function of a community without isolating organisms.Finally,the environmental proteomics
literature is summarized as it pertains to the specific application areas of wastewater treatment,metabolic engi-
neering,microbial ecology and environmental stress responses.
Keywords:environmental microbiology;metaproteomics;microbial community
In this review,we describe the application of pro-
teomics in studies of microbial physiology,metabo-
lism,and ecology in the context of natural and
engineered soil and water environments.These
habitats often contain a very diverse population
(e.g.,ca.104 prokaryotic species in 30 cm
of forest
soil [1]) with total population sizes that vary over
many orders of magnitude.Despite a growing
knowledge of the range of microbial diversity,
most of the microorganisms seen in natural environ-
ments are uncultivated,and their functional roles and
interactions are unknown.The metabolic capabilities
of microorganisms,including dehalogenation,
methanogenesis,denitrification and sulfate reduc-
tion,are studied for their applications in environ-
mental biotechnology.In addition,the abilities of
some microorganisms to tolerate radiation and toxic
chemicals,to use many different electron donors and
acceptors,or to survive at extremes of environmental
conditions are all of interest.Furthermore,micro-
organisms in both natural and engineered environ-
ments generally function in communities,allowing
them to benefit from syntrophism,exchange of
genes and cell–cell communication,among other
phenomena.However,few details are known about
these interactions.Similarly,little is known about
how naturally occurring microbial communities
respond to perturbations such as starvation,desicca-
tion,or freeze-thaw cycles.
Using proteomics,one can determine protein
expression profiles related to these research questions
for both microbial isolates and communities.
Proteomics provides a global view of the protein
complement of biological systems and,in combina-
tion with other omics technologies,has an important
Corresponding author.Kenneth F.Reardon,Department of Chemical and Biological Engineering,Colorado State University,Fort
Collins,CO 80523-1370,USA.Tel:þ1 970 491 6505;Fax:þ1 970 491 7369;E-mail:kenneth.reardon@colostate.edu
Carla M.R.Lacerda,PhD is a postdoctoral researcher in the Veterinary Teaching Hospital at Colorado State University.
Kenneth F.Reardon,PhD is professor of Chemical and Biological Engineering at Colorado State University.
BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS.page 1 of 13 doi:10.1093/bfgp/elp005
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role in helping uncover the mechanisms of these
cellular processes and thereby advance the develop-
ment of environmental biotechnologies.
As a field,environmental proteomics is much less
developed than other proteomics applications areas.
Most published proteomics studies focus on one
organism or cell type,and the effects of the growth
environment are investigated by comparing different
controlled conditions.One challenge of environ-
mental proteomics is that the environment of interest
is not controlled,and is difficult to emulate in the
laboratory.Furthermore,issues related to uncultured
and/or unsequenced organisms and protein extrac-
tion from native samples are key to the success of
environmental proteomics studies.An important
recent advance in environmental proteomics is the
ability to identify proteins from unsequenced
organisms with the use of modern bioinformatics
techniques.Cross-species protein identification [2,3]
and protein sequence similarity searches [4] are the
most common strategies used to identify proteins
when the genomic sequence is not available.
However,caution must be used since these
approaches can have low rates of success [5] and
require careful statistical analysis in order to avoid
false positive identifications.
An important recent development in environ-
mental proteomics that introduces new promises and
challenges is the analysis of the collective proteome
of microbial communities,known as metaproteo-
mics.Here,the community is viewed as a ‘metaor-
ganism’,in which population and meta-proteome
shifts are forms of functional responses.This
approach has been used by a few research groups
and has shown great potential in the evaluation of
biological processes in a community without isolat-
ing organisms.It also allows for a view of organism
interactions,which are impossible to determine
using pure cultures.Metaproteomic samples are
biologically highly complex,which makes these
studies especially challenging.If one considers that a
typical bacterium contains approximately 3000 genes
(e.g.3200 ORFs in Escherichia coli [6]),then a
metaorganism constituted by 100 species would
have about 3x 10
genes and a proteome of
corresponding complexity.Some of the main
challenges in metaproteomics are the difficulties
related to evaluating such a large number of gene
products as well as the lack of genome sequences
for the large majority of environmental bacteria.
Nonetheless,important progress has been made
with fascinating results [7],including advances that
allowed for the extraction and identification of
proteins directly from soil [8] or seawater [9].
Environmental proteomics,including metapro-
teomics,yields better results in combination with
other omics approaches such as metabolomics and
transcriptomics.In addition,proteomics allows
one to confirm the existence of gene products
predicted from a DNA sequence,providing a major
contribution to genomic science and an effective
complement to nucleic-acid-based methods as a
problem-solving tool in molecular biology [10].In
addition,proteomics can be used for phylogenetic
classification of bacterial species,either by using 2D
maps [11] or peptide sequences obtained from mass
spectrometry [12].Proteomics has the advantage of
not being limited to organisms for which the
genomic sequence is available.In addition,the
proteome represents the actual enzyme content in
a system,going beyond potential gene expression as
determined by microarrays,and can provide infor-
mation about post-translational modifications.
Another technique of great potential,especially
when combined with metaproteomics,is the
recently developed pyrosequencing approach,
which has already been applied in some metage-
nomics projects [13–15].Current proteomics
methods have limitations;for example,it is not yet
possible to acquire data on all the proteins present in
a sample,due mainly to their large concentration
range and the lack of a method for amplifying
low-abundance proteins.Thus,it is often advanta-
geous to complement proteomics with other omics
In this review,we present the current state of
environmental proteomics,including studies
of microorganisms isolated from the environment
as well as investigations of microbial communities.
These are considered first by the type of micro-
organism studied and the biological complexity
of the system.A general comparison of the
major research questions and tradeoffs involved in
investigations of microbial isolates or communities
is illustrated in Figure 1,and the examples
summarized here demonstrate that valuable informa-
tion has been obtained at all levels of complexity.In
the second part of this review,we consider
environmental proteomics research according to
application area in order to provide a sense of
the information that has been obtained thus far in
this field.
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Microbial isolates in the laboratory
The large majority of proteomic investigations of
environmental microorganisms focus on model
microorganisms cultured in the laboratory.These
species have been studied because of their interesting
traits such as the ability to tolerate,degrade,or
precipitate toxic compounds,or their versatility in
the use of electron donors,electron acceptors,or
carbon and energy sources.These qualities make
these organisms attractive for environmental bio-
technology applications,and proteomics can lead to
a better understanding of their functions in specific
habitats.One technical advantage of using model
organisms in the laboratory is that their genome may
be sequenced,which almost always improves the
quality of protein identifications.The study of model
organisms cultured in the laboratory allows for
flexibility during method development for more
complete proteome profiling.Some sequenced
bacterial species are well known for their unique
abilities.For example,Shewanella oneidensis strain
MR1 can use more than ten electron acceptors,
Deinococcus radiodurans can tolerate radiation,and
Burkholderia strain LB400 is an effective tetrachlor-
obenzene degrader.Here,we review several groups
of microorganisms and the issues studied using
Several species within the Bacillus genus are
models for Gram-positive bacteria.These organisms
are of interest due to their ubiquity,their ability to
sporulate and to formbiofilms,the virulence of some
species,and the tools available for their genetic
manipulation.Sporulation and biofilm formation are
key mechanisms of survival during environmental
stresses.Applications of proteomics to study the
physiology of B.subtilis have been reviewed [16].
Extensive proteomic work has been performed to
understand the tolerance of bacilli to extreme
environments [17–20],the allocation of stimulons
and regulons [21,22],and biofilm formation [23,
24],as well as full proteome [25–27] and secretome
[28–30] mapping.
The genera Halobacteria and Haloarchaea are of
great interest due to their atypical metabolism.They
are usually aerobes or facultative anaerobes that use
photosynthesis to create a proton gradient,which
allows themto survive in highly saline environments.
The archaeon genus Halobacterium is possibly the
most-studied halophile.These archaea are adapted to
be active and stable in hypersaline environments,
making them especially interesting for industrial
bioprocesses.There are a few literature reviews of
the post-genomic research on Halobacterium sp.
NRC-1,especially concerning its physiological
capabilities and the role of lateral gene transfer in
its evolution [31],the acidity of its proteome for
function at high salinity [32],and efficient proce-
dures for discovering novel halophilic enzymes [33].
The bacterial genus Pseudomonas and related
genera have been extensively studied because of
the ease of culturing these organisms from environ-
mental samples and their versatility with regard to
carbon and energy utilization,which allows them to
be very active in aerobic decomposition and
contaminant biodegradation.Pseudomonads may
be autotrophic or lithotrophic,planktonic or sessile,
aerobic or anaerobic.Most have a broad ability to
degrade aliphatic and aromatic hydrocarbons,as well
as to tolerate non-degradable toxic pollutants.
Proteomic studies of pseudomonads have mainly
been used to characterize their ability to degrade
toxic compounds [34–37],their ability to form
biofilms [38–41],their low nutritional requirements
[42] and flexible metabolism [43].
Shewanella oneidensis is a Gram-negative bacterium
that inhabits oxic–anoxic surfaces in nature.Its
bioremediation potential relates to its diverse respira-
tory capabilities,giving it the ability to reduce a wide
range of organic compounds,metal ions and radio-
nuclides.Several proteomics investigations have
been conducted to learn more about the bacterium,
and to improve the performance of different
Figure1:Relationship betweenfeasibilityof proteomic
studies and sample complexity.Different complexity
levels canbe used to accomplish certain studygoals.
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chromatographic and mass spectrometric proteomics
methods,quantification alternatives and database
annotations [44–53].
Sulfate-reducers are also of particular interest in
bioremediation.The proteome of Geobacter sulfurredu-
cens has been described in the presence of a range of
electron donors and acceptors [54].Around 90% of
the total predicted gene products were identified
in this study,and most differentially regulated
proteins were either cytochromes or were annotated
as hypothetical.Khare et al.[55] used proteomics to
understand the metabolic processes involved in metal
reduction in the same organism with either fumarate
or ferric citrate as the electron acceptor.Their results
suggested adjustments in membrane transport and
specific metabolic pathways in response to different
electron acceptors,as well as distinct differences
in the oxidative environment within the cell.The
metabolism of different carbon sources in
Desulfovibrio vulgaris was described by Zhang et al.
[56].Almost 1000 gene products were identified,
including proteins involved in ATP biosynthesis
and substrate-level phosphorylation.A large number
of hypothetical proteins were also found,leading to
a more detailed study [57] that aimed at assigning
functions to these proteins according to several
non-homology based methods.
Methanogenesis plays an important role in both
natural and engineered environmental systems.
Kao et al.[58] investigated the Methylococcus capsulatus
(Bath) response to different copper concentrations.
More than 100 proteins were differentially regulated,
including methane and carbohydrate metabolic
enzymes,and cellular signaling proteins.The
Vorholt laboratory compared the proteome of
Methylobacteriumextorquens AM1 grown under methy-
lotrophic and nonmethylotrophic conditions [59]
(methanol versus succinate as sole carbon source).
The majority of the differentially expressed proteins
were involved in methanol oxidation to CO
assimilation of one carbon units.A more recent
study from the same group evaluated the effects of
plant colonization by the same bacterium [60].They
compared colonization of roots,leaves and synthetic
medium growth.More than 50 proteins were found
to be either leaf- or root-specific,including
methanol utilization and stress proteins.They also
found a two-domain response-regulator essential for
epiphytic growth.
Bacteria with denitrification and dehalogenation
potential have also been the subjects of proteomic
investigations.Rabus and collaborators studied the
responses of the denitrifying bacterium Strain EbN1
to a variety of environmental stresses.They evaluated
anaerobic growth on different carbon sources and
focused on the expression of two toluene-related
operons in toluene-adapted cells [61].They also
confirmed the up-regulation of an ethylbenzene
pathway in the presence of toluene.In a comple-
mentary study,they used proteomics and bioinfor-
matics to uncover different mechanisms of regulation
of toluene and ethylbenzene pathways [62].More
recently,the same group proposed a genus name for
this strain—’Aromatoleum’ sp.strain EbN1,in a study
where a total of 556 different proteins were
identified [63].They were able to identify a broad
collection of pathway-specific subproteomes,reflect-
ing the metabolic versatility as well as the regulatory
potential of this bacterium.The Rabus laboratory
also worked on a Pirellula sp.strain 1 proteome after
growth on glucose and N-acetylglucosamine [64].
A number of proteins were unique to cells grown on
N-acetylglucosamine,and those included mostly
proteins related to carbohydrate metabolism.
Reductive dehalogenases were detected in proteo-
mics studies of Dehalococcoides ethenogenes strain
195 during anaerobic reductive dechlorination of
tetrachloroethene (PCE),trichloroethene (TCE),
or 2,3-dichlorophenol (2,3-DCP)[65].In another
study,it was observed that different strains of this
organism are capable of dehalogenating diverse
ranges of compounds,depending largely on the
suite of reductive dehalogenases that each strain
expresses [66].
Fermenting organisms,such as bacteria and yeast,
have also had their proteomes explored.In addition
to biotechnological interest,such studies allow for a
better understanding of survival strategies in these
organisms when faced with extreme environments
generated by fermentation products,e.g.,acids and
alcohols.Lactic acid bacteria and other fermenting
bacteria were analyzed while fermenting pyruvate to
lactate [67] and formate [68].Syntrophic bacteria had
their proteomes described when fermenting propio-
nate to acetate while living in association with a
methanogen [69].Detailed proteomic analysis of
ethanol production by Saccharomyces cerevisiae under
high-gravity glucose fermentation conditions
showed up-regulation of glycolysis and gluconeo-
genesis pathways [70–73].
Cyanobacteria are photosynthetic bacteria,pos-
sessing different lifestyles,including aquatic or
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terrestrial and unicellular or filamentous,some of
which formsymbiotic associations with plants.These
microorganisms are of interest for biotechnology
because of their abilities to fix nitrogen and carbon
dioxide,and to produce hydrogen and secondary
metabolites.The most commonly studied genera are
Synechocystis,Nostoc and Anabaena.An overview of
proteomics and other omics studies of cyanobacteria
was written by Burja et al.[74].Proteomic studies
typically focus on stress conditions [75–78] and
comparisons between lifestyles [79–83].
Microalgae are unicellular photosynthetic organ-
isms of particular interest due to their ability to
produce hydrogen and lipids under stress conditions,
and because they produce biomarkers of environ-
mental contamination [84].Proteomic investigations
have been published for Chlamydomonas sp.[85,86],
Nannochloropsis oculata [87],Dunaliella parva [88] and
Haematococcus pluvialis [89].These studies concern
structural changes in algal physiology under stress
conditions.More recently,there has been increased
interest in the study of salt stress,which has been
shown to increase lipid production [90].Proteomic
analysis can help uncover the mechanisms involved
in differential protein expression that leads to lipid
Communities in the laboratory
Metaproteomics refers to the proteomic study of
communities as metaorganisms.A metaorganism
is defined as a collection of organisms evolving as a
whole,sharing genes and metabolic capacities.
Metaproteomics studies are fundamentally more
complicated than those of pure cultures.While the
protocols for community analysis involve much
more complex protein and peptide separation
methods,the largest intricacy of metaproteomics
resides in the in silico analyses involved in protein
identification.Laboratory-based investigations of
microbial communities provide an intermediate
stage of complexity between isolates and complex
environmental samples,allowing information to be
obtained about a community rather than a single
microorganism but without the challenges of protein
extraction from environmental samples.
Wilmes and Bond investigated a community of
microorganisms from a laboratory-scale sequencing
batch reactor optimized for enhanced biological phos-
phorus removal and enriched for polyphosphate-
accumulating organisms.In their first study [91],
they used a two-dimensional electrophoresis/mass
spectrometry (2DE-MS) approach to detect many
proteins and were able to identify three.In their
subsequent studies [92,93],they compared the
proteome profiles of activated sludges with different
degrees of phosphorus removal performance as well as
profiles of communities in sludges that removed
phosphorus versus those that did not.Our laboratory
reported on a 2DE-MS/MS metaproteomics study of
anunsequenced bacterial community [94],identifying
more than 100 proteins differentially expressed in the
presence of cadmium.
Communities in the environment
Proteomic investigations of microbial communities
in their native environments provide the most
realistic information about their function but also
pose the greatest experimental and bioinformatic
challenges.Valenzuela et al.[95] have discussed the
use of metagenomics and high-throughput proteo-
mic technologies to study biomining communities,
and Schweder et al.[9] presented a similar discussion
for marine bacteria.A comprehensive review of
metaproteomics developments and expected out-
comes was written by Maron et al.[96].The Banfield
laboratory has performed pioneering work in this
area and presented a review of proteogenomic
approaches used recently for the molecular char-
acterization of bacterial communities [97].
Most investigations have focused on microbial
communities in surface waters.Initially,SDS-PAGE
(1-D electrophoresis,1DE) was used to generate
protein fingerprints of communities,with no attempt
made to identify proteins [98,99].Kan et al.used
a 2DE-MS workflow to study Chesapeake Bay
microbial communities and identified eight proteins
In 2005,the Banfield group published a landmark
study on a natural acid mine drainage microbial
biofilm community [7].With a shotgun proteomics
approach,they identified more than 2000 proteins
through the use of a database created from the
sequencing of a microbial community sampled from
the same mine but at a different location and time.
Recently,a strain-resolved community proteomics
study [101] was published by this group.Community
genomic data were used to identify proteins from
dominant community members,with strain specifi-
city.These findings provided evidence of exchange
of genes during adaptation to specific ecological
niches.Another study from the same group
evaluated how shotgun proteomics is affected by
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amino acid divergence between the sample and the
genomic database using a probability-based model
and a random mutation simulation model con-
strained by experimental data [102].
Proteomic analysis of microorganisms in soil has
been hampered by the lack of effective methods
for extracting proteins directly from soil in a
manner that is compatible with proteomic techni-
ques.Despite this difficulty,some results have been
obtained.For example,Schulze et al.[8,103] studied
proteins isolated from dissolved organic matter using
mass spectrometry and demonstrated the ability to
determine a proteome fingerprint of soil.Among the
proteins identified to be abundant in dissolved organic
matter were cellulases and laccases,which composed a
proteomic fingerprint of the presence and activity of
organisms in an ecosystem.Verberkmoes et al.[104]
used proteomics to evaluate biological threat agents in
complex environmental matrices.They determined
the ability of current mass spectrometric-based
methods to detect target species in different matrices
at concentrations as low as 6%.More recently,
Benndorf et al.[105] developed a proteome protocol
that enabled the analysis of the metaproteome of soil
and groundwater samples,addressing functional
community aspects more directly than metagenome
or even metatranscriptome analysis.
Wastewater treatment
To date,protein profiling related to wastewater
treatment has primarily used SDS–PAGE
(1D electrophoresis,1DE) to characterize the
organisms involved in this process and their ecology.
Such studies have focused on the diversity of
organisms in a treatment system and the influence
of environment on protein profiles rather than the
identification of interactions or specific metabolic
pathways.For example,MacRae and Smit [99]
described 33 different strains of Caulobacter present in
wastewater.The strains were distinguished based on
colony characteristics,DNA,and protein profiles
using 1DE.They also point out the increasing
antibiotic resistance of these strains,indicating
environmental adaptation.Jacob et al.[106] used
1DE to characterize 24 different strains of
Campylobacter present in a wastewater treatment
plant.Their work detected the presence of different
strains,based on evidence of different protein
band patterns.A similar study was conducted by
Niemi et al.[107] and involved 371 environmental
isolates of fecal streptococci samples.Samples were
collected from domestic and industrial wastewater,
and were characterized and clustered into seven
groups according to their 1DE protein profiles.
Samples from each environment had typical species
compositions,and their protein profiles varied
according to their environments.Maszenan et al.
[108] performed a similar analysis for strains of the
species Acinetobacter,together with a range of isolates
from a biological nutrient-removal activated sludge
plant.These studies show that the idea of studying
the proteome of a community of organisms,i.e.
metaproteomics,has been in development by
environmental researchers for at least two decades,
even though the laboratory and bioinformatic
methodologies available were limiting.
Wagner-Dobler et al.[109] pursued the goal of
better understanding bacterial communities capable
of degrading biphenyl for future bioremediation
applications.Different species were identified using
16S rDNA methods,and 1DE of whole-cell proteins
was used to provide information on the similarity
to strains of the same species.Comparison of
normalized protein patterns revealed that all of
the representative isolates were very similar to each
other,thus likely coming from the same species.
More recently,Francisco et al.[98] studied the
proteome of a microbial community under chronic
chromate stress in an effort to better understand
and improve microbial metal remediation of a
chromium-contaminated activated sludge.Using
numerical analysis of protein patterns and correlating
these with lipid profiles,they were able to cluster the
organisms in the community into subgroups that
shared similar metabolic abilities.The main findings
here were that the protein and lipid clusters were in
good agreement and that,within the same protein
and lipid cluster,there were functional differences in
the chromium resistance and reducing abilities of the
strains in the community.
Metabolic engineering
Although not native to soil and water environments,
E.coli has been studied in the environmental context
because of its role as a platform for metabolic
engineering.Pferdeort et al.[110] investigated
the proteome of E.coli metabolically engineered
for trichloroethene biodegradation by the introduc-
tion of six genes of an evolved toluene
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ortho-monooxygenase from Burkholderia cepacia G4.
The cellular physiology of the engineered strain was
significantly altered due to the insertion of the
toluene ortho-monooxygenase genes,with differ-
ential regulation of 45 proteins.Another study by
Lee et al.[111] analyzed strains from the next
stage of the metabolic engineered strategy in which
protective enzymes (glutathione S-transferase or
epoxide hydrolase) were inserted.Using a quantita-
tive proteomics approach,they found that some of
the induced proteins were involved in the oxidative
defense mechanism,pyruvate metabolism and
glutathione synthesis.Proteins involved in indole
synthesis,fatty acid synthesis,gluconeogenesis and
the tricarboxylic acid cycle were repressed.
Proteomic studies of the effects of metabolic
engineering are essential for the identification and
quantification of the changes in host cell physiology
reflected by protein production or other cellular
processes.Since most bacterial cellular processes are
either regulated or directly carried out by proteins or
protein complexes,physiological responses to new
genes can be expected to result in altered production
of various host cell proteins other than those
introduced in the genetic manipulation.
Microbial ecology
Ecological studies focus on naturally occurring
bacterial adaptation to their environments.
Proteomics has been used in several studies to
provide insights into the mechanisms of adaptation,
especially to extremes of temperature.Proteins
of hyperthermophilic organisms are of particular
importance since they have an enhanced conforma-
tional stability,allowing them to be active at high
temperatures.This property can be used to investi-
gate the molecular basis of protein folding and
conformational stability.Prosinecki et al.[112]
studied hyperstable proteins from Sulfurispharea sp.,
a hyperthermophilic archaeon that is able to grow
between 70

C and 97

C.They dynamically per-
turbed the proteome and identified proteins with
enhanced stabilities,involved in key cellular pro-
cesses such as detoxification,nucleic-acid processing
and energy metabolism.These proteins were still
biologically active after extensive thermal treatment
of the proteome.
Other ecological studies have focused on cold
adaptation of bacteria.Proteomic analysis was used
by Qiu et al.[114] to investigate the cold adapta-
tion of Exiguobacterium sibiricum 255-15,a strain
isolated from Siberian permafrost sediment.They
used an alternative approach involving chromatofo-
cusing coupled to mass spectrometry to identify 256
proteins preferentially or uniquely expressed at 4

Among these were 39 cold acclimation proteins,
including chaperones,and three cold shock proteins.
These results indicated that the adaptive nature of E.
sibiricum 255-15 at near-freezing temperatures could
be regulated by cellular physiological processes
through the regulation of specific cellular proteins.
The researchers concluded that the proteins that
were upregulated at the lower temperatures may
enable the cells to adapt to near or below-freezing
temperatures.Here it was shown that in order to
understand the biological context of bacterial cold
adaptation,large-scale proteomic studies are neces-
sary to uncover all cellular processes and not only
small sets of proteins isolated in specific functional
et al.[113],in a similar study,used
Colwellia psychrerythraea 34H and found changes to
the cell membrane fluidity,uptake and synthesis of
cryotolerance compounds,and strategies to over-
come temperature-dependent barriers to carbon
uptake.The salt and cold adaptation of Psychrobacter
273-4 was evaluated by Zheng et al.[115].Different
proteins were identified in cold adaptation in the
presence of salt,showing a combination effect of salt
and cold on protein expression.
Environmental stress responses
Proteomics approaches have often been used to gain
insights into the physiological responses of micro-
organisms to temperature,chemical and other stresses
(Table 1).The choice of proteomics as the primary
experimental tool is a reflection of the ability to
obtain system-wide information for non-model
organisms (e.g.given the cost of procuring DNA
microarrays for these species) and to obtain protein
identifications without a genomic sequence.Thus,
both 2DE- and chromatography-based proteomics
methods have been used to investigate the mecha-
nisms of tolerance to such stresses as low tempera-
tures [116,117],high temperatures [118,119],acidic
conditions [120,121],organic solvents [122],heavy
metals [123–125] and oxidizing chemicals [126,127].
While the up-regulation of known stress-response
proteins was frequently observed in these studies,
there were also discoveries of proteins involved in
other detoxification or adaptation strategies,includ-
ing novel transporter proteins,lipid biosynthesis
pathways and osmoprotectants.Moreover,the
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regulation of stress responses could be discerned,
particularly when the same species was exposed
to different stresses (e.g.nitrate,salinity and high
temperature for D.vulgaris [118,127,128]).In the
response of D.vulgaris Hildenborough to growth
inhibitory levels of nitrate stress [127],it was found
that proteins involved in central metabolism
and sulfate reduction were unaffected.However,
up-regulation was observed in nitrate reduction
systems,transport systems for proline,glycine-
betaine and glutamate,oxidative stress proteins,
ABC transport systems as well as in iron-sulphur-
cluster-containing proteins.In the case of increased
salinity [128],D.vulgaris responded with up-
regulated efflux systems,ATPases,RNA and DNA
helicases,and chemotaxis genes.Down-regulated
systems included flagellar biosynthesis,lactate uptake
permeases and ABC transport systems.These results
demonstrated that D.vulgaris responded similarly to
NaCl and KCl stresses.In the case of the response
of D.vulgaris to heat shock [118],proteomic
analysis revealed the up-regulation of heat shock
proteins,protein turnover and chaperones,and
down-regulation of energy production and conver-
sion,nucleotide transport,metabolism,translation
and ribosomal structure.The proteomics study also
suggested the possibility of posttranslational mod-
ifications in the chaperones and in several periplasmic
ABC transporters.It is clear from this set of studies
that proteomic analysis not only reveals system-wide
stress responses but also has the ability to identify
specific mechanisms of defense that characterize each
stress condition.
There are still many challenges for the field of
environmental proteomics to overcome.In the
laboratory,the primary challenge lies in the extraction
of cellular proteins from soil and other high-solids
matrices.In these samples,the presence of surfaces
(on which proteins can adsorb) and high concentra-
tions of interfering compounds with properties similar
to proteins (e.g.tannins) means that newstrategies for
protein extraction,purification and separation must
Table 1:Examples of proteomics studies related to stress responses of microorganisms
Microorganism Stress Findings fromproteomics
Deinococcus radiodurans [129] Gamma-irradiation Transcription and translation,replication and repair,general metabolism and
signal transduction are affected
Desulfovibrio vulgaris [127] Nitrate Up-regulation of nitrate reduction proteins,transport systems and oxidative
stress response proteins
Desulfovibrio vulgaris [118] High temperature Up-regulation of heat shock proteins,including DnaK,HtpG,HtrA and
AhpC,chaperones DnaK,AhpC,GroES and GroEL and also several periplas-
mic ABC transporters
Desulfovibrio vulgaris [128] Salinity Up-regulation of ATP synthesis and efflux systems,as well as
helicases,chemotaxis genes and osmoprotectants
Enterobacter liquefaciens strain [124] Cobalt Increased levels of twelve proteins involved with cellular antioxidant
response and resistance to heavy metals
Escherichia coli [126] Seleniumoxides Up-regulation of eight enzymes with antioxidant properties
Escherichia coli [120] Hypochlorous acid Nineteen proteins were identified as differentially expressed under
this condition
Ferroplasma acidarmanus [123,130] Copper and arsenic Protein folding and DNA repair,thermosome group II HSP60 family
chaperonin and HSP70 DnaK type heat shock proteins
Methanococcoides burtonii [117] Low temperature Alteredlipidbiosynthesis andspecific changes inmembranelipidunsaturation
Methylocystis sp.M[131] Carbon starvation,
heat and cold
Different stress proteins respond to different stress conditions
Pseudoalteromonas haloplanktis [116,132] Low temperature Amino-acid distributions in mesophilic and psychrophilic species
were different;changes observed in cell envelope and membrane-
associated,intermediary metabolismand information transfer proteins
Ralstonia metallidurans [125] Heavy metals Copper-resistance proteins and several regulatory gene clusters are involved
in multiple metal resistance
Stenotrophomonas maltophilia [133] Selenite Nucleotide synthesis and metabolism,protein and amino-acid metabolism,
and carbohydrate metabolism,cell division,oxidative stress and cell wall
Thermoanaerobacter tengcongensis [119] Low and high
Two groups of temperature-sensitive proteins noted:specific expression at
certain temperatures and consistent changes of expression responsive to
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be developed.Improved bioinformatics tools are also
needed to aid in the identification of proteins from
unsequenced microorganisms and especially from
unsequenced microbial communities.While the
cost of new sequencing technologies has fallen and
the number of published metagenomic databases has
increased,the complexities of analyzing environ-
mental microbial communities still requires advances
in bioinformatics.However,advances in the field
represent major steps towards a systems view of
organisms and metaorganisms.The proteome analyses
presented here are invaluable for the progress of
environmental biotechnology,despite being focused
on low complexity samples.The potential applica-
tions are wide,including improvement of wastewater
treatment,bioremediation and environmental mon-
itoring.In a broader view,the development of
environmental proteomics represents a major advance
in the fields of environmental biotechnology and
microbial ecology.Furthermore,while this review
has focused on microorganisms in soil and water
environments,the same approaches can be applied
with success in studying microorganisms in environ-
ments of interest in medicine (e.g.gastrointestinal
system) as is the focus of the Human Microbiome
Project (http://nihroadmap.nih.gov/hmp/) and
other efforts.In association with proteomics,tran-
scriptomics and metabolomics are also powerful tools
for acquiring information on gene function and
regulatory networks [134,135].Only combined
studies can correlate metabolic fluxes and physiolo-
gical changes in organisms.As research in this field
progresses,we will be able to make accurate
predictions about microbial activities and apply
this knowledge to improve environmental quality
and human health.
This work was supported in part by a National
Science Foundation Grant (BES-0329514) to K.F.R.
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