Abstract
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 important 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 engineering, microbial ecology and environmental stress responses.
INTRODUCTION
In this review, we describe the application of proteomics in studies of microbial physiology, metabolism, 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 cm3 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 environments are uncultivated, and their functional roles and interactions are unknown. The metabolic capabilities of microorganisms, including dehalogenation, methanogenesis, denitrification and sulfate reduction, are studied for their applications in environmental 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, microorganisms in both natural and engineered environments 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, desiccation, 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 combination with other omics technologies, has an important role in helping uncover the mechanisms of these cellular processes and thereby advance the development 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 environmental 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 extraction 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 environmental proteomics that introduces new promises and challenges is the analysis of the collective proteome of microbial communities, known as metaproteomics. Here, the community is viewed as a ‘metaorganism’, 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 isolating 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 3 x 105 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 metaproteomics, 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 information 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 metagenomics 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 advantageous to complement proteomics with other omics tools.
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 microorganism 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 information 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.
Relationship between feasibility of proteomic studies and sample complexity. Different complexity levels can be used to accomplish certain study goals.
TYPES OF ENVIRONMENTAL PROTEOMICS STUDIES
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 biotechnology 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 tetrachlorobenzene degrader. Here, we review several groups of microorganisms and the issues studied using proteomics.
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 form biofilms, 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 them to 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 procedures 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 environmental 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 respiratory capabilities, giving it the ability to reduce a wide range of organic compounds, metal ions and radionuclides. Several proteomics investigations have been conducted to learn more about the bacterium, and to improve the performance of different 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 sulfurreducens 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 Methylobacterium extorquens AM1 grown under methylotrophic and nonmethylotrophic conditions [59] (methanol versus succinate as sole carbon source). The majority of the differentially expressed proteins were involved in methanol oxidation to CO2 and 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 complementary study, they used proteomics and bioinformatics 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, reflecting 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 proteomics 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 propionate 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 gluconeogenesis pathways [70–73].
Cyanobacteria are photosynthetic bacteria, possessing different lifestyles, including aquatic or terrestrial and unicellular or filamentous, some of which form symbiotic 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 organisms of particular interest due to their ability to produce hydrogen and lipids under stress conditions, and because they produce biomarkers of environmental 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 overproduction.
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 phosphorus 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 an unsequenced 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 proteomic 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 outcomes 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 characterization 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 [100].
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 specificity. 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 amino acid divergence between the sample and the genomic database using a probability-based model and a random mutation simulation model constrained 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 techniques. 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.
RESEARCH QUESTIONS AND APPLICATION AREAS
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 introduction of six genes of an evolved toluene 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 differential 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 quantitative 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 conformational stability, allowing them to be active at high temperatures. This property can be used to investigate 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 perturbed the proteome and identified proteins with enhanced stabilities, involved in key cellular processes 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 adaptation of Exiguobacterium sibiricum 255-15, a strain isolated from Siberian permafrost sediment. They used an alternative approach involving chromatofocusing coupled to mass spectrometry to identify 256 proteins preferentially or uniquely expressed at 4°C. 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 necessary to uncover all cellular processes and not only small sets of proteins isolated in specific functional contexts. Methé 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 overcome 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 microorganisms 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 mechanisms of tolerance to such stresses as low temperatures [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, including novel transporter proteins, lipid biosynthesis pathways and osmoprotectants. Moreover, the 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 conversion, nucleotide transport, metabolism, translation and ribosomal structure. The proteomics study also suggested the possibility of posttranslational modifications 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.
Examples of proteomics studies related to stress responses of microorganisms
| Microorganism | Stress | Findings from proteomics |
|---|---|---|
| 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 periplasmic 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] | Selenium oxides | 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 | Altered lipid biosynthesis and specific changes in membrane lipid unsaturation |
| 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 metabolism and 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 synthesis |
| Thermoanaerobacter tengcongensis [119] | Low and high temperatures | Two groups of temperature-sensitive proteins noted: specific expression at certain temperatures and consistent changes of expression responsive to temperature |
| Microorganism | Stress | Findings from proteomics |
|---|---|---|
| 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 periplasmic 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] | Selenium oxides | 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 | Altered lipid biosynthesis and specific changes in membrane lipid unsaturation |
| 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 metabolism and 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 synthesis |
| Thermoanaerobacter tengcongensis [119] | Low and high temperatures | Two groups of temperature-sensitive proteins noted: specific expression at certain temperatures and consistent changes of expression responsive to temperature |
Examples of proteomics studies related to stress responses of microorganisms
| Microorganism | Stress | Findings from proteomics |
|---|---|---|
| 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 periplasmic 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] | Selenium oxides | 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 | Altered lipid biosynthesis and specific changes in membrane lipid unsaturation |
| 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 metabolism and 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 synthesis |
| Thermoanaerobacter tengcongensis [119] | Low and high temperatures | Two groups of temperature-sensitive proteins noted: specific expression at certain temperatures and consistent changes of expression responsive to temperature |
| Microorganism | Stress | Findings from proteomics |
|---|---|---|
| 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 periplasmic 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] | Selenium oxides | 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 | Altered lipid biosynthesis and specific changes in membrane lipid unsaturation |
| 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 metabolism and 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 synthesis |
| Thermoanaerobacter tengcongensis [119] | Low and high temperatures | Two groups of temperature-sensitive proteins noted: specific expression at certain temperatures and consistent changes of expression responsive to temperature |
FRONTIERS IN ENVIRONMENTAL PROTEOMICS
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 concentrations of interfering compounds with properties similar to proteins (e.g. tannins) means that new strategies for protein extraction, purification and separation must 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 environmental 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 applications are wide, including improvement of wastewater treatment, bioremediation and environmental monitoring. 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 environments 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, transcriptomics 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 physiological 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.
Proteomics has the potential to contribute significantly to the fields of environmental microbiology, microbial ecology and environmental biotechnology.
To date, the majority of environmental proteomics investigations have focused on microorganisms isolated from their environment and studied in the laboratory.
Important insights have been gained from the small number of metaproteomics investigations, which demonstrate the increasing ability to identify proteins from microbial communities, even in the absence of genomic sequences or phylogenetic characterization.
Important challenges facing the further development of environmental proteomics are the need to develop methods for extraction of proteins from microbial communities in soil and the need for protein bioinformatics tools that are better able to identify peptides and proteins from shotgun metaproteomics workflows.
FUNDING
This work was supported in part by a National Science Foundation Grant (BES-0329514) to K.F.R.
