1. INTRODUCTION
The protein equivalent of genomics is called proteomics. The term proteomics was defined for the first time in 1996 by Wilkins et al. to describe the entire protein content in a cell, tissue, or organism through a systematic approach. Proteomics, as a molecular biology branch, has focused the researchers’ attention since it delivers a general overview of biological systems proteins. Proteomics comprises a set of technologies that allow the identification and quantitative determination of the proteins expressed in the cell. These techniques provide for the proteins’ three-dimensional structure and interaction manners. Since protein-protein interactions are the fundamentals of signal transduction in various regulatory events, systemically classifying these interactions may contribute to understanding the modelling of protein complexes and the principles of their recognition at the molecular level.
Proteomics, combined with the rest of omics technologies, also contributes to the discovery of the mechanisms of essential cellular processes (in physiology, metabolism, and ecology context) and the promotion of practical applications of these findings in the fields of biomedicine, clinical surveys, environmental microbiology, microbial ecology, and environmental biotechnology.
Proteomics helps predict a gene product based on its DNA sequence, thus facilitating the enlargement of molecular biology technical tools and complementing nucleic acids-based approaches in genomics. Proteomics can be exploited for microbial phylogenetic studies and classification using 2D maps of protein sequences obtained by MS analyses.
Environmental proteomics is one of the proteomics application areas with great promise for studying the effects of growth environments on organism development in natural, non-controlled conditions. Although this branch of proteomics is relatively less developed than other applications, it is an indispensable tool that uses protein chemistry to solve environmental obscurities.
Environmental proteomics faces severe challenges in performance and data interpretation. It acts in an uncontrolled environment without standardized laboratory conditions. Environmental proteomics deals with issues related to non-culturable organisms whose genomes are not sequenced. Furthermore, protein extraction from soil or aquatic samples is another challenge that overcoming is an essential prerequisite for proteomic studies.
Environmental proteomics predominantly studies microbial physiology, metabolism, and ecology in natural environments. These are habitats populated by dense and multifarious populations. Most microbial species in natural environments are unculturable, and their physiology and interaction are almost known. However, their biochemical potential in the form of specific metabolic capabilities (i.e., methanogenesis, denitrification, dehalogenation, sulfate reduction, etc.) is attracting the attention of researchers and biotech practitioners as potential participants in environmental biotechnology activities. On the other hand, the capabilities of some species to use diverse electron donors and acceptors, tolerate toxic substances and radiation, or survive under severe environmental extremities (e.g., desiccation, starvation, freezing/thawing processes, etc.) are of particular interest. Environmental proteomics brings insight at the molecular level into phenomena like syntrophy, gene exchange, and cell-to-cell communication due to the potential to operate in microbial communities, the form in which microbial populations naturally function.
Environmental proteomics is a powerful strategy and methodological approach toward revealing the full microbial potential in these directions, studying the protein expression profiles in different ecological conditions. The high throughput techniques for protein separation (such as 2D PAGE, LC, isotope-coded affinity tag labelling (ICAT)), developed during the last decade together with the advancement of the techniques for protein analysis (such as MS and database searching) enhanced and facilitated the procedures of protein identification rapidly and reliably.
The progress in bioinformatics also significantly contributes to the protein identification from unculturable organisms. Strategies to apply cross-species protein identification and search for protein sequence similarity help identify proteins without the availability of relevant genomic sequences.
2. ECOSYSTEMS AND COMMUNITIES PROTEIN DIVERSITY
The technological advancement in exploiting methods for characterizing proteins of unculturable organisms (the so-called metaproteomics) opened the prospects for cataloguing proteins, thus constituting two proteomics sub-branches that reveal protein abundance and diversity:
- The community proteomics: cataloguing the proteins of component species of assemblages;
- Environmental proteomics: cataloguing the proteins of ecosystem parts, even lacking the knowledge about the organisms producing those proteins.
The first such studies focussed on relatively uncomplex assemblages, comprising mainly microorganisms that inhabited extreme habitats such as foliar microflora or the microflora of some seawater and soil samples. The results of these surveys are of crucial importance for the advancement of environmental proteomics. They shape differential protein production and expression profiles that reflect the organism’s physiological and metabolic responses to fluctuations in ecological conditions. Those fluctuations (both in norma and under stress, including climate change) lead to changes in the protein composition and abundance, which, although best analysed in microbial populations, could shed light on the changes in the global material and energy fluxes within or among ecosystems.
Besides these essential findings, it is still not clear if the results from microbial assemblages can be extrapolated to more complex by the number and variety of elements of terrestrial and aquatic ecosystems. For instance, microbial assemblages lack the components of the complex food webs that comprise interacting prokaryotes and multicellular eukaryotes (the so-called macrobial eukaryotes). On the other hand, the macrobial systems face technical challenges that have to be overcome, in particular:
- Protein identification from species whose genomes are not sequenced;
- Differences between physical and biological conformation of proteins;
- Differences in the activities of the native and the isolated proteins;
- Technical difficulties in extracting proteins from complex matrices (see water or soil) reliably enough to fit the research purpose.
However, the high-throughput techniques and bioinformatics approaches answer these challenges, generating valuable information at the protein level, the most direct level of measuring molecular phenotype patterns of the microbe-macrobe ecosystem.
3. ENVIRONMENTAL PROTEOMICS STUDIES
3.1. Model environmental microorganisms’ laboratory surveys
Currently, most proteomics studies of environmental microorganisms are performed with well-known model microbes that are readily culturable under controlled conditions in the laboratory. These species are chosen because of their environmentally essential characteristics, such as the capability of withstanding, degrading, or precipitating substances toxic to terrestrial/water habitats and profound flexibility toward electron donors/acceptors, carbon, and energy sources. Environmental proteomics could contribute to elucidating their functions in specific habitats and enhance their potential application in environmental biotechnology. Besides, the genomes of all these microbes are sequenced or can be readily sequenced, facilitating the understanding of their functions in the above-mentioned particular habitats and easily getting their proteome profiles. The proteomic studies of several groups of microbial species and the research achievements revealing their unique metabolic abilities are listed in Table 3.1.
Table 3.1. Proteomic studies of different microbial species and the research achievements.
| Microbial group | Point of interest | Proteomic studies |
| g. Bacillus |
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| g. Pseudomonas |
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| g. Halobacterium |
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| Sulfate-reducing bacteria of g. Geobacter and Desulfovibrio |
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| Methylotrophic bacteria |
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| Denitrifying bacteria / dehalogenators |
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| Fermenting bacteria and yeast |
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| Cyanobacteria / photosynthetic bacteria |
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3.2. Proteomic studies of natural microbial communities
3.2.1. Metaproteomics
Proteomic studies upgrade the genomics data since the proteome of a living system reflects its operating enzymes under certain conditions. Moreover, the proteomic analyses enrich the gene expression profiles obtained by microarray application with information about the proteins’ posttranslational modifications. Thus, proteomics can operate for all microorganisms, not only for those in which genetic information is sequenced.
The microbial natural habitats represent complex communities for which the environmental proteomics study approach can contribute significantly to highlighting the structure and function of microbial consortia. Namely, such global analysis of the microbial communities in the environment is called metaproteomics. The metaproteomic analysis approach considers the microbial communities a living meta-organism. Any physiological change in this meta-organism registered at the proteomic (i.e., population) level can be interpreted as a functional response to environmental alterations. Another reason to consider the microbial consortia a whole organism rather than a simple collection of individual microbes is the ability of these meta-organisms to exchange genes and share metabolic capabilities.
From technical point of view, the metaproteomic approach provides great advantage compared to standard biochemical/molecular biology analyses since it omits the laborious steps of isolation and maintenance of pure cultures and all the species comprising a certain microbial consortium. Additionally, metaproteomics spreads light on the multifaceted relationships among microorganisms within a community. Such information is not possible to be generated with pure cultures.
It is true that proteomic studies are more challenging than those of the pure cultures. The community samples subjected to proteomic analyses are complex in content and structure, comprising a big volume of ORF to be studied for finding putative genes. This tremendous number of gene products makes the data processing and interpretation quite difficult since there aren’t genome sequencing data for the majority of the consortia representatives. The in silico analysis that mediates the gene product annotation is also crucial for the success of the metaproteomic studies.
At present, the advancement of the protein extraction methods and their identification directly in the environmental samples (from water or soil habitats) overcomes the hurdles imposed by the laborious traditional approaches and guarantees faster and better results. The pyrosequencing approach that is progressively gaining application in metagenomic studies is a good example of such modern methods.
3.2.2. Natural microbial communities
The metaproteomic studies of microbial communities in their natural habitats help to elucidate valuable functional peculiarities of these communities. Soil and aquatic (marine) microbial communities have been studied using a metaproteomic approach, and valuable information with potential practical application has been accumulated.
For instance, the surface water microbial communities were initially studied through 1D and 2D SDS-PAGE. These methodological approaches resulted in protein fingerprint generation and, for some of the proteins – in their functional characterization. Following the technological advancement, the shotgun technique was explored for studying microbial biofilms of acid-mine drainages. This approach resulted in the identification of more than 2000 proteins using a sequences data set generated from the same environmental habitat. Metaproteomic studies were used to identify the protein repertoire of individual strains, which proved to dominate in the community. Thus, evidence is accumulated for horizontal gene transfer that contributed to the successful microbial adaptation to the harsh environmental conditions in the acid-mine habitat.
The metaproteomic studies of soil microbial communities are also advancing. The bottleneck step in the methodological approaches is the lack of technology that allows direct protein extraction from the complex soil medium. One has to keep in mind that the extracted proteins have to be of sufficient quantity and quality to generate reliable proteomic data. Searching to resolve this hampering problem, the scientists succeeded in generating soil proteome fingerprints on a model matrix of dissolved organic matter. Using this approach, they determined the presence of abundant laccase and cellulase hydrolytic enzymes. They proposed their use as indicators for the presence of active microorganisms in a defined ecosystem. Another model of complex environmental matrices has been used to assess the presence of target microorganisms in very low concentrations (about 6%).
These data are recommended for evaluation of the presence of potential biothreats agents that reflect the scarce species abundance.
3.2.3. The microbial communities’ functional diversity seen through proteomics
The study of the protein reservoir in living systems is gaining increasing popularity. Accordingly, the bioinformatics data input is enlarged. Besides these robust information flows regarding protein structure and function, the direct relationship of microbial community structure and operation with an ecosystem and the individual protein profiles’ estimation is difficult. To reveal the functional significance of protein diversity in an ecosystem, 1) the protein diversity must be characterized, and 2) the proteomic assemblage must be analyzed. For this purpose, model systems are used under controlled conditions that match the experimental data to the particular statistical model. Protein identification and relative quantitative abundance in the model systems are the obligatory first step in this approach. It is not possible to determine the absolute quantity and the entire protein diversity in an ecosystem. Data estimate figures like 1 x 104 – 109, even 1010 in proteome assemblages, containing both pro- and eukaryotes. Since there is a remarkable correspondence between biodiversity data and environmental proteomics data compared through the indices sampling procedure, raw analytic data, and reference data, the statistical approaches commonly applied for biodiversity data analyses can also be applicable to environmental proteomics data. This mutual analytical approach will help further explore the proteomic diversity patterns and evaluate their functional significance.
Benndorf et al. (2007) have established a protocol for metaproteomic analysis of groundwater and soil directed towards revealing the functional aspects of a certain microbial community.
4. ENVIRONMENTAL PROTEOMICS RESEARCH AND APPLICATION
4.1. Metabolic engineering
Metabolic engineering is a multidisciplinary branch of science that deals with the generation of desirable products through the manipulation of the metabolic characteristics of a host organism. As an essential element of effective protein design, metabolic engineering contributes to value-added protein biosynthesis at high productivity. Besides its great advantage in the design and production of targeted products through the optimization of genetic and regulatory processes within the cell producers, metabolic engineering faces certain obstacles that underpin its practical application. For instance, along with the production of exocellular protein, the host cell undergoes physiological shifts that impact other cellular processes, especially the production of the other host cell proteins. Proteomic analyses can significantly contribute to elucidating the host cell response to heterologous gene expression since the process is catalysed and/or regulated by proteins or protein complexes, and alterations in their production can be predicted along the exogenous genes’ expression. The proteomic analysis provides valuable data during the heterologous expression of intracellular enzymes grounded on this fundamental action pattern during metabolic engineering. Examples are the expression of glutathione S-transferase and epoxide hydrolase (essential components of the cellular enzymatic antioxidant defence system). Through a quantitative proteomics approach, Lee et al. (2006) found that the application of this metabolic engineering strategy led to the induction of the enzymes from the pyruvate metabolism, glutathione synthesis, and antioxidant defence, and the repression of the enzymes from the gluconeogenesis, TAC, indole synthesis and fatty acids synthesis.
The gut microbe Escherichia coli, although not native to soil and aquatic environments but used as a s classical metabolic engineering host, has also been extensively studied with the tools of environmental proteomics. The analyses of the E. coli behaviour in an ecological context, based on the proteome study of the E. coli strain, metabolically engineered for biodegradation of trichloroethene with introduced six genes of Burkholderia cepacia G4 strain, showed that the physiology of the metabolically altered host strain is changed because of the insertion of the genes.
4.2. Microbial ecology
Traditional ecological studies search for naturally occurring adaptation of bacteria to their environments. Environmental proteomics contributes to these studies due to the provision of insights into the adaptation mechanisms, especially under extreme environmental conditions (e.g., hyperthermophilic ones). Thus, the hyperthermophiles’ proteins focus the research due to their enhanced conformational stability and high-temperature operability. Studying this phenomenon sheds light on the molecular mechanisms of protein folding. Prosinechki et al. (2006) have studied the hyperthermophilic archaeon belonging to g. Sulfurispharea, which can grow within the thermal range of 70 – 97oC. Using the dynamic proteome perturbation technique, they have identified proteins with higher stability involved in essential cellular processes – detoxification, nucleic acid processing, energy metabolism, etc.
The study of the other temperature extremity, the cold, has also contributed to the microbial adaptation mechanism revealing. Applying proteomic analysis, Qiu et al. (2006) studied the adaptation of an Exiguobacterium strain isolated from Siberian permafrost sediment to low temperatures. Combining chromatofocusing and MS, the authors identified over 250 proteins uniquely or preferentially expressed at 4 oC. They identified cold-acclimation and cold shock proteins that indicated a cold-adaptation cellular strategy that includes physiological processes regulation through individual cellular protein regulation. It can be speculated that the proteins upregulated at lower temperatures enable the cells to adapt to temperatures near or below zero. Proteomic studies can contribute to proving the hypothesis about bacterial cold adaptation. Extensive proteomic studies are needed to provide information about the whole protein assemblage of the cell and its operation. The small sets of proteins studied in a specific functional context do not make the big picture. This fact is proved by another study with Colwellia psychrerythraea, in which changes to the cell membrane fluidity cryotolerance compounds synthesis and uptake and strategies for overcoming temperature-dependent barriers to C-uptake have been proved.
A combinatory effect of two different abiotic stress factors – salt and cold, was proven in Psychrobacter sp. Zheng et al. (2006). Various proteins were identified in cold adaptation in the presence of salt, showing a combination effect of salt and cold on protein expression.
4.3. Environmental stress tolerance
The big advantage of proteomics to protein identification without genomic sequencing makes this molecular approach a versatile tool to gain insights into physiological responses to environmental abiotic stress conditions. The proteomics capability to offer system-wide information for microorganisms in their native environment urges its application in microbes’ response assessment to thermal, chemical, oxidative, and other stresses. The 2D SDS PAGE and the chromatography-based proteomic studies are the two most explored methods for studying the mechanisms of low and high temperature, low pH, heavy metals, and oxidative stress tolerance. These studies proved the upregulation of well-known stress-response proteins and registered proteins involved in other adaptation/detoxification activities, such as lipid biosynthetic pathways, osmoprotectants’ accumulation, novel transporter proteins, etc.
In addition, it became clear that the stress response regulation might be differentiated in case one species is subjected to different stresses. A typical example of this observation is Desulfovibrio vulgaris exposed to high salt concentrations, nitrate stress, and elevated temperatures. Proteomic analyses showed the up- and down-regulated proteins/protein systems patterns under different stress conditions. It was found that D. vulgaris responded to NaCl and KCl stresses similarly. However, they react differently to nitrate stress and high temperatures. Moreover, the proteomics study speculates posttranslational modification of the proteins’ chaperones. All these data indicate that proteomic analysis discloses system-wide stress responses. In addition, this approach reveals the specific defense mechanisms attributed to each stress condition.
5. ENVIRONMENTAL PROTEOMICS POTENTIAL
5.1. Methodological and technical innovations
Within its quarter-century history, proteomics has undergone a transformation from a biochemical analysis of individual proteins to a technique for matching many diverse protein characteristics, such as expression patterns, structure, localization, interactions, and modifications, at any stage of development of the living systems. Proteomics objective is to study the organism’s proteome dynamic along its development.
From a technological point of view, proteomics relies on recent technological advances to reach its scientific objective. The development of 2D electrophoresis in the late 90s of last century allowed for mapping a whole proteome for the first time. Later, protein sequencing technologies enhanced proteomics development and helped to reveal its full capacity in protein identification and characterization. The high sensitivity of analysis offered by mass spectrometry (MS) and its coupling with complementary gas-phase technologies (e.g., ion mobility spectrometry) turned MS into a principal analytical methodology in proteomic analysis.
The protein extraction and derivatization methods coupled with the above-mentioned separation approaches contribute to researchers’ ability to resolve complex protein mixtures into their elements.
Finally, the processing and interpretation of the vast amount of raw proteomic data accumulated through the experimental stages of research are supported by the constantly improved methods of bioinformatics analysis that facilitated the gaining of deeper insight into biological functions and their regulation in the living systems. Bioinformatics, as an integrated branch of mathematics, biostatistics, and computing analyses of genomics and proteomics studies’ bio-data through sophisticated tools, assists in experimental results’ annotation and interpretation. In addition, this methodological approach helps in the molecule design that can interact with proteins of interest and in protein-protein interaction prediction. Thus, bioinformatics is essential for converting primary proteomics data into knowledge and its application.
However, besides these technological advancements, the proteomics methodology has limitations that hamper its universal application. For instance, it is still impossible to get data for the total protein pool presented in a sample because of their concentration range that is quite large and also due to the lack of appropriate methodology for the amplification of proteins whose concentration is very low.
Table 3.2 presents the recent technological developments of the major groups of proteomics methodology with emphasis on their advantages, bottle-neck steps in performance, and new developments in the state of the art. The chart in Fig. 3.1 outlines how these methodological approaches are organized in a proteomics workflow.
Table 3.2. Synopsis of the recent technological developments of the major groups of proteomics methodology.
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Method |
Advantages |
Bottle-neck steps in performance |
New developments |
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Individual methods |
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| 2D polyacrylamide gel electrophoresis (2D-PAGE) – method for proteins separation based on their mass and charge (isoelectric points) |
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| Liquid chromatography (LC) – a method for quantitative separation and identification of compounds in a complex mixture |
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| Isotope-coded affinity tag labelling (ICAT-labelling) – a method for relative quantification of peptides based on labelled peptides as internal standards |
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| Mass spectrometry (MS) – a technique for mass determination adapted for protein identification based on mass-to-charge (m/z) ratio approach and use of different ionization methods: |
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| Phage display – making peptide/protein libraries on viral surfaces and screening them for activity |
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| Cloning of ligand (peptide) targets that bind to larger domains within proteins |
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| Coupled methodologies approaches | |||
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Figure 3.1. Schematic presentation of the proteomics technologies work flow. Source: Cho, 2007.
5.2. Experimental environmental proteomics at a glance
Environmental proteomics can enlarge our knowledge of how proteins interact in an ecosystem or, at least, in a species assemblage. Understanding the mechanisms of this interaction will shed light on the performance of the ecological processes. To understand the functional significance of proteins in an ecosystem, an approach that groups proteins based on their biochemical properties, subcellular localization, and bioprocesses they take part in seems reasonable and informative. The gene ontology databases that classify proteins in functional categories are a good option in this context, and proteomics might contribute to the generation of new ecological insights since it provides information on functionally equivalent proteins produced by (micro)organisms living in similar environments. Thus, instead of characterizing the whole protein pool in an assemblage, it is better to focus on a smaller number of proteins identified to change their quantification as a response to environmental conditions. Experimentally, an ecologist could add or remove a species from an assemblage and find out which one is responsible for responding to a given environmental factor. The described approach, named molecular phenotyping of an assemblage, contributes to our knowledge of how proteins function and their dynamics in a temporal perspective. This will help ecologists predict which proteins play an essential role in the ecosystem.
Furthermore, the experimental throughputs in synthesizing proteins in vitro will assist ecologists in the design of specific experimental mesocosms as models of food webs and following protein destiny within them under various environmental conditions or enriching the ecosystems with potentially essential proteins and metabolites and measuring the food-web responses. Thus, ecological proteome quantification and measuring the biodiversity response when species are added or removed from an ecosystem or artificially synthesized proteins are added to it will provide deep insight into the proteins’ functions in ecosystem organization.
6. CHALLENGES, FRONTIERS, AND PERSPECTIVES
Proteomics is a powerful omics branch that possesses the potential to expand further microbiology, microbial ecology, and environmental biotechnology studies. Currently, most of the environmental proteomics studies with microorganisms are laboratory-based. However, the first metaproteomics investigations shed light on the capability of proteomics to contribute to proteins’ identification and characterization within microbial consortia besides the lack of sequencing data or phylogenic peculiarities. One of the most essential challenges that threaten the development of environmental proteomics is the necessity of protein extraction methods development from microbial communities in different media (soil, water). Moreover, progress in bioinformatics science and its tools is also anticipated better to identify peptides and proteins from metaproteomics raw data.
The proteins give the cellular structure, generate energy, underpin communication, and allow reproduction. Thus, they provide the structural and functional networks of the living cell. While genetic information is static, the protein component of a cell is dynamic. It is well known that proteins are much more complex and difficult to work with than nucleic acids (DNA and RNA). This challenge triggered the research on peptides/proteins and shaped the perspectives of proteomics regarding the:
- Protein physical/chemical properties. It is the secondary and tertiary structures that have to be minded and maintained during the analysis; the process of denaturation that has to be regarded, and the relevant potential denaturing factors: enzymes, temperature, irradiation, mechanic forces, etc.; the problems with protein solubility to be solved.
- Protein quantification. One should be concerned that proteins can not be amplified like DNA, and when they are not abundant, they can not be detected. The recently developed technology by Thulasiraman et al. (2005) explores ligand library beads that allow access to many proteins at minor concentrations and are practically undetectable by classical analytical techniques. This approach leads to trace proteins’ concentration on their specific affinity ligands.
- Microarray and protein sensitivity. Nettikadan et al. (2006) proposed a technique that combines microarray and screening of small sample volumes to design the so-called ultramicroarrays that offer high specificity and high sensitivity that help to overcome the problem with the small volumes and protein quantities. This strategic approach yields reliable techniques to deal with limited protein quantities.
- Multiomics integration. The integration of proteomics with genomics and metabolomics is still a challenge but possesses the potential of revealing proteins functional diversity. Thus, proteomics contributes to the development of functional genomics for the benefit of biomedical research and impacts the development of innovative products for diagnostics and therapy.
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