Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function. To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH \u3c 1) and hot (temperature \u3e 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment. The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the \u3e 100,000 mass spectra collected during an MS experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth

Comparative Proteomics Reveals Core vs. Unique Molecular Signatures for Dissimilatory Metal Reducing Bacteria Grown with Various Electron Acceptors

Dissimilatory metal reducing bacteria (DMRB) are probably one of the most respiratory versatile microorganisms on earth. Their ability to use metals as terminal electron acceptor allows them to survive in severe environments (e.g. radionuclide contaminated soil). In addition to metals, many other organic and inorganic substrates can be utilized as electron acceptors for DMRB respiration, including fumarate, nitrate, oxygen, etc. Genome information for many DMRB species is available, which reveals large numbers of c-type cytochrome encoding genes present in their genomes. For example, the genomes of three DMRBs, Anaeromyxobacter dehalogenans strain 2CP-C, Shewanella oneidensis strain MR-1, and Geobacter daltonii strain FRC-32, contain 69, 40, and 72 putative c-type cytochrome genes, respectively. Although mutagenesis techniques have determined the respiratory roles of several c-type cytochromes, gene disruption for majorities of the putative c-type cytochromes does not generate visible phenotypical alterations, and is not able to functionally link them to specific respirational activities. Thus, comprehensive proteome characterization for DMRBs is needed to elucidate the molecular mechanisms underlying their respirational versatilities. In this dissertation, a mass spectrometry-based proteomics approach was used to interrogate the proteomes of A. dehalogenans strain 2CP-C, S. oneidensis strain MR-1, and G. daltonii strain FRC-32. The proteomic responses of DMRBs to a wide range of electron acceptors were tested in this dissertation, including soluble and insoluble ferric iron, manganese oxide, fumarate, nitrate, oxygen, and nitrous oxide. The in-depth proteomic characterizations comparatively revealed the c-type cytochrome profiles of DMRBs, providing evidence for the identities and expressions of putative c-type cytochromes, and established the linkage between specific electron acceptor and individual c-type cytochromes. The entire proteome complements of DMRBs were also characterized, generating metabolic maps reflecting pathway-level activities responding to various electron acceptors. The results identified the core proteome carrying out the essential cellular machineries for each tested DMRB, and demonstrated clearly elevated energy metabolism for A. dehalogenans strain 2CP-C during respiration of metal electron acceptors. Comparative proteomics analysis between tested DMRB strains revealed the commonalities and differences of proteomic phenotypes displayed by different strains, and shed light into deeper understandings for DMRB metabolic activities

Characterization of the Human Host Gut Microbiome with an Integrated Genomics / Proteomics Approach

The new field of ‘omics’ has spawned the development of metaproteomics, an approach that has the ability to identify and decipher the metabolic functions of a proteome derived from a microbial community that is largely uncultivable. With the development and availabilities of high throughput proteomics, high performance liquid chromatography coupled to mass spectrometry (MS) has been leading the field for metaproteomics. MS-based metaproteomics has been successful in its’ investigations of complex microbial communities from soils to the human body. Like the environment, the human body is host to a multitude of microorganisms that reside within the skin, oral cavity, vagina, and gastrointestinal tract, referred to as the human microbiome. The human microbiome is made up of trillions of bacteria that outnumber human genes by several orders of magnitude. These microbes are essential for human survival with a significant dependence on the microbes to encode and carryout metabolic functions that humans have not evolved on their own. Recently, metaproteomics has emerged as the primary technology to understand the metabolic functional signature of the human microbiome. Using a newly developed integrated approach that combines metagenomics and metaproteomics, we attempted to address the following questions: i) do humans share a core functional microbiome and ii) how do microbial communities change in response to disease. This resulted in a comprehensive identification and characterization of the metaproteome from two healthy human gut microbiomes. These analyses have resulted in an extended application to characterize how Crohn’s disease affects the functional signature of the microbiota. Contrary to measuring highly complex and representative gut metaproteomes is a less complex, controlled human-derived microbial community present in the gut of gnotobiotic mice. This human gut model system enhanced the capability to directly monitor fundamental interactions between two dominant phyla, Bacteroides and Firmicutes, in gut microbiomes colonized with two or more phylotypes. These analyses revealed membership abundance and functional differences between phylotypes when present in either a binary or 12-member consortia. This dissertation aims to characterize host microbial interactions and develop MS-based methods that can provide a better understanding of the human gut microbiota composition and function using both approaches

Genomic and Proteomic Characterisation of the European House Dust Mite, Dermatophagoides pteronyssinus

House dust mites are major causative agents in the pathogenesis of allergy. Their proximity with human habitats, association with development of allergenic diseases, and resistance to physical and chemical control measures; make them some of the most medically important mites. Understanding of house dust mites has been hampered by a lack of genomic sequence data and limited to a discrete number of proteins. The work presented here is a detailed characterisation of the European house dust mite, Dermatophagoides pteronyssinus airmid strain, at the genomic and proteomic level. Genomic sequencing and assembly resulted in a high-quality assembly of 70.76 Mb in size with 96.86% coverage. A comprehensive bioinformatic and proteomic examination was conducted on the 12,530 predicted proteins, validating the expression of 4,002. A small group of D. pteronyssinus airmid proteins showed significant homology to known allergens from other species. A large scale comparative proteomic investigation of the mite body and spent growth medium allowed for: (i) qualitative assessment of allergen localisation and (ii) the identification of numerous enzymes that may be involved in key physiological activities. The characterisation of protein extracts from house dust also identified a substantial number of uncharacterised D. pteronyssinus proteins in addition to known and putative allergens. The genes encoding novel β-1,3 glucanases were identified within a trigene cluster in D. pteronyssinus airmid. Recombinant protein expression, biochemical and proteomic analysis revealed Glu1 and Glu2 to exhibit hydrolytic activity toward β-1,3 glucans and have increased expression in the mite body and excretome of D. pteronyssinus in response to yeast diet. Further proteomic and enzymatic analysis correlated glucanase activity in house dust with presence of Glu1 and Glu2. These findings provide evidence that active β-1,3 glucanases are expressed and excreted in the faeces of D. pteronyssinus in response to fungal diet, in both the laboratory and the wild-type environment

QUANTITATIVE CHARACTERIZATION OF PROTEINS AND POST-TRANSLATIONAL MODIFICATIONS IN COMPLEX PROTEOMES USING HIGH-RESOLUTION MASS SPECTROMETRY-BASED PROTEOMICS

Mass spectrometry-based proteomics is focused on identifying the entire suite of proteins and their post-translational modifications (PTMs) in a cell, organism, or community. In particular, quantitative proteomics measures abundance changes of thousands of proteins among multiple samples and provides network-level insight into how biological systems respond to environmental perturbations. Various quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling. This dissertation starts with systematic comparison of these three methods, and shows that isobaric chemical labeling provides accurate, precise, and reproducible quantification for thousands of proteins. Based on these results, we applied this approach to characterizing the proteome of Arabidopsis seedlings treated with Strigolactones (SLs), a new class of plant hormones that modulate various developmental processes. Our study reveals that SLs regulate the expression of a range of proteins that have not been assigned to SL pathways, which provides novel targets for follow-up genetic and biochemical characterization of SL signaling. The same approach was also used to measure how elevated temperature impacts the physiology of individual microbial groups in an acid mine drainage (AMD) microbial community, and shows that related organisms differed in their abundance and functional responses to temperature. Elevated temperature repressed carbon fixation by two Leptospirillum genotypes, whereas carbon fixation was significantly up-regulated at higher temperature by a third member of this genus. Further, we developed a new proteomic approach that harnessed high-resolution mass spectrometry and supercomputing for direct identification and quantification of a broad range of PTMs from an AMD microbial community. We find that PTMs are extraordinarily diverse between different growth stages and highly divergent between closely related bacteria. The findings of this study motivate further investigation of the role of PTMs in the ecology and evolution of microbial communities. Finally, a computational approach has been developed to improve the sensitivity of phosphopeptide identification. Overall, the research presented in the dissertation not only reveals biological insights with existing quantitative proteomics methods, but also develops novel methodologies that open up new avenues in studying PTMs of proteins (e.g. PTM cross-talk)

DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY-BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as plants or humans, remains challenging because of the incongruity between the complexity of the biological samples and the analytical techniques available. In this dissertation, analytical methods utilizing state-of-the-art MS instrumentation have been developed to address challenges associated with both qualitative and quantitative characterization of eukaryotic organisms. In particular, these efforts focus on characterizing Populus, a model organism and potential feedstock for bioenergy. The effectiveness of pre-existing MS techniques, initially developed to identify proteins reliably in microbial proteomes, were tested to define the boundaries and characterize the landscape of functional genome expression in Populus. Although these approaches were generally successful, achieving maximal proteome coverage was still limited by a number of factors, including genome complexity, the dynamic range of protein identification, and the abundance of protein variants. To overcome these challenges, improvements were needed in sample preparation, MS instrumentation, and bioinformatics. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: 1) network-wide, 2) pathway-specific, and 3) protein-level viewpoints. In addition, we implemented two bioinformatic approaches that were capable of decoding the plasticity of the Populus proteome, facilitating the identification of single amino acid polymorphisms and generating a more accurate profile of protein expression. Though the methods and results presented in this dissertation have direct implications in the study of bioenergy research, more broadly this dissertation focuses on developing techniques to contend with the notorious challenges associated with protein characterization in all eukaryotic organisms

Causes and consequences of mine waste microbial community structure

Acid mine drainage (AMD) is a widely studied environment in microbiology and geochemistry. However, there have been far fewer detailed studies of the microbiology and biogeochemistry of historic sulfidic mine wastes giving rise to AMD. Key questions have yet to be answered about the ecological mechanisms underlying the relationship between microbial communities and mineral substrates, the environmental features imposing selective pressure on such communities compared to nearby soils and the main ecological principles that can be used to explain such complex relationships. The South West of England has been subject to intensive mining activity, resulting in a variety of mine wastes and disused underground tunnels left undisturbed for decades. The microbial consortia inhabiting these environments make an interesting case study, as they derive from the same region and yet their similarity is unknown. Samples of mine waste and nearby soils were collected from twelve sites in Cornwall and West Devon. Geochemistry and microbial ecology were analysed to study the environmental drivers of microbial community composition. Metals from different fractions of the samples were analysed (total, readily extractable and pore water) and their compositions related to the microbial community. The microbial ecology of most sites appeared to be largely associated with pH, and to a lesser extent to the bulk metals composition. and communities were more diverse in waste sites than nearby soils. This suggested the possibility of strong local adaptation or dispersal limitation. Information on local adaptation of consortia is potentially useful for further manipulations as it provides insights into their performance in defined conditions. Therefore, inocula prepared from the twelve mine wastes were assessed for local adaptation to sympatric and allopatric substrates via a reciprocal transplant experiment. Results revealed that, with the exemption of a few sites, microbial communities were not generally locally adapted. Bioleaching performance (pyrite dissolution) was further analysed to understand how this is improved (or not) through community mixing and coalescence. Four inocula were mixed in all possible sixteen combinations to form new coalesced inocula whose performance was tested in pyrite, showing that coalescence potentially increases performance. The results give insights for the use of communities in biotechnologies such as biohydrometallurgy, as well as the microbial ecology of AMD-generating wastes. This study contributes to the knowledge of the microbial ecology of acidophiles in the scenario of whole communities coalescence and transplant