Proteome characterizations of microbial systems using MS-based experimental and informatics approaches to examine key metabolic pathways, proteins of unknown function, and phenotypic adaptation

Microbes express complex phenotypes and coordinate activities to build microbial communities. Recent work has focused on understanding the ability of microbial systems to efficiently utilize cellulosic biomass to produce bioenergy-related products. In order to maximize the yield of these bioenergy-related products from a microbial system, it is necessary to understand the molecular mechanisms.The ability of mass spectrometry to precisely identify thousands of proteins from a bacterial source has established mass spectrometry-based proteomics as an indispensable tool for various biological disciplines. This dissertation developed and optimized various proteomics experimental and informatic protocols, and integrated the resulting data with metabolomics, transcriptomics, and genomics in order to understand the systems biology of bio-energy relevant organisms. Integration of these various omics technologies led to an improved understanding of microbial cell-to-cell communication in response to external stimuli, microbial adaptation during deconstruction of lignocellulosic biomass and proteome diversity when an organism is subjected to different growth conditions.Integrated omics revealed Clostridium thermocellum's accumulate long-chain, branched fatty acids over time in response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. A striking feature implies a restructuring of C. thermocellum's cellular membrane as the culture progresses. The membrane remodulation was further examined in a study involving the swarming and swimming phenotypes of Paenibacillus polymyxa. The possible roles of phospholipids, hydrolytic enzymes, surfactin, flagellar assembly, chemotaxis and glycerol metabolism in swarming motility were investigated by integrating lipidomics with proteomics.Extracellular proteome analysis of Caldicellulosiruptor bescii revealed secretome plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. This study further opened the avenue for research to characterize proteins of unknown function (PUFs) specific to growth conditions.To gain a better understanding of the possible functions of PUFs in C. thermocellum, a time course analysis of C. thermocellum was conducted. Based on the concept of "guilt-by-association," protein intensities and their co-expressions were used to tease out the functional aspect of PUFs. Clustering trends and network analysis were used to infer potential functions of PUFs. Selected PUFs were further interrogated by the use of phylogeny and structural modeling.