SUBPROJECT 3
The objective of this subproject is to correlate the hypothetical proteins encoded in the composite genome of the AMD microbial community with characterized proteins and with environmental and cellular activities. A central feature of the biofilm community that thrives in an acidic, hot, and toxic metal environment is that it is rich in novel proteins and biochemical pathways that are intimately linked with local geochemistry. The unusually well characterized genome and proteome of this consortium, its defined geochemical context, and the abundance of biomass make it an ideal system to develop methods for isolating and characterizing proteins directly from environmental samples. Our approach will combine several technologies into a unified proteogenomic function analysis for a comprehensive description of complexes that include novel proteins in this microbial community. Initially, we will identify all of the hypothetical proteins and their distributions within community composite proteomes from different growth stages, nearby locations, and mixtures of cultivated microorganisms. Physical properties predicted from the genome and proteome datasets will be used for targeted purification of specific novel proteins directly from environmental samples. Using surface labeling and highly resolved subcellular fractionation, we will further discriminate the localization of novel proteins within source organisms. We will develop new separation methods for isolating high molecular mass protein complexes containing novel proteins, and use amino acid sequencing and mass spectrometry analysis to identify the individual complex members. Proteins thus identified will be correlated with genomic context for indications of coordinated gene expression under different environmental conditions or stages of biofilm growth. Distinguishing features from these analyses will be combined with bioinformatics, metabolic models, and advanced computational modeling to predict detailed protein structural and functional characteristics, including properties related to environmental adaptation that are relevant to biotechnology applications. Predictions of biochemical function will then be tested directly, using both new and conventional biochemical assays. The integration of these several methods into an overall proteogenomic function analysis that is centered on naturally occurring, novel proteins from a defined model ecosystem is certain to solve many longstanding questions and accelerate research on metabolic and signaling networks, both in this community and in other microorganisms where such novel proteins are conserved.