Cloning of Identified Pathways

The goal of the Functional Genomics Core is to develop the experimental methods to elucidate the regulatory networks in the stress responses of Desulfovibrio vulgaris, Shewanella oneidensi, and Geobacter metallireducens. Specifically, we propose to:

• use existing DNA arrays for S. oneidensis, and to develop DNA arrays for D. vulgaris and G. metallireducens to measure the transcript profile (transcriptome) during the response to various environmental stresses;
• use HPLC-MS-MS to measure the protein profile (proteome) of D. vulgaris, G. metallireducens, and S. oneidensis during the response to various environmental stresses;
• measure the metabolite profile (metabolome) of D. vulgaris, G. metallireducens, and S. oneidensis during the response to various environmental stresses;
• determine protein-protein interactions in the signaling cascade of D. vulgaris, G. metallireducens, and S. oneidensis during the response to various environmental stresses;
• generate mutants in the various genes whose gene products are found to be responsible for a particular stress response, and to compare transcript, protein, and metabolite profiles in the mutant and isogenic wild-type strains;
• produce small-molecule inhibitors that will interrupt the interaction between key proteins in the stress response, and to compare transcript, protein, and metabolite profiles in the treated and untreated strains; and
• biopan the environment for stress response pathways.

A diagram explaining how these goals relate to each other the overall GTL project is shown in Figure 3.1. In general, samples (from bioreactors or the environment, subjected to a stress or grown in the absence of stress) will be obtained from the Applied Environmental Microbiology Core. These samples will be analyzed to elucidate the changes in transcript, protein, and metabolite levels in response to a particular stress. The data from these analytical techniques is sent to the Computational Core where it is analyzed to determine potential stress response networks. The information about potential stress response networks is used to design genetic mutants or chemical inhibitors to further elucidate the stress response. These chemically-inhibited wild-type strains and mutant strains are then sub­jected to the stress, and their response is compared to that of the wild-type strain. Further, mutations and/or chemical inhibitors may be needed depending on the nature of the network controlling the stress response. The Functional Genomics Core is highly dependent on the Applied Environmental Microbiology Core and the Computational Core for a variety of products. To see where the interaces are see Figure 1.9. For details with the AEM Core see Figure 2.1.