Geobacter species are of interest because of their role in in situ bioremediation of uranium and harvesting electricity from waste organic matter and renewable biomass. As part of our involvement in the Genomics:GTL Geobacter Project, we are investigating networks of regulatory interactions in this versatile group of microorganisms in order to elucidate molecular mechanisms of their regulatory response to environmental changes.

Figure 1. An example of GSEL query output listing predicted regulatory sites and operon structure in a region of the G. sulfurreducens genome.

Individual regulatory sequence elements are predicted using analyses of individual microarray data sets or sequence data alone. For example, G. sulfurreducens has molybdate-responsive transcription factor, ModE (GSU2964). We identified 80 likely ModE binding sites in the genome of G. sulfurreducens, including likely functional sites in the upstream regions of (1) the modABC operon; (2) an operon containing gene NP_954455, a distant homolog of the moaA gene that encodes the molybdenum cofactor biosynthesis protein A in Archaeoglobus fulgidus and Sulfolobus tokodaii; (3) an operon that encodes a putative membrane protein (NP_954447) with homology to permeases of the drug/metabolite transporter (DMT) family (COG0697); and (4) glycine (CCC) tRNA.

Similarly, G. sulfurreducens contains RpoS (σ^S), a global regulator of gene expression in G. sulfurreducens, in addition to major housekeeping sigma factor RpoD (σ⁷⁰). Our analysis of conservation and divergence of possible functional RpoD amino acid residues suggested that most of them are substantially conserved between E. coli and G. sulfurreducens. However, some degree of sequence variation between the two species was observed in several amino acid sites of RpoS proteins that might be important for promoter recognition. In addition, the similarity or identity of a number of residues between G. sulfurreducens RpoS and RpoD that might be functionally important suggests that the differences between the promoters recognized by RpoS and RpoD in G. sulfurreducens may be subtle. Using microarray gene expression information, we have been able to suggest a number of RpoS-regulated promoter elements as well as elements regulated by other sigma factors including RpoD. Experimental analysis of several promoters predicted to be RpoS-regulated and those predicted to be RpoD-regulated fully validated computational predictions.

In another example of analysis, we predicted gene regulatory interactions using information from the Rel_Gsu regulon. Rel_Gsu is the single Geobacter sulfurreducens homolog of RelA and SpoT proteins found in other organisms to be involved in regulation of levels of guanosine 3’, 5’ bispyrophosphate, ppGpp, a molecule that signals slow growth and stress response under nutrient limitation in bacteria. We used information obtained from genome wide expression profiling of the rel_Gsu deletion mutant to identify putative regulatory sites involved in transcription networks modulated by Rel_Gsu or ppGpp. We identified likely sites regulated by Fur (ferric uptake repressor) in the upstream regions of upregulated operons and RpoS-regulated promoters in the upstream regions of the downregulated operons of the rel_Gsu deletion mutant. These findings suggest that Fur- and RpoS-dependent gene expression in G. sulfurreducens is affected by ppGpp-mediated signaling.

Among multiple other sequence and gene expression analyses by our group of regulatory interactions influenced by specific transcription factors, our most recent analyses involve prediction of the promoter elements regulated by alternative sigma factor RpoN (σ54). We predicted 467 RpoN-regulated promoter elements that had the same orientation with their downstream target genes or operons, including 110 such elements in the noncoding regions. We identified those promoters for which the expression of their target genes was significantly altered in the RpoN gene overexpression microarrays. Further analyses focus on the function of the specific genes whose regulation may be significantly affected by RpoN and on their possible role in different environmental conditions.

Data mining of predicted regulatory interactions allowed us to identify genome regulatory regions and their target operons that are involved in a variety of regulatory pathways. This powerful approach allows us to identify gene products that may be central to G. sulfurreducens response to a variety of trigger conditions, to find genes and operons whose expression may be altered in response to very specific sets of conditions, and to suggest the molecular mechanisms of their regulation.