Interplay of ScoC, CodY and AbrB in the regulation of the oligopeptide transporter OppA in G. stearothermophilus

The extracellular xylanase gene, xynA, is regulated by quorum sensing and this regulation depends on a functional oligopeptide transport system (oppABCDF), suggesting that the transporter is essential for importing the extracellular cell density factor. CodY and ScoC are global transcriptional regulators in Gram-positive bacteria. Gel retardation assays and isothermal titration calorimetry indicated that ScoC binds specifically to the oppA operator, and CodY was shown to bind in vitro to the -35 region of scoC gene promoter. These results suggest that oppA is regulated directly by ScoC and indirectly by CodY. Using oppA-lacZ fusions in B. subtilis, we show that oppA expression is lower in a codY null strain, probably due to the higher levels of ScoC in this background. Surprisingly, in a scoC/codY double mutant, oppA expression remains low, suggesting that an additional factor is involved, and in fact, potential AbrB binding sites are located in the promoter region of oppA. The 3D crystal structure of ScoC was recently determined at 3.0 Å resolution, revealing a novel tetrameric X-shape assembly with two pseudo-symmetric DNA binding sites on the two opposite sides. To demonstrate that ScoC binds two sites we performed a fluorescence resonance energy transfer (FRET) assay. Indeed, in the presence of ScoC and 248 bp dsDNA labeled with Cy3 and Cy5 at both ends of DNA, the fluorescence intensity was enhanced. Insertion of specific nucleotide replacements in the binding sites abolished the fluorescence resonance energy transfer. These results suggests that ScoC could induce DNA looping and empower DNA repression.