Promoter selectivity of the Bacillus subtilis response regulator DegU, a positive regulator of the fla/che operon and sacB

<p>Abstract</p> <p>Background</p> <p>The response regulator DegU and its cognate histidine kinase DegS constitute a two-component system in the Gram-positive soil bacterium <it>Bacillus subtilis</it>. Unphosphorylated and phosphorylated forms of DegU are known to activate target gene transcription in <it>B. subtilis</it>. Although phosphorylated DegU (DegU-P) regulates more than one hundred and twenty genes, the targets of unphosphorylated DegU are unknown, except for <it>comK</it>.</p> <p>Results</p> <p>We found that the <it>fla/che </it>(flagella and chemotaxis) operon is positively regulated by unphosphorylated DegU. The effect was most prominent in a strain bearing the functional <it>swrAA </it>gene, a positive regulator of <it>fla/che</it>. Unphosphorylated DegU bound to two regions in the <it>fla/che </it>regulatory region containing an inverted repeat-like sequence that resembles the inverted repeat (IR) in the <it>comK </it>promoter. Mutational analysis revealed that positive regulation of <it>fla/che </it>by SwrAA requires DegU-binding. An analysis of the DegU-P-regulated gene <it>sacB </it>(levansucrase gene) by footprint and mutational analyses revealed that DegU-P bound to a direct repeat (DR) of the DegU-recognition motifs, which has been shown to be functional in vivo, while unphosphorylated DegU did not. These results strongly suggest that the arrangement of the DegU-binding motifs determines whether unphosphorylated DegU or DegU-P binds to the <it>sacB </it>promoter. The hypothesis was confirmed by observing <it>degS</it>-independent expression when the DR in the <it>sacB-lacZ </it>fusion was changed to an IR, suggesting that unphosphorylated DegU regulates the <it>sacB </it>promoter through the newly created IR. This was confirmed by binding of unphosphorylated DegU to the IR in the <it>sacB </it>promoter.</p> <p>Conclusion</p> <p>This study demonstrated that DegU positively regulates <it>flgB </it>and <it>sacB </it>through its binding to the promoter regions. We demonstrated that DegU-P prefers binding to DR but not to IR in the <it>sacB </it>promoter.</p