Dataset Information

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

ABSTRACT:

Background

The response regulator DegU and its cognate histidine kinase DegS constitute a two-component system in the Gram-positive soil bacterium Bacillus subtilis. Unphosphorylated and phosphorylated forms of DegU are known to activate target gene transcription in B. subtilis. Although phosphorylated DegU (DegU-P) regulates more than one hundred and twenty genes, the targets of unphosphorylated DegU are unknown, except for comK.

Results

We found that the fla/che (flagella and chemotaxis) operon is positively regulated by unphosphorylated DegU. The effect was most prominent in a strain bearing the functional swrAA gene, a positive regulator of fla/che. Unphosphorylated DegU bound to two regions in the fla/che regulatory region containing an inverted repeat-like sequence that resembles the inverted repeat (IR) in the comK promoter. Mutational analysis revealed that positive regulation of fla/che by SwrAA requires DegU-binding. An analysis of the DegU-P-regulated gene sacB (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 sacB promoter. The hypothesis was confirmed by observing degS-independent expression when the DR in the sacB-lacZ fusion was changed to an IR, suggesting that unphosphorylated DegU regulates the sacB promoter through the newly created IR. This was confirmed by binding of unphosphorylated DegU to the IR in the sacB promoter.

Conclusion

This study demonstrated that DegU positively regulates flgB and sacB through its binding to the promoter regions. We demonstrated that DegU-P prefers binding to DR but not to IR in the sacB promoter.

SUBMITTER: Tsukahara K

PROVIDER: S-EPMC2245950 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P).Growing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase.Under physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.