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.

Rap proteins regulate the activity of response regulators including Spo0F, DegU and ComA. We found that overexpression of either RapG or RapH severely downregulated the expression of srfA, which belongs to the ComA regulon. Disruption of those genes, however, showed small effects on srfA expression. These observations suggested that Bacillus subtilis cells possess a repressor for rapG and rapH. To identify candidate repressors we developed a novel transcription factor array (TF array) assay, in which disruptions of 287 genes encoding regulatory proteins were independently transformed into a strain carrying rapH-lacZ and the resultant transformants were grown on agar plates containing Xgal to detect beta-galactosidase activity. We identified a yvaN disruptant which showed a rapH-overproducing phenotype. DNA microarray analysis of the yvaN mutant suggested that both rapG and rapH were overproduced, leading to inhibition of srfA expression. In a gel retardation assay, purified His-tagged YvaN specifically bound to promoter sequences of rapG and rapH. Further footprint and gel retardation analyses using various deleted probes uncovered critical sequences for YvaN binding. In addition, a lacZ fusion analysis confirmed the significance of YvaN binding for transcription regulation of rapG and rapH. Thus, YvaN was renamed RghR (rapG and rapH repressor). As the rapH gene is activated by ComK and RapH inhibits comK indirectly, this constitutes an autoregulatory loop modulated by RghR.

pgsB encodes gamma-poly glutamic acid (gamma-PGA) synthetase and constitutes an operon with pgsC, pgsAA, and pgsE. Genetic analysis revealed that degQ and swrA, the known regulators of pgsB, are not required for pgsB expression when high cellular concentrations of phosphorylated form of the response regulator DegU (DegU-P) are present. However, swrA appeared still to be required for gamma-PGA synthesis under the conditions we tested. Since genetic analysis suggested that DegU-P activates pgsB directly, we performed gel retardation and footprint analyses using purified His-tagged DegU and the pgsB promoter. The in vitro experiments revealed that His-tagged DegU bound to the immediate upstream region of the -35 region of the pgsB promoter. A six-base deletion within the sequence (the -44 to -39 region) abolished DegU-binding to the pgsB promoter and pgsB transcription, confirming the importance of the sequence for DegU-dependent regulation of pgsB. Hence we conclude that DegU is a direct activator of the pgsB operon.

SummaryThe response regulator DegU and its cognate kinase DegS constitute a two-component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU-lacZ fusions, we showed that phosphorylated DegU (DegU-P) binds to two direct repeats (DR1 and DR2) of the consensus DegU-binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATPdependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild-type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU-P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU-P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N-terminal region of DegU is important for proteolysis.

No abstract

During the course of screening for competence-deficient mutants in the mutant collection constructed by the Japan Consortium of Bacillus Functional Genomics, a disruption mutant of sodA encoding superoxide dismutase was identified as a mutant with decreased transformation efficiency. In fact, in the sodA mutant we observed a severe decrease in the expression of srfA required for the development of genetic competence. Northern and primer extension analyses revealed inhibition of the transcription of the comQXP quorum-sensing locus in the sodA mutant, thereby preventing srfA expression. Furthermore, an excess amount of superoxide anion induced by the addition of paraquat also resulted in a decrease in comQXP transcription. Thus, it was concluded that high levels of superoxide are able to inhibit specifically the transcription of the comQXP operon. In support of this conclusion, the effect of added paraquat was significantly alleviated in a comX-independent srfA expression system.

The response regulator DegU and its cognate histidine kinase DegS constitute a two-component system in the Gram-positive soil bacterium Bacillus subtilis. The phosphorylated form of DegU is known to activate transcription of more than 120 genes in B. subtilis, including the bpr gene encoding bacillopeptidase F. To characterize DegU-dependent regulation of bpr, the interaction of the bpr regulatory region with His-tagged DegU was analyzed using gel retardation and footprint analyses. This revealed that DegU bound three direct repeats of a motif that is known to be arranged as an inverted repeat in the comK promoter, to which DegU binds. Mutational analysis using a bpr-lacZ fusion revealed that the three direct repeats in bpr are needed for DegU-dependent transcription activation.

The Bacillus subtilis yclJ gene encodes an OmpR-type response regulator of a two-component regulatory system with unknown function. A previous DNA microarray experiment suggested that multicopy yclJ greatly enhances the expression of several operons in a cognate kinase (YclK)-deficient strain. To confirm this, lacZ fusion analysis was performed in the yclK background with overexpressed yclJ. As a result, yclHI, ykcBC, and yngABC were indeed positively regulated by YclJ. Gel retardation and DNase I footprint analyses revealed that YclJ binds to the promoter regions of yclHI, ykcBC, and yngABC. Nucleotide sequence analysis of the binding regions suggested that YclJ recognizes a direct repeat of the consensus sequence TTCATANTTT, the upstream half of which has close similarity to the consensus binding sequence of the other OmpR family response regulator PhoP. LacZ fusion analysis of the control region of yngA with deletion or point mutation confirmed that the YclJ-binding sequence is required for the YclJ-mediated activation of yngA. Furthermore, we identified two more YclJ-regulated genes, yycA and yfjR, using bioinformatic analysis of the B. subtilis genome, and it was shown that YclJ binds to those promoters and controls the expression of those genes.