Hayashi_2006_Mol.Microbiol_59_1714

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.