Regulation of therhaEWRBMAOperon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis
ABSTRACTTheBacillus subtilisrhaEWRBMA(formerlyyuxG-yulBCDE) operon consists of four genes encoding enzymes forl-rhamnose catabolism and therhaRgene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of therhaEWgene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited byl-rhamnulose-1-phosphate, an intermediate ofl-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control inB. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site.In vivoanalysis of therhaEWpromoter-lacZfusion in the background ofccpAdeletion showed that thel-rhamnose-responsive induction of therhaEWpromoter was negated by the disruption ofrhaAorrhaBbut notrhaEWorrhaM, whereasrhaRdisruption resulted in constitutiverhaEWpromoter activity. Thesein vitroandin vivoresults clearly indicate that RhaR represses the operon by binding to the operator site, which is detached byl-rhamnulose-1-phosphate formed froml-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, thelacZreporter analysis using the strains with or without theccpAdeletion under the background ofrhaRdisruption supported the involvement of CcpA in the carbon catabolite repression of the operon.IMPORTANCESincel-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested thatl-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance ofl-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system ofEscherichia coli. In this study, we show that, inBacillus subtilis, one of the plant growth-promoting rhizobacteria, therhaEWRBMAoperon forl-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms ofl-rhamnose catabolism in other bacterial species.