Molecular characterization of the mycobacterial SenX3-RegX3 two-component system: evidence for autoregulation

Environmental regulation of bacterial gene expression is often mediated by two-component signal transduction systems, which are themselves tightly reg ulated. The response regulator RegX3 and the cytoplasmic portion of the his tidine kinase SenX3 from Mycobacterium bovis BCC were overproduced in Esche richia coli and purified as N-terminally (His)(6)-tagged proteins. Phosphor ylation assays demonstrated autophosphorylation of the cytoplasmic portion of SenX3 and a phosphotransfer from SenX3 to RegX3, involving conserved his tidine and aspartate residues, respectively. In addition, as shown by elect rophoretic mobility shift assays, (His),RegX3 was able to specifically bind to the promoter region of the senX3-regX3 operon. Furthermore, operon fusi on analyses indicated that the overexpression of the senX3-regX3 operon inc reases the activity of the senX3 promoter in Mycobacterium smegmatis. Toget her, these results indicate that the mycobacterial SenX3-RegX3 two-componen t system is positively autoregulated.