The two-component regulatory system senX3-regX3 regulates phosphate-dependent gene expression in Mycobacterium smegmatis

Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate ([P.sub.i]) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in [P.sub.i] acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a [P.sub.i]-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon [P.sub.i] availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3~P) as a transcriptional activator of phoA. Furthermore, purified RegX3~P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3~P. Based upon our findings, we propose a model for [P.sub.i]-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.