The Two-Component Regulatory System senX3-regX3 Regulates Phosphate-Dependent Gene Expression in Mycobacterium smegmatis

Abstract

ABSTRACT Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (P i ) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in P 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 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 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 i -regulated gene expression mediated by SenX3-RegX3 in mycobacteria.