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

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 (RegX3ϳP) as a transcriptional activator of phoA. Furthermore, purified RegX3ϳP 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 RegX3ϳP. Based upon our findings, we propose a model for P i -regulated gene expression mediated by SenX3-RegX3 in mycobacteria.

Differential regulation of the two-component regulatory system senX3-regX3 in Mycobacterium tuberculosis

“…Accumulating evidence has suggested that SenX3-RegX3 is directly involved in the mycobacterial phosphate starvation response (Glover et al, 2007;James et al, 2012;Rifat et al, 2009). P i limitation is believed to be a physiologically relevant microenvironment encountered by Mtb within the arrested macrophage phagolysosome (Rengarajan et al, 2005).…”

“…To study the promoter activities of senX3 and the IR, the Escherichia coli-mycobacterium shuttle vector pYUB76 harbouring the lacZ gene (kind gift of Dr William R. Jacobs, Jr, Albert Einstein College of Medicine, NY, USA) was used (Barletta et al, 1992;Glover et al, 2007). Briefly, a 199 bp segment of the 59 UTR of senX3 was PCR-amplified using primers dl9-F and dl10-R (Table 1) from Mtb CDC1551 genomic DNA.…”

“…Mycobacterial SenX3-RegX3, like other 2CRSs, has been described as an auto-regulatory operon (Glover et al, 2007;Himpens et al, 2000). Interestingly, the intergenic region (IR) between senX3 and regX3 consists of several mycobacterial interspersed repetitive units (MIRU) (Supply et al, 1997).…”

“…In Mycobacterium smegmatis, SenX3-RegX3 has been shown to control expression of IP: 54.191.190.102 On: Sat, 12 May 2018 06:44:34 phosphate-dependent genes (Glover et al, 2007), and is required for optimal growth under P i -limiting conditions (James et al, 2012). Mtb SenX3-RegX3 is also required for bacillary survival during P i limitation (Rifat et al, 2009), within THP-1 cells and activated murine macrophages (Parish et al, 2003), and in mammalian lungs (Parish et al, 2003;Rickman et al, 2004;Rifat et al, 2009).…”

“…Significant attention has focused on regulation of senX3-regX3 and its role in Mtb virulence (Glover et al, 2007;Himpens et al, 2000;James et al, 2012;Parish et al, 2003;Rickman et al, 2004;Rifat et al, 2009;Supply et al, 1997;Tischler et al, 2013). In Mycobacterium smegmatis, SenX3-RegX3 has been shown to control expression of IP: 54.191.190.102 On: Sat, 12 May 2018 06:44:34 phosphate-dependent genes (Glover et al, 2007), and is required for optimal growth under P i -limiting conditions (James et al, 2012).…”