Dual control of RegX3 transcriptional activity by SenX3 and PknB

The mycobacterial SenX3-RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system involved in sensing environmental P_i levels and induction of genes required for P_i acquisition under P_i-limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KD^Mtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3^Mtb and RegX3^Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3^Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3^Mtb and that Thr-100 phosphorylation likely prevents RegX3^Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental P_i signals with the cellular replication state to adjust gene expression in response to P_i availability.