Abstract
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 Pi levels and induction of genes required for Pi acquisition under Pi-limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KDMtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3Mtb and RegX3Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3Mtb 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 RegX3Mtb and that Thr-100 phosphorylation likely prevents RegX3Mtb 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 Pi signals with the cellular replication state to adjust gene expression in response to Pi availability.
Highlights
The mycobacterial SenX3–RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator
For the Y2H assay, the regX3Mtb gene was cloned into the prey vector pGADT7linker, whereas the gene portions encoding the KDs of PknBMtb, PknEMtb, and SenX3Mtb were cloned into the bait vector pGBKT7
The pknB gene forms an operon with pknA, pbpA, rodA, pstP, and two genes coding for forkhead-associated domain– containing proteins
Summary
Mycobacterium tuberculosis; TCS, two-component system; HK, histidine kinase; RR, response regulator; PAS, Per– ARNT–Sim; STPK, Ser/Thr protein kinase; KD, kinase domain; Y2H, yeast two-hybrid; qPCR, quantitative PCR; IPTG, isopropyl 1-thio--D-galactopyranoside; PASTA, protein and Ser/Thr kinase–associated; SD, synthetic defined dropout; CBB, Coomassie Brilliant Blue. The kinase/phosphatase activity of SenX3 has been suggested to be regulated by the functional state of the Pst uptake system with the assistance of other auxiliary proteins (PhoU in Mycobacterium smegmatis and PhoY in Mtb). This suggestion was made on the basis of the finding that inactivation of either Pst transporter or PhoU (PhoY) by mutation brings about constitutive activation of the SenX3–RegX3 TCS and constitutive expression of the RegX3 regulon independent of Pi availability [20, 24, 25]. As an extension of our previous study, here we report that overexpression of PknB-KDMtb significantly inhibits the transcriptional activity of RegX3 of Mtb (RegX3Mtb) by phosphorylating Thr-100, Thr191, and Thr-217
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