Regulatory mechanism of the SenX3-RegX3 two-component system in Mycobacterium smegmatis: Roles of PhoU in sensing inorganic phosphate levels.

The SenX3-RegX3 two-component system (TCS) in mycobacteria, consisting of the SenX3 histidine kinase and the RegX3 response regulator, regulates gene expression related to inorganic phosphate (Pi) acquisition in response to environmental Pi availability. In this study, we investigated how SenX3 senses environmental Pi levels and examined the role of the two PhoU paralogs, PhoU1 and PhoU2, in Pi sensing in association with the SenX3-RegX3 TCS in Mycobacterium smegmatis. Our findings revealed that while membrane localization of SenX3 is not required for its sensory function, its Per-ARNT-Sim domain is essential for Pi sensing. Contrary to previously proposed models, the Pst system, a high-affinity Pi transporter, does not directly participate in Pi sensing or signal transduction through the SenX3-RegX3 TCS. Instead, the PhoU paralogs likely sense intracellular Pi levels independent of the Pst system and modulate the balance between the kinase and phosphatase activities of SenX3. Using in vitro kinase and phosphatase assays, we demonstrated that purified PhoU1 shifts SenX3 activity toward its phosphatase function in the presence of Pi, thereby promoting the dephosphorylation of phosphorylated RegX3. We further confirmed that PhoU1 and PhoU2 are functionally redundant in M. smegmatis. Finally, we found that the expression of phoU1 and phoU2 is induced under Pi-deficient conditions: phoU1 upregulation is mediated by the SenX3-RegX3 TCS, whereas phoU2 expression is induced by SigF, an alternative sigma factor.