Seung Youjin, Baik Ha-Eun, Park Eun-Jin, Oh Jeong-Il
Department of Integrated Biological Science, Pusan National University, Busan, South Korea.
Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, South Korea.
J Biol Chem. 2025 Jun 28;301(8):110435. doi: 10.1016/j.jbc.2025.110435.
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.
分枝杆菌中的SenX3-RegX3双组分系统（TCS）由SenX3组氨酸激酶和RegX3反应调节因子组成，可根据环境中无机磷酸盐（Pi）的可用性调节与Pi获取相关的基因表达。在本研究中，我们研究了SenX3如何感知环境Pi水平，并研究了两个PhoU旁系同源物PhoU1和PhoU2在耻垢分枝杆菌中与SenX3-RegX3 TCS相关的Pi感知中的作用。我们的研究结果表明，虽然SenX3的膜定位对其传感功能不是必需的，但其Per-ARNT-Sim结构域对Pi感知至关重要。与先前提出的模型相反，Pst系统是一种高亲和力的Pi转运体，它不直接参与Pi感知或通过SenX3-RegX3 TCS进行信号转导。相反，PhoU旁系同源物可能独立于Pst系统感知细胞内Pi水平，并调节SenX3激酶和磷酸酶活性之间的平衡。通过体外激酶和磷酸酶测定，我们证明在Pi存在的情况下，纯化的PhoU1将SenX3活性转向其磷酸酶功能，从而促进磷酸化RegX3的去磷酸化。我们进一步证实，PhoU1和PhoU2在耻垢分枝杆菌中功能冗余。最后，我们发现phoU1和phoU2的表达在Pi缺乏条件下被诱导：phoU1的上调由SenX3-RegX3 TCS介导，而phoU2的表达由替代sigma因子SigF诱导。
