Mycobacterium smegmatis PhoU proteins have overlapping functions in phosphate signaling and are essential

Many bacteria regulate gene expression in response to phosphate availability using a two-component signal transduction system, the activity of which is controlled by interaction with the Pst phosphate specific transporter and a cytoplasmic protein PhoU. Mycobacterium tuberculosis, the causative agent of tuberculosis, requires its phosphate sensing signal transduction system for virulence and antibiotic tolerance, but the molecular mechanisms of phosphate sensing remain poorly characterized. M. smegmatis serves as a model for studying mycobacterial pathogens including M. tuberculosis. M. smegmatis encodes two proteins with similarity to PhoU, but it was unknown if both proteins participated in signal transduction with the phosphate-responsive SenX3-RegX3 two-component system. We constructed phoU single and double deletion mutants and tested expression of genes in the RegX3 regulon. Only the ΔphoU1ΔphoU2 mutant exhibited constitutive activation of all the RegX3-regulated genes examined, suggesting that M. smegmatis PhoU1 and PhoU2 have overlapping functions in inhibiting activity of the SenX3-RegX3 two-component system when phosphate is readily available. The ΔphoU1ΔphoU2 mutant also exhibited decreased tolerance to several anti-tubercular drugs. However, a complex plasmid swapping strategy was required to generate the ΔphoU1ΔphoU2 mutant, suggesting that either phoU1 or phoU2 is essential for in vitro growth of M. smegmatis. Using whole-genome sequencing, we demonstrated that all five of the ΔphoU1ΔphoU2 mutants we isolated had independent suppressor mutations predicted to disrupt the function of the Pst phosphate transporter, suggesting that in the absence of the PhoU proteins phosphate uptake by the Pst system is toxic. Collectively, our data demonstrate that the two M. smegmatis PhoU orthologs have overlapping functions in both controlling SenX3-RegX3 activity in response to phosphate availability and regulating phosphate transport by the Pst system. Our results suggest that M. smegmatis can serve as a tractable model for further characterization of the molecular mechanism of phosphate sensing in mycobacteria and to screen for compounds that would interfere with signal transduction and thereby increase the efficacy of existing anti-tubercular antibiotics.