Analysis of phosphate-regulated gene expression in mycobacteria

Abstract

Phosphate transporters are required for the virulence of Mycobacterium tuberculosis and are regulated by environmental inorganic phosphate (Pi) concentrations, although the mechanism regulation is unknown. The expression of genes involved in high affinity Pi acquisition is frequently regulated by two-component regulatory systems (2CR), consisting of a sensor histidine kinase (HK) and DNA-binding response regulator (RR). Here, the senX3-regX3 2CR is identified as a Pi-dependent regulator of alkaline phosphatase (phoA) expression in M. smegmatis. Transposon insertion mutations and targeted deletions of the HK senX3 resulted in the constitutive expression of phoA in high P i growth conditions. The PhoA+ phenotype observed in senX3 mutants was complemented by the senX3-regX3 genes from M. smegmatis and M. tuberculosis H37Rv, suggesting a shared function between species. Disruptions within regX3 were only obtained in merodiploid strains and suggest an essential function for RegX3 in M. smegmatis. Upon Pi starvation, senX3-regX3 and phoA mRNA were rapidly induced and lacZ reporter constructs confirmed transcriptional regulation of phoA. Furthermore, phosphorylated RegX3 bound to the phoA promoter, consistent with a direct role in gene regulation. RegX3-P also bound directly to the promoter region of the high affinity phosphate transporter encoded by pstSCAB-phoU and to its own promoter, suggesting that many genes involved in Pi acquisition are coordinately regulated by senX3-regX3. Importantly, although M. tuberculosis does not encode a phoA homolog, the M. smegmatis phoA promoter is active and transcriptionally regulated by available Pi in M. tuberculosis H37Ra. The identification of Pi as a stimulus for the senX3-regX3 2CR will facilitate the identification of additional Pi-regulated genes and help determine their significance in the virulence of M. tuberculosis. .