A specificity determinant for phosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphate

Bacterial two-component systems (TCSs) sense stimuli and transduce signals intracellularly through phosphotransfer between cognate histidine kinases (HKs) and response regulators (RRs) to alter gene expression or behavioral responses. Without high phosphotransfer specificity between cognate HKs and RRs, cross-phosphorylation or cross-talk between different TCSs may occur and diminish responses to appropriate stimuli. Some mechanisms to reduce cross-talk involve HKs controlling levels of cognate RR phosphorylation. Conceivably, some RRs may have evolved HK-independent strategies to insulate themselves from cross-talk with acetyl phosphate (AcP) or other small phosphodonor metabolites. Initial steps in flagellar biosynthesis in Campylobacter jejuni stimulate phosphotransfer from the FlgS HK to the FlgR RR to promote σ54-dependent flagellar gene expression. We discovered that the FlgR C-terminal domain (CTD), which commonly functions as a DNA-binding domain in the NtrC RR family, is a specificity determinant to limit in vivo cross-talk from AcP. FlgR lacking the CTD (FlgRΔCTD) used FlgS or AcP as an in vivo phosphodonor and could be reprogrammed in ΔflgS mutants to respond to cellular nutritional status via AcP levels. Even though exclusive AcP-mediated activation of FlgRΔCTD promoted WT flagellar gene expression, proper flagellar biosynthesis was impaired. We propose that the FlgR CTD prevents phosphotransfer from AcP so that FlgR is solely responsive to FlgS to promote proper flagellar gene expression and flagellation. In addition to mechanisms limiting cross-talk between noncognate HKs and RRs, our work suggests that RRs can possess domains that prevent in vivo cross-talk between RRs and the endogenous metabolite AcP to ensure signaling specificity.