Microbiology Preventing Inappropriate Activation

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

Phosphotransfer between bacterial histidine kinases and their target response regulators mediates bacterial response to stimuli. Boll and Hendrixson found that, unlike other response regulators of the NtrC family, the flagellar biosynthesis response regulator FlgR did not require its C-terminal domain (CTD) for induction of gene expression. Instead, this domain appeared to function as an insulator, preventing inappropriate phosphorylation of the protein by the phosphodonor metabolite acetyl phosphate (AcP). In cells with wild-type histidine kinase FlgS, which mediates the phosphotransfer to FlgR, induction of the ⁵⁴ regulon (the target of FlgR) occurred in cells expressing a FlgR lacking the CTD (FlgR_CTD) to a similar extent as that in cells expressing wild-type FlgR. In cells lacking FlgS, FlgR_CTD still enabled expression of the ⁵⁴ regulon, albeit at a reduced amount compared with that in cells with wild-type FlgS. Electrophoretic mobility shift assays showed that FlgR_CTD did not bind to DNA, despite its ability to promote ⁵⁴-dependent gene expression from a reporter plasmid. Transposon mutagenesis screening for genes that reduced FlgR_CTD-mediated expression of the reporter in cells lacking FlgS identified mutations in genes associated with the acetogenesis pathway, which controls the abundance of AcP. Increased FlgS-independent, FlgR_CTD-mediated expression of the reporter was observed in mutant bacteria (knockout of ackA) that produced higher amounts of AcP. Mutation of the aspartic acid that is phosphorylated in FlgR abolished the ability of increased AcP to promote FlgR_CTD-mediated expression of the reporter and its phosphorylation by AcP in vitro. In bacteria with wild-type FlgS and FlgR, deletion of ackA to enhance AcP abundance failed to stimulate expression of the reporter but modestly stimulated reporter expression in cells with FlgS and FlgR_CTD. In vitro autophosphorylation assays showed that the FlgR_CTD was phosphorylated more efficiently than was wild-type FlgR and that phosphotransfer from FlgS to FlgR_CTD was much less efficient than transfer to wild-type FlgR. Despite activation of FlgR with AcP by deletion of ackA or supplementation with pyruvate, bacteria expressing FlgR_CTD failed to develop the wild-type number of flagella, suggesting that optimal flagellation requires coupling to the upstream histidine kinase FlgS. The authors suggest that the CTD serves as an insulator preventing FlgR from inappropriate activation by metabolic phosphodonors and this is one part of a mechanism controlling the appropriate induction of flagellar genes and formation of flagella in Campylobacter jejuni.

J. M. Boll, D. R. Hendrixson, A specificity determinant for phosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphate. Proc. Natl. Acad. Sci. U.S.A. 108, 20160–20165 (2011). [PubMed]