Abstract
The histidine-containing phosphotransfer protein-B (HptB; PA3345) is an intermediate protein involved in transferring a phosphoryl group from multiple sensor kinases to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. The objective of this study was to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription profiling analysis of an hptB knock-out mutant showed that the expression of a number of motility-related genes was altered consistent with the non-swarming phenotype observed for the mutant. Domain analysis indicated that the PA3346 C-terminal region (PA3346C) exhibits ∼30% identity with the anti-σ factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity targeting an anti-σ antagonist, PA3347, at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-σ 28 factor FlgM were found to interact with PA3347 individually both in vivo and in vitro. FlgM displaced PA3346C in binding of PA3347 and was then competitively displaced by σ 28 from the PA3347-FlgM complex, forming a phosphorylation-dependent partner-switching system. The significance of PA3347 phosphorylation in linking the partner-switching system and swarming motility was established by analyzing the swarming phenotype of the PA3347 knock-out mutant and its complement strains. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.