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 is to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription-profiling analysis of an hptB knockout 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 indicates that the PA3346 C-terminal region (PA3346C) exhibits approximately 30% identity with the anti-sigma factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity-targeting an anti-sigma antagonist PA3347 at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-σ28 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 σ28 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 knockout mutant and its complement strain. The over expression of phosphodiesterase in hptB mutant restore its swarming motility defect showing the role of intracellular signaling molecule cyclic-di-GMP in regulating the motility behavior of HptB mediated signaling pathway.