Abstract
The histidine-containing phosphotransfer protein-B (HptB, PA3345) regulates swarming motility in Pseudomonas aeruginosa PAO1 through a partner-switching mechanism involving PA3346 (Ser/Thr protein kinase/phosphatase), PA3347 (anti-σ28 factor antagonist), and anti-σ28 factor FlgM. The ΔhptB mutant is defective in swarming, although its flagellum remains intact. Overexpression of PA2133 and PA4367, both encode a c-di-GMP phosphodiesterase (PDE), could rescue the swarming defect of the ΔhptB. To investigate whether σ28 (FliA) regulates P. aeruginosa motility through modulating c-di-GMP level, this study generated a fliA-deletion mutant and a fliA overexpressing stain. The deletion of fliA resulted in a decreased swarming motility which could be partially restored by overexpressing either PA2133 or PA4367. Surprisingly, overexpressing fliA also led to defective swimming and swarming, suggesting that an optimal concentration of FliA is essential for the bacterium to achieve the highest motility. Using transmission electron microscope to examine the bacterial morphology has revealed that ΔfliA had no flagella, and the flagella of the fliA overexpressing strain were significantly shorter than that of wild-type. Transcriptome profiling of PAO1 and ΔfliA showed that the expression levels of several flagella biogenesis and PDE genes decreased in the ΔfliA strain. Further analysis the promoter activity of the PDE genes using the GUS reporter assay confirmed that the activity of PA4367 promoter was indeed decreased in ΔfliA. Finally, this study demonstrated that the intracellular concentration of c-di-GMP was increased in the ΔfliA strain as determined using a GFP-based c-di-GMP reporter system. These findings suggest that FliA participates in HptB-mediated swarming regulation through both affecting flagella biogenesis and modulating c-di-GMP levels. Altogether our findings contribute to a better understanding of how the HptB partner switching system cooperates with FliA to regulate bacterial motility, an important pathogenesis feature of P. Aeruginosa.