Abstract:[Objective] To identify the PilZ domain-containing receptor(s) that sense the second messenger cyclic di-GMP (c-di-GMP) produced by the diguanylate cyclase SadC in Pseudomonas aeruginosa and investigate the functions and regulatory mechanisms of the identified receptor(s). [Methods] We constructed the strains in which sadC gene was deleted or overexpressed and tested their ability to swim by using a plate-based approach. We then added sadC in multicopy in each deletion mutant of the eight PilZ domain-containing receptors and screened for the mutants with alleviated swimming repression compared to the wild-type PA14 overexpressing SadC. For the mutations screened out, single gene knockout and overexpression strategies were used to explore the function of the identified receptor(s). Furthermore, site-directed mutagenesis and genetic complementation were employed to test whether the identified receptor's role in SadC-mediated swimming repression requires its c-di-GMP-binding motif. [Results] The SadC-mediated repression of swimming motility was associated with flagellar malfunction rather than flagellum formation. Two PilZ domain-containing receptors, PilZ and FlgZ, were identified to be involved in SadC-mediated swimming repression. The deletion of gene pilZ or flgZ increased the swimming motility, while overexpression of them significantly impaired swimming. A R10A substitution in the conserved c-di-GMP-binding motif of PilZ, or a R140A substitution in FlgZ, resulted in a variant that was no longer able to repress swimming in ΔpilZ or ΔflgZ overexpressing SadC, indicating that the conserved residue required for c-di-GMP binding is critical for PilZ or FlgZ to repress swimming in response to SadC-derived c-di-GMP. [Conclusion] PilZ and FlgZ are the effector relay proteins that respond to SadC c-di-GMP signaling to mediate swimming repression in P. aeruginosa.
