FlhF affects the subcellular clustering of WspR through HsbR in Pseudomonas aeruginosa.

In bacteria, the second messenger cyclic di-GMP (c-di-GMP) is synthesized and degraded by multiple diguanylate cyclases (DGCs) and phosphodiesterases. A high level of c-di-GMP induces biofilm formation and represses motility. WspR, a hybrid response regulator DGC, produces c-di-GMP when it is phosphorylated. FlhF, a signal recognition particle-type GTPase, is initially localized to the cell poles and is indispensable for polar flagellar localization in Pseudomonas aeruginosa. In this study, we report that deletion of flhF affected biofilm formation and the c-di-GMP level in P. aeruginosa. Phenotypic analysis of a flhF knockout mutant revealed increased biofilm formation, wrinkled colonies on Congo red agar, and an elevated c-di-GMP level compared to the wild-type strain, PAO1. Yeast and bacterial two-hybrid systems showed that FlhF binds to the response regulator HsbR, and HsbR binds to WspR. Deletion of hsbR or wspR in the ΔflhF background abolished the phenotype of ΔflhF. In addition, confocal microscopy demonstrated that WspR-GFP was distributed throughout the cytoplasm and formed a visible cluster at one cell pole in PAO1 and ΔhsbR, but it was mainly distributed as visible clusters at the lateral side of the periplasm and with visible clusters at both cell poles in ΔflhF. These findings suggest that FlhF influences the subcellular cluster and localization of WspR and negatively modulates WspR DGC activity in a manner dependent on HsbR. Together, our findings demonstrate a novel mechanism for FlhF modulating the lifestyle transition between motility and biofilm via HsbR to regulate the DGC activity of WspR.IMPORTANCECyclic di-GMP (c-di-GMP) is a second messenger that controls flagellum biosynthesis, adhesion, virulence, motility, exopolysaccharide production, and biofilm formation in bacteria. Recent research has shown that distinct diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) produce highly specific outputs. Some DGCs and PDEs contribute to the total global c-di-GMP concentration, but others only affect local c-di-GMP in a microenvironment. However, the underlying mechanisms are unclear. Here, we report that FlhF affects the localization and DGC activity of WspR via HsbR and is implicated in local c-di-GMP signaling in Pseudomonas aeruginosa. This study establishes the link between the c-di-GMP signaling system and the flagellar localization and provides insight for understanding the complex regulatory network of c-di-GMP signaling.

Characterization of a Conjugative Multidrug Resistance IncP-2 Megaplasmid, pPAG5, from a Clinical Pseudomonas aeruginosa Isolate.

The spread of resistance genes via horizontal plasmid transfer plays a significant role in the formation of multidrug-resistant (MDR) Pseudomonas aeruginosa strains. Here, we identified a megaplasmid (ca. 513 kb), designated pPAG5, which was recovered from a clinical multidrug-resistant P. aeruginosa PAG5 strain. The pPAG5 plasmid belonged to the IncP-2 incompatibility group. Two large multidrug resistance regions (MDR-1 and MDR-2) and two heavy metal resistance operons (merEDACPTR and terZABCDE) were identified in the pPAG5 plasmid. Genetic analysis demonstrated that the formation of MDR regions was mediated by several homologous recombination events. Further conjugation assays identified that pPAG5 could be transferred to P. aeruginosa but not Escherichia coli. Antimicrobial susceptibility testing on transconjugants demonstrated that pPAG5 was capable of transferring resistance genes to transconjugants and producing a multidrug-resistant phenotype. Comparative analysis revealed that pPAG5 and related plasmids shared an overall similar backbone, including genes essential for replication (repA), partition (par), and conjugal transfer (tra). Further phylogenetic analysis showed that pPAG5 was closely related to plasmids pOZ176 and pJB37, both of which are members of the IncP-2-type plasmid group. IMPORTANCE The emergence and spread of plasmid-associated multidrug resistance in bacterial pathogens is a key global threat to public health. It is important to understand the mechanisms of the formation and evolution of these plasmids in patients, hospitals, and the environment. In this study, we detailed the genetic characteristics of a multidrug resistance IncP-2 megaplasmid, pPAG5, and investigated the formation of its MDR regions and evolution. To the best of our knowledge, plasmid pPAG5 is the largest multidrug resistance plasmid ever sequenced in the Pseudomonas genus. Our results may provide further insight into the formation of multidrug resistance plasmids in bacteria and the molecular evolution of plasmids.