The GTPase Activity of FlhF Is Dispensable for Flagellar Localization, but Not Motility, in Pseudomonas aeruginosa

Abstract

The opportunistic human pathogen Pseudomonas aeruginosa uses two surface organelles, flagella and pili, for motility and adhesion in biotic and abiotic environments. Polar flagellar placement and number are influenced by FlhF, which is a signal recognition particle (SRP)-type GTPase. The FlhF proteins of Bacillus subtilis and Campylobacter jejuni were recently shown to have GTPase activity. However, the phenotypes associated with flhF deletion and/or mutation differ between these organisms and P. aeruginosa, making it difficult to generalize a role for FlhF in pseudomonads. In this study, we confirmed that FlhF of P. aeruginosa binds and hydrolyzes GTP. We mutated FlhF residues that we predicted would alter nucleotide binding and hydrolysis and determined the effects of these mutations on FlhF enzymatic activity, protein dimerization, and bacterial motility. Both hydrolytically active and inactive FlhF point mutants restored polar flagellar assembly, as seen for wild-type FlhF. However, differential effects on flagellar function were observed in single-cell assays of swimming motility and flagellar rotation. These findings indicate that FlhF function is influenced by its nucleotide binding and hydrolytic activities and demonstrate that FlhF affects P. aeruginosa flagellar function as well as assembly.

Convergent Evolution of Hyperswarming Leads to Impaired Biofilm Formation in Pathogenic Bacteria

“…Previous studies have shown that both P. aeruginosa PAK and P. putida became polar multi-flagellates when FlhF was overexpressed (Pandza et al, 2000; Schniederberend et al, 2013). Since FlhF and FleN are binding partners (Balaban and Hendrixson, 2011; Kazmierczak and Hendrixson, 2013; Kusumoto et al, 2006; Murray and Kazmierczak, 2006), it is possible that if their interaction is hampered by the fleN mutations, the cells may be experiencing a relatively higher activity of FlhF.…”

The role of FlhF and HubP as polar landmark proteins in Shewanella putrefaciens CN‐32

“…In addition, we could provide evidence that Sp FlhF and Sp FlhG interact in vitro and that the N‐terminal section of Sp FlhG stimulates the GTPase activity of Sp FlhF. Thus, Sp HubP‐mediated interaction between FlhF and FlhG and control of the GTPase activity might affect FlhF‐related functions such as polar accumulation and recruitment of flagellar components, but also flagellar performance, as has recently been suggested for P. aeruginosa (Schniederberend et al ., ). Accordingly, a deletion of hubP in both V. cholerae and S. putrefaciens CN‐32 resulted in a significantly decreased ability to navigate through soft agar.…”

A new route for polar navigation

“…Clean deletions confirmed that all three genes were necessary for normal swimming, and for polar placement of the flagellum. The GTPase FlhF was previously considered the topmost component in the polar localization hierarchy for the flagellum of P. aeruginosa, controlling not just localization, but also function (Kazmierczak and Hendrixson, 2013;Schniederberend et al, 2013). However, FlhF has now been dethroned by the Poc complex, as a FlhF-GFP fusion traded its wild-type bipolar localization for non-polar sites -correlated with the positions of non-polar flagella -when any of TonB3, PocA or PocB was missing.…”