Mutational Analysis of the GTP-binding Motif of FlhF which Regulates the Number and Placement of the Polar Flagellum in Vibrio alginolyticus

Abstract

Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutations in the GTP-binding motif affected the ability to interact with FlhG. In contrast to our prediction, no significant difference was detected in the interaction with FlhG between the wild-type and mutant FlhFs. We showed that the GTP-binding motif of FlhF is important for polar localization of the flagellum but not for the interaction with FlhG.

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

“…Because PaFlhF is not required for flagellar assembly per se, it is difficult to compare our findings to those of Green et al, who reported that mutations analogous to K222A and D294A (VcFlhF K295A and D367A) resulted in the absence of flagellar assembly in V. cholerae (35). These mutations were also introduced into V. alginolyticus FlhF (K305A and D377A), where they partially restored swarming motility and supported flagellar assembly in a small proportion of bacterial cells (38). It is unclear whether the differences between these Vibrio sp.…”

“…FlhF plays distinct roles in flagellar assembly and positioning in bacteria with polar flagella. In C. jejuni and V. alginolyticus, flhF deletion results in nonflagellated cells that are nonmotile (14,38). In contrast, P. aeruginosa ⌬flhF bacteria assemble flagella at nonpolar sites and swim, albeit via circular rather than straight paths (7).…”

HubP, a Polar Landmark Protein, Regulates Flagellar Number by Assisting in the Proper Polar Localization of FlhG in Vibrio alginolyticus

“…FlhF is a GTPase protein and is similar to the signal recognition particle receptor FtsY (8,9). The GTPase motif of FlhF influences the intracellular location of this protein (10,11), and GTPase activity is stimulated through interactions with FlhG (12)(13)(14). FlhG is an ATPase protein and is similar to MinD, a cell division regulator in prokaryotes (15)(16)(17).…”

De- and repolarization mechanism of flagellar morphogenesis during a bacterial cell cycle

“…The FlhF GTPase of vibrios, pseudomonads, and Campylobacter jejuni may function akin to the TipF and c-di-GMP-dependent flagellar polarization pathway (Pandza et al 2000;Salvetti et al 2007;Balaban et al 2009;Kusumoto et al 2009). FlhF is polarized and recruits the MS ring protein FliF to the polar site of flagellation in Vibrio.…”