The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na ؉ -driven flagellar motors may have similar structure and function as proton-driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.

The marine bacterium

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.

For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross‐complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH‐fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH‐fliI flhB(P28T) ΔflhA mutant. Pull‐down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN–FlgK chaperone–substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN–FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly.

The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The ⌬flhF mutant makes no flagellum, whereas the ⌬flhFG double-deletion mutant usually lacks a flagellum. However, the ⌬flhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ⌬flhFG strain (⌬flhFG-sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA (suppressor of ⌬flhFG), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced into the suppressor strain, motility was impaired. Introducing a mutant version of the sflA gene into the ⌬flhFG strain conferred the suppressor phenotype. Thus, we conclude that loss of the sflA gene is responsible for the suppressor phenotype and that the wild-type SflA protein plays a role in preventing polar-type flagella from forming on the lateral cell wall. Vibrio alginolyticus, ubiquitously found in marine and estuarine environments, is a halophilic Gram-negative bacterium that is occasionally the cause of human infections. Indeed, reports have shown that this bacterium is responsible for a large portion of wound infections (1, 2), ear infections (otitis media and otitis externa) (3), and some instances of gastroenteritis and chronic diarrhea among immunocompromised patients (4).Flagella are the most common way to facilitate motility in bacteria; they are composed of more than 20 proteins and typically exploit 30 other proteins for their precise regulation and assembly (reviewed elsewhere [5-8]). One obvious application of such a complex flagellar apparatus is to propel bacterial cells through liquids (swimming) and on surfaces (swarming) in response to favorable/unfavorable signals and/or to allow the bacteria to successfully compete with other microorganisms. In addition, bacterial flagella are widely associated with adhesion to surfaces, biofilm formation, and the virulent behaviors of a variety of pathogenic bacteria, such as V. cholerae (9-11) and Helicobacter pylori (12)(13)(14). Although many bacteria possess such organelles, mixed patterns of flagellation are observed in different bacterial species. While some bacteria, such as Escherichia coli (15), have peritrichous (lateral) flagella, others have single (e.g., V. cholerae) or multiple (e.g., H. pylori) polar flagella. Several species have been reported to have a dual flagellar system where they express both polar and lateral flagella on the same cell for distinct purposes, i.e., polar flagella for swimming and lateral flagella for swarming (16). Such organisms include V. parahaem...

A fragment of DNA was cloned which complemented a polar flagellum-defective (pof) mutation of Vibrio alginolyticus. The fragment contained two complete and two partial open reading frames (ORFs) (ORF2 and -3 and ORF1 and -4, respectively). The presumed product of ORF2 has an amino acid sequence with a high degree of similarity to that of RpoN, which is an alternative sigma factor ( 54 ) for other microorganisms. The other ORFs are also homologous to the genes adjacent to other rpoN genes. Deletion analysis suggests that ORF2 complements the pof mutation. These results demonstrate that RpoN is involved in the expression of polar flagellar genes.Many bacteria move by means of rotating flagella, located either peritrichously or at a pole on the cell body. Certain marine Vibrio species are unique in that they have two types of flagella: a single polar flagellum suited for swimming in a liquid environment and numerous lateral flagella suited for swarming over the surfaces of animate or inanimate objects (2,4,20,24). In Vibrio parahaemolyticus and the closely related Vibrio alginolyticus, it has been shown that polar and lateral flagella are powered by different ion-motive forces: the coupling ion of the polar flagellar motor is sodium and that of the lateral flagellar motor is a proton (5, 15).The lateral flagella are synthesized under viscous conditions (2,6,24,31). An increase in viscosity is a cue for induction of lateral flagellar expression (6). Surprisingly, a decrease in the rotation of the polar flagellum is sensed and lateral flagellar expression is induced (14, 23). Thus, the polar flagellum functions not only as a locomotive organelle but also as a mechanosensor which couples viscous drag to cell differentiation. The polar flagella of V. alginolyticus rotate very fast, up to 1,700 rps, and the rotation is stable (22,27). Furthermore, strictly speaking, the synthesis of polar flagella may not be constitutive. It should be more or less coupled with the cell division cycle, since most of the Vibrio cells have a single polar flagellum at one of the cell poles.To investigate these unique features of the polar flagellum in V. alginolyticus, we wanted to clone the genes involved. From a lateral flagellum-defective mutant, we have isolated many polar flagellum-defective mutants which are defective in both polar and lateral flagellar formation (12,15,28). In this study, using one of those strains as a recipient for shotgun cloning by electroporation (16), we cloned a pof gene involved in polar flagellar formation. Nucleotide sequencing of the gene and the flanking region revealed that the pof gene encodes an alternative sigma factor, 54 (RpoN).Cloning of the pof gene. Most of the strains and plasmids used in this work have been listed previously (28). Plasmid pSU18 and chromosomal DNA from V. alginolyticus 138-2 were digested with EcoRI and BamHI and ligated. These DNA libraries were transferred into YM14 cells by electroporation as described previously (16), and the cells were inoculated onto 0.3% agar VC plates (...

Precise regulation of the number and positioning of flagella are critical in order for the mono-polarflagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no flhF cells have flagella, while a very small fraction of flhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of flhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.Key words bacterial flagellum, flagellar localization, flagellar number, polar flagellum.The bacterial flagellum is a locomotive organelle which is composed of a motor and a screw part. The motor is embedded in the cell envelope and driven by ion motive force. The screw part is composed of a helical filament which is connected to the motor via a hook. This hook acts as a universal joint between the two structures. Flagellar assembly begins with formation of the membrane-embedded motor part, after which the hook structure is constructed. Finally the filament is polymerized from the proximal end towards the distal tip. It is believed that formation of an ‡ These authors contributed equally to this work. MS ring by the membrane protein FliF is the first step towards flagellar morphogenesis. After this soluble proteins (FliG, FliM, and FliN) are attached to the cytoplasmic side of the MS ring, forming the C ring. Then a specific apparatus used for flagellar protein export is assembled inside the C ring, which acts as the entrance to the channel for flagellar proteins (1). The number and localization of flagella vary between species (2). For example, V. alginolyticus and V. parahaemolyticus have both peritrichous (or lateral) flagella 76

Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-). Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella.