Noriko Nishioka

Mutational analysis of the GTP-binding motif of FlhF which regulates the number and placement of the polar flagellum in Vibrio alginolyticus

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.

FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella

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.

A Novel dnaJ Family Gene, sflA, Encodes an Inhibitor of Flagellation in Marine Vibrio Species

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 ΔflhF mutant makes no flagellum, whereas the ΔflhFG double-deletion mutant usually lacks a flagellum. However, the ΔflhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ΔflhFG strain (ΔflhFG-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 ΔflhFG), 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 ΔflhFG 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.

Conversion of mono-polar to peritrichous flagellation in Vibrio alginolyticus

Precise regulation of the number and positioning of flagella are critical in order for the mono‐polar‐flagellated 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 ΔflhF cells have flagella, while a very small fraction of ΔflhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of ΔflhFG 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.

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

The marine bacterium Vibrio alginolyticus has a single polar flagellum, the number of which is regulated positively by FlhF and negatively by FlhG. FlhF is intrinsically localized at the cell pole, whereas FlhG is localized there through putative interactions with the polar landmark protein HubP. Here we focused on the role of HubP in the regulation of flagellar number in V. alginolyticus Deletion of hubP increased the flagellar number and completely disrupted the polar localization of FlhG. It was thought that the flagellar number is determined primarily by the absolute amount of FlhF localized at the cell pole. Here we found that deletion of hubP increased the flagellar number although it did not increase the polar amount of FlhF. We also found that FlhG overproduction did not reduce the polar localization of FlhF. These results show that the absolute amount of FlhF is not always the determinant of flagellar number. We speculate that cytoplasmic FlhG works as a quantitative regulator, controlling the amount of FlhF localized at the pole, and HubP-anchored polar FlhG works as a qualitative regulator, directly inhibiting the activity of polar FlhF. This regulation by FlhF, FlhG, and HubP might contribute to achieving optimal flagellar biogenesis at the cell pole in V. alginolyticus IMPORTANCE: For regulation of the flagellar number in marine Vibrio, two proteins, FlhF and FlhG, work as positive and negative regulators, respectively. In this study, we found that the polar landmark protein HubP is involved in the regulation of flagellar biogenesis. Deletion of hubP increased the number of flagella without increasing the amount of pole-localizing FlhF, indicating that the number of flagella is not determined solely by the absolute amount of pole-localizing FlhF, which is inconsistent with the previous model. We propose that cytoplasmic FlhG and HubP-anchored polar FlhG negatively regulate flagellar formation through two independent schemes.

Suppression by the DNA Fragment of the motX Promoter Region on Long Flagellar Mutants of Vibrio alginolyticus

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 μm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild‐type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long‐Pof strain VIO578. The swimming speed and flagellar length of these transformants were almost equal to the wild‐type values. The amounts of PF47 flagellin and PF60 sheath‐associated protein, which increased in the long‐Pof mutants, were retrieved to almost the wild‐type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V. parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long‐Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.

Characterization of polar-flagellar-length mutants in Vibrio alginolyticus

Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof+Laf-), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5.0.9 μm, 2.5.0.6 μm and 11.2.3.6 μm, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. In the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 μm and 0.062 μm, respectively, whereas those of the wild-type flagella were 1.5 μm and 0.16 μm. The relative amounts of the various molecular components of the long Pof were different from those of the wild-type or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.

Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na+-driven polar flagellum of Vibrio alginolyticus

The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.

Construction of functional fragments of the cytoplasmic loop with the C-terminal region of PomA, a stator component of the Vibrio Na+ driven flagellar motor

The membrane motor proteins, PomA (polar flagellar motility protein A) and PomB (polar flagellar motility protein B), of Vibrio alginolyticus form a stator complex that converts energy from the ion flow to mechanical work in bacterial flagellar motors. The cytoplasmic domain of PomA is believed to interact with the rotor protein FliG to make a torque. In this study, to investigate the function of the cytoplasmic domain of PomA, we constructed a series of fragments that flank the cytoplasmic loop of PomA between the second and third transmembrane (TM) domains (A-loop) and the C-terminal region, and expressed them in Escherichia coli together with PomA and PotB (a chimeric protein of PomB and MotB). We observed a dominant-negative effect of one PomA fragment on motility. We confirmed that these PomA fragments localized both in the membrane fraction and in the cytoplasmic fraction, and induced bacterial growth delay. Effect of additional point and deletion mutations into this fragment implies that the C-terminal region and TM domains used as a linker play a significant part in these observations. From these results, we conclude that the PomA fragments retain the structure important for functions. We expect that further constructions will provide a variety of experimental approaches to characterize the interaction between PomA and FliG.

Mutation in the a-subunit of F1FO-ATPase causes an increased motility phenotype through the sodium-driven flagella of Vibrio

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion as energy source and are composed of stator and rotor proteins. Vibrio alginolyticus has a Na(+)-driven motor and its stator is composed of PomA and PomB. Recently, we isolated increased motility strains (sp1-sp4) from the PomA-N194D/PomB-D24N mutant whose motility was quite weak. To detect the responsible mutation, we have used a next-generation sequencer and determined the entire genome sequences of the sp1 and sp2 strains. Candidate mutations were identified in the gene encoding the a-subunit of F1Fo-ATPase (uncB). To confirm this, we constructed a deletion strain, which gave the increased motility phenotype. The amount of membrane-bound ATPase was reduced in the sp2 and ΔuncB mutants. From these results, we conclude that a mutation in the uncB gene causes the increased motility phenotype in V. alginolyticus. They confer faster motility in low concentrations of sodium than in the parental strain and this phenotype is suppressed in the presence of KCN. Those results may suggest that the proton gradient generated by the respiratory chain is increased by the uncB mutation, consequently the sodium motive force is increased and causes the increased motility phenotype.