Various species of marine Vibrio produce two distinct types of flagella, each adapted for a different type of motility. A single, sheathed polar flagellum is suited for swimming in liquid medium, and numerous unsheathed lateral flagella, which are produced only under viscous conditions, are suited for swarming over viscous surfaces. Both types of flagella are driven by reversible motors embedded in the cytoplasmic membrane. Here we report that the energy source for the polar flagellar motor of Vibrio parahaemolyticus is the sodium-motive force, whereas the lateral flagellar motors are driven by the proton-motive force. This is evidence that two distinct types of flagella powered by different energy sources are functionally active in one cell.

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof ؉ Laf ؊ ) or only lateral flagella (Pof ؊ Laf ؉ ). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H ؉ and Na ؉ motive forces, respectively, as in the related species V. parahaemolyticus.Vibrio alginolyticus and V. parahaemolyticus, which are closely related, have similar flagellar systems. They have two types of flagella, named polar flagella (Pof) and lateral flagella (Laf), in one cell (1, 4). When they are grown in a liquid environment, they express mainly a single polar flagellum at their cell poles and swim by it rapidly. When they are transferred to a solid agar medium, they express lateral flagella peritrichously in addition to polar flagella, and the cells elongate and begin to move and spread on the surface (3, 9, 12, 13). Recently, by using those flagellar mutants with only polar flagella and with only lateral flagella, it has been shown that in V. parahaemolyticus, the energy sources of polar and lateral flagellar motors are Na ϩ and H ϩ motive forces, respectively (2). Results of experiments using wild-type cells have suggested that the polar and lateral flagella of V. alginolyticus are also powered by different ion motive forces (2, 5). Although some flagellum-defective mutants have been isolated from V. alginolyticus (12), the organism has not yet been characterized in this regard. In this study, we isolated mutants with only polar or with only lateral flagella from V. alginolyticus, which is less pathogenic than V. parahaemolyticus (4) and is well characterized in terms of respiration-coupled primary sodium pumps (10, 11). Furthermore, an energetic analysis of the polar and lateral motors was carried out.Isolation of lateral or polar flagellum-defective mutants. The parental strain V. alginolyticus 138-2 (Pof ϩ Laf ϩ ) (11) was exposed to UV light at the 0.01% survival level. The cells were spotted on the center of 1.5% agar HI plates (1.5% agar in HI broth, which contained [per liter of distilled water] 25 g of heart infusion broth [Difco] and 20 g of NaCl) and incubated at 30ЊC overnight. To enrich nonswarming cells on 1.5% agar, the cells from the original spot location were grown in HI broth to stationary phase. After the enrichment procedure was repeated, the diluted cultures from the center were spread on 2.5% agar HI plates (2.5% agar in HI broth). As a result, we isolated the three independent mutants which could not swarm on the 1.5% agar plate. The lateral flagellum-defective mutants (Pof ϩ Laf Ϫ ) were named YM4, YM5, and YM6. Strains YM4 and YM6 did not give rise to Laf ϩ revertants when the cells were inoculated on 1.5% agar plates and incubated at 30ЊC for 3 days, but YM5 did. Next, we tried to isolate polar flagellum-defective mutants (Pof Ϫ Laf ϩ ), which were expected to swarm on either 1.5 or 0.3% agar plates by using lateral flagella. Therefore, first we isolated Pof Ϫ...

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

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Amiloride, a specific inhibitor for the Na -driven flagellar motors of alkalophilic BacUlus strains, was found to cause growth inhibition; therefore, the use of amiloride for the isolation of motility mutants was difficult. On the other hand, phenamil, an amiloride analog, inhibited motor rotation without affecting cell growth. A concentration of 50 ,IM phenamil completely inhibited the motility of strain RA-1 but showed no effect on the membrane potential, the intracellular pH, or Na+-coupled amino acid transport, which was consistent with the fact that there was no effect on cell growth. Kinetic (7,(10)(11)(12) and is powered by the electrochemical potential gradient of sodium across the membrane, the so-called Na+ motive force.To clarify the mechanism of energy coupling in the flagellar motors, an important step is the identification of the force-generating unit, which is assumed to have a channel activity for the coupling ion in the motor. In the case of the H+-driven flagellar motors of Escherichia coli and Salmonella typhimurium, genetic approaches revealed that the products of the motA and motB genes are required for the motor rotation (21,26 In the instance of the Na+-driven flagellar motors, the force-generating unit of the motor is the essential site for the specific interaction with Na+. We therefore assumed that the drugs which interfere with the interaction of Na+ with the motor could cause the inhibition of motor rotation. A search for such drugs was successful, and Sugiyama et al. (27) found that amiloride, a potent inhibitor for the Na+ channel in animal cells (2, 4, 18), specifically inhibits the rotation of the Na+-driven flagellar motors of alkalophilic Bacillus spp. by competing with Na+ in the medium. The results suggest that the site of action of amiloride on the motor is the Na+ interaction site located at the outer side of the forcegenerating unit. Thus, amiloride is expected to be a useful tool for elucidating the energy conversion mechanism at the force-generating unit of the motor.During the studies with amiloride for the isolation of mutants with an altered sensitivity of motility to amiloride, we found that this agent caused an inhibition in the growth of alkalophilic Bacillus spp. Here we report that an amiloride analog, phenamil, specifically inhibits the Na+-driven flagellar motors without any growth inhibition and that the drug is quite useful for the isolation of motor-specific mutants. MATERIALS AND METHODSBacterial strains and growth conditions. The alkalophilic Bacillus strain used for most experiments was a streptomycin-resistant mutant of Bacillus firmus RAB (19), which was isolated as a spontaneous mutant on an agar plate containing 200 ,ug of streptomycin per ml and was named RA-1. Alkalophilic Bacillus sp. strains 202-1, YN-1, and 8-1 and Bacillus alcalophilus ATCC 27647 have been described previously (13). The other alkalophilic Bacillus strains used in this study were Bacillus sp. strains YN-2000 (29), C59-2 (16), M-29 (1), and N-6 (17). Neutrophilic strains used i...

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