Flagellar Morphology of Vibrio parahaemolyticus (Fujino et al) Sakazaki,Iwanami and Fukumi 19631

The flagellar morphology of 88 Vibrio parahaemolyticus strains, including a strain descended from Fujino's original strain EB101 (= ATCC17802 = KM1339) was studied. EB101 and 83 other strains (95%) showed mixed polar and peritrichous type of flagellation when grown on modified MOF (MMOF) agar after 16-hr incubation at 20 C. Cultures containing numerous peritrichous cells showed wiggly movements in moist preparations and rapidly spreading growth in semisolid agar plates. Peritrichous flagella were easily removed mechanically from the soma. The mean wavelengths of polar and peritrichous flagella were 2.53 μm (normal type) and 1.72 μm (atypical curly type) respectively. Peritrichous cells on solid media appeared after incubation for 2.5 hr at 37 C and 7 hr at 20 C. Overnight incubation at 37 C and acidity of the medium due to fermentation of carbohydrate markedly ruined peritrichous flagella. Electron micrograph of cells grown on MMOF agar revealed a sheathed polar flagellum and unsheathed peritrichous flagella. A hook structure was demonstrated at the proximal end of the latter. Polar monotrichous cultures in MMOF broth sometimes contained some cells having several or many peritrichous flagella of atypical curly type. Seven strains of Vibrio cholerae were exclusively polar monotrichous on solid and in liquid media. The flagellation of V. parahaemolyticus is concluded as being a mixed polar-peritrichous type. This fact would indicate that V. parahaemolyticus should be excluded from the genus Vibrio, since the genus Vibrio was defined as polar monotrichous.     

A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella.

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.     

Cell orientation of swimming bacteria: From theoretical simulation to experimental evaluation

The motion of small bacteria consists of two phases: relatively long runs alternate with intermittent stops, back-ups, or tumbles, depending on the species. In polar monotrichous bacteria, the flagellum is anchored at the cell pole inherited from the parent generation (old pole) and is surrounded by a chemoreceptor cluster. During forward swimming, the leading pole is always the pole recently formed in cell division (new pole). The flagella of the peritrichous bacterium Escherichia coli often form a bundle behind the old pole. Its cell orientation and receptor positioning during runs generally mimic that of monotrichous bacteria. When encountering a solid surface, peritrichous bacteria exhibit a circular motion with the leading pole dipping downward. Some polar monotrichous bacteria also perform circular motion near solid boundaries, but during back-ups. In this case, the leading pole points upward. Very little is known about behavior near milieu-air interfaces. Biophysical simulations have revealed some of the mechanisms underlying these phenomena, but leave many questions unanswered. Combining biophysics with molecular techniques will certainly advance our understanding of bacterial locomotion.     

A Phase Variant of Azospirillum lipoferum Lacks a Polar Flagellum and Constitutively Expresses Mechanosensing Lateral Flagella

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.     

Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

Comparison of the flagellins from different flagellar morphotypes of Escherichia coli.

The molecular weights of the flagellins of 13 strains of Escherichia coli, each with a different H antigen, were estimated using polyacrylamide gel electrophoresis. In each case only one major polypeptide was demonstrated, although some strains possessed apparently sheathed flagella. Considerable differences in the molecular weight of flagellin accompanied the previously described structural differences between flagella from strains with different H antigens. The relationship between flagellar diameter and the molecular weight of the corresponding flagellins was similar for both unsheathed and apparently sheathed flagella. Crosss-polymerization occurred between seed consisting of fragment of unsheathed flagella and flagellin solution from apparently sheathed flagella and vice versa. Co-polymerization of flagellin from unsheathed flagella and flagellin from apparently sheathed flagella was also demonstrated. These polymerization experiments indicate that the assembly pattern of flagellin molecules is probably the same in all E. coli flagella. The above and other evidence suggests that there is no true sheath, but that the differences in flagellar surface structure between different E. coli flagella are the result of differences in the superficial parts of the flagellin molecules.     

Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.     

Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei.

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.     

Isolation and characterization of Campylobacter flagellins.

Sequential acid pH dissociation, differential ultracentrifugation, and neutral pH reassociation were used to partially purify serotypically distinct flagella from three strains of Campylobacter jejuni and the two antigenic phases of flagella of Campylobacter coli VC167. Each C. jejuni flagellin and C. coli VC167 antigenic phase 1 flagellin were purified to homogeneity by reverse-phase high-performance liquid chromatography with a C8 Spheri-10 column. C. coli VC167 antigenic phase 2 was purified to homogeneity by ion-exchange chromatography with a Mono-Q column. Amino acid compositional analysis put the C. jejuni flagellin molecular weight in the range 63,200 to 63,800 and the C. coli antigenic phase 1 and 2 flagellins at 61,500 and 59,500, respectively. The amino acid compositions of the C. jejuni were similar to each other and to the C. coli VC167 antigenic phase 1 and phase 2 flagellins. One-dimensional peptide mapping of the C. jejuni flagellins by partial digestion with trypsin or chymotrypsin confirmed the structural similarities of the C. jejuni flagellins and the C. coli VC167 antigenic phase 1 flagellin and showed that C. coli VC167 antigenic phase 2 flagellin was structurally distinct from the phase 1 flagellin. The antigenic phase 2 flagellin was especially sensitive to digestion by chymotrypsin. Amino-terminal sequence analysis showed that the 20 N-terminal amino acids of the Campylobacter flagellins were highly conserved. The Campylobacter flagellins also shared limited sequence homology with the N-terminal sequences reported for Salmonella and Bacillus flagellins.     

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449.

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.     

Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7.

Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.     

Lateral flagella are required for increased cell adherence,invasion and biofilm formation by Aeromonas spp

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.     

The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica.

The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.     

pH-dependent flagella formation by facultative alkaliphilic Bacillus sp. C-125.

A facultative alkaliphilic strain of Bacillus sp. C-125 grown at alkaline pH had many sinuous peritrichous flagella and was highly motile. However, most of the cells grown initially at pH 7 were non-motile and possessed few straight flagella. The amount of flagellin was low when the organism was grown at pH 7, suggesting that non-motility is due to poor synthesis of flagellin. The molecular mass of the flagellin was 37 kDa and the isoelectric point was pH 5.0. The amino acid composition of the flagellin was similar to that found in the flagellin from neutrophilic Bacillus subtilis 168.     

Recent advances in the structure and assembly of the archaeal flagellum

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

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.     

Effects of pH of the medium on flagellation of Vibrio parahaemolyticus.

Formation of the lateral flagella by Vibrio parahaemolyticus was inhibited under an alkaline condition of the medium. However, flagellation of the polar monotrichous flagellum was not affected in the same condition. Flagellation of the lateral flagella depended on the pH of the medium.     

Effects of pH of the medium on flagellation of Vibrio parahaemolyticus.

Formation of the lateral flagella by Vibrio parahaemolyticus was inhibited under an alkaline condition of the medium. However, flagellation of the polar monotrichous flagellum was not affected in the same condition. Flagellation of the lateral flagella depended on the pH of the medium.     

A method for the purification of bacterial flagellin that allows simple upscaling

There is a growing interest in enterobacterial flagellins that may result in a demand to produce flagellin on an industrial scale for possible applications as an adjuvant, immunomodulatory agent or vaccine antigen. Traditionally, small-scale production of flagellin has occurred in the laboratory by flagellar shearing of bacterial surfaces and subsequent ultracentrifugation. The main drawback of this method is the need to use low-agitation cultures to avoid the loss of flagella due to shearing during culture. In the present work, we describe a scalable protocol for the production of flagellin with higher yields than traditional laboratory-scale protocols. The use of cross-flow filtration to concentrate bacterial cultures combines extensive shearing of flagella with a reduction in volume, greatly simplifying downstream processing. This technique also allows the use of highly-agitated culture conditions because any sheared flagella are retained in the bacterial concentrate. Flagella obtained with this procedure showed in vivo and in vitro innate activating capacities similar to those of flagella produced at laboratory scale. This procedure is flexible, allowing an increase in production scale, an enhancement of flagellin yield and no requirement for expensive equipment.