Structure and Arrangement of Flagella in Species of the Genus Beneckea and Photobacterium fischeri

Species of marine bacteria belonging to the genus Beneckea and strains of Photobacterium fischeri were negatively stained and examined by means of the electron microscope to determine the structure and arrangement of their flagella. All of the species of the genus Beneckea had single, polar, sheathed flagella when grown in liquid medium. When grown on solid medium, most strains of B. campbellii and B. neptuna and all strains of B. alginolytica and B. parahaemolytica had unsheathed, peritrichous flagella in addition to the single, sheathed, polar flagellum. The remaining species, B. nereida, B. pelagia, and B. natriegens, had a single, polar, sheathed flagellum when grown on solid medium. Strains of P. fischeri had sheathed flagella arranged in polar tufts. Only one group (B-2) of marine bacteria included in this study was found to have polar, unsheathed flagella.     

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.     

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.     

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.     

Taxonomy of Marine Bacteria: the Genus Beneckea

One-hundred-and-forty-five isolates of marine origin were submitted to an extensive physiological, nutritional, and morphological characterization. All strains were gram-negative, facultatively anaerobic, straight or curved rods which were motile by means of flagella. Glucose was fermented with the production of acid but no gas. Sodium but no organic growth factors were required. None of the strains were able to denitrify or fix molecular nitrogen. The results of nutritional and physiological tests were submitted to a numerical analysis. On the basis of phenotypic similarity, nine groups were established. These groups could be distinguished from one another by multiple, unrelated, phenotypic traits. Six groups which had deoxyribonucleic acid (DNA) containing 45 to 48 moles per cent guanine plus cytosine (GC) were assigned to a redefined genus Beneckea. All of the strains in this genus, when grown in liquid medium, had a single, polar flagellum. When grown on a solid medium, many strains had peritrichous flagella. Two groups were similar to previously described species and were designated B. alginolytica and B. natriegens. The remaining four groups were designated B. campbellii, B. neptuna, B. nereida, and B. pelagia. An additional group of phenotypically similar strains having the properties of the genus Beneckea was not included in the numerical analysis. These strains were readily separable from species of this genus and were designated B. parahaemolytica. Of the remaining groups, one was identified as Photobacterium fischeri. The other group (B-2) which had about 41 moles% GC content in its DNA could not be placed into existing genera.     

Effects of some chemical factors on flagellation and swarming ofVibrio alginolyticus

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0–9.0% (w/v) NaCl, cells with polar tufts of 2–4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3–9.8% (w/v) Na₂SO₄ had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1–0.3% boric acid or 0.05–0.1% aluminium hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na-deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01–0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05–0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.     

Giant flagellar bundles ofVibrio alginolyticus (NCMB 1803)

Summary Following swarming ofVibrio alginolyticus on solid medium a large number of giant flagellar bundles appear behind the growth front. The suggested sequence of events leading to bundle formation is as follows. After inoculation from liquid to solid media the short rods with a single polar sheathed flagellum develop peritrichous nonsheathed flagella and elongate into long filamentous swarmers. After division into short rods, some of the cells become spherical in shape with many peritrichous flagella concentrated at one pole in close association with the sheathed polar flagellum. These tufted spherical bodies form the template upon which masses of loose peritrichous flagella spontaneously aggregate.Flagellar bundles formed when bacteria are grown at pH 8.5 are longer than those formed at pH 7.2 and shorter when grown at pH 6.5. In distilled water the flagellar bundles disintegrate into masses of flagellar fragments.     

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.     

Na(+)- and H(+)-dependent motility in the coral pathogen Vibrio shilonii

In this work, we analyzed motility and the flagellar systems of the marine bacterium Vibrio shilonii. We show that this bacterium produces lateral flagella when seeded on soft agar plates at concentrations of 0.5% or 0.6%. However, at agar concentrations of 0.7%, cells become round and lose their flagella. The sodium channel blocker amiloride inhibits swimming of V. shilonii with the sheathed polar flagellum, but not swarming with lateral flagella. We also isolated and characterized the filament–hook–basal body of the polar flagellum. The proteins in this structure were analyzed by MS. Eight internal sequences matched with known flagellar proteins. The comparison of these sequences with the protein database from the complete genome of V. shilonii allows us to conclude that some components of the polar flagellum are encoded in two different clusters of flagellar genes, suggesting that this bacterium has a complex flagellar system, more complex possibly than other Vibrio species reported so far.     

Flagellation and taxonomy ofAcetobacter andAcetomonas

Summary Leifson's findings, that motile, acetate-oxidizing acetic acid bacteria (Acetobacter) have peritrichous flagella, and that motile, non-acetate oxidizing ones (Acetomonas) have polar flagella, of notably short wavelength, are fully confirmed and photographically illustrated. It is not confirmed, however, that the peritrichous flagella ofAcetobacter are always of “orthodox” type with a wavelength of about 2.9 μ, nor that they always tend to be few in number. In one strain ofA. aceti they were numerous, and the wavelength was as short (1.4 μ) as that considered byLeifson to be uniquely confined to the polar flagella ofAcetomonas. Furthermore the polar flagella of the latter genus seem not always to be multitrichous, strains having been found with only a single polar flagellum.     

Luminous bacteria synthesize luciferase anaerobically

Four species of luminous bacteria, Photobacterium phosphoreum, P. leiognathi, P. fischeri and Beneckea harveyi (two strains of each), were shown to synthesize luciferase anaerobically. One of these, P. phosphoreum, produced as much luciferase anaerobically as it did aerobically, and all four species were found to grow almost equally rapidly under the two sets of conditions. Previous work with B. harveyi and P. fischeri had shown that aerobic luciferase synthesis can proceed only after an inhibitor in the complex medium has been removed and a species-specific autoinducer secreted. All strains tested also removed the inhibitor and secreted an autoinducer anaerobically. The small amount of luciferase produced anaerobically by some strains is thus apparently not due either to lack of removal of inhibitor or to insufficient production of autoinducer but may involve an oxygen-dependent control mechanism.Abbreviations LU light units - OD optical density     

Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus.

Scanning electron microscopy was used to study the production of lateral flagella and the swarming phenomenon in Vibrio parahaemolyticus. Differences in the size and diameter of the sheathed, polar flagellum and lateral flagella were apparent in these preparations. Swarming of V. parahaemolyticus was found to be similar to the swarming of Proteus spp. in that swarm cells which are heavily flagellated and elongated are formed.     

Isolation and characterization of a visibly luminous variant ofVibrio fischeri strain ES114 from the sepiolid squidEuprymna scolopes

Vibrio fischeri strains isolated from light organs of the sepiolid squid Euprymna scolpes are non-visibly luminous and fast growing in laboratory culture, whereas in the symbiosis they are visibly luminous and slow growing. A spontaneous, visibly luminous, slow-growing variant was isolated from a laboratory culture of the squid-symbiotic V. fischeri strain ES114. Taxonomic and DNA-homology analyses demonstrated that the variant was V. fischeri and was very similar to the original form. However, the variant grew at one-fourth the rate of the original form, produced 30,000-fold more luminescence, induced luminescence at a lower cell density, and produced a higher level of V. fischeri luminescence autoinducer. Regulation of luminescence, nonetheless, was similar in the two forms and typical of V. fischeri with respect to responses to autoinducer, glucose, the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid), and 3′:5′-cyclic AMP. Compared to the original form, cells of the variant were smaller, exhibited from zero to two polar, sheathed flagella instead of a tuft of three to eight flagella, produced a deeper yellow-orange pigment, did not acidify media containing glycerol, and produced a more distinct pellicle. The two forms also differed in the levels of several outer membrane and soluble proteins. These results establish a distinctive physiological, morphological, and biochemical dimorphism in V. fischeri ES114 in which the variant exhibits several traits similar to V. fischeri cells in the symbiotic state. The variant and its conversion from the original form in laboratory culture may provide insight into the properties of V. fischeri cells in the symbiosis and may serve as a model for elucidating the mechanism for their pleiotropic conversion upon colonization of the squid. Received: 10 January 1995 / Accepted: 24 May 1995     

Attachment of Vibrio alginolyticus to Glass Surfaces Is Dependent on Swimming Speed

The attachment of Vibrio alginolyticus to glass surfaces was investigated with special reference to the swimming speed due to the polar flagellum. This bacterium has two types of flagella, i.e., one polar flagellum and numerous lateral flagella. The mutant YM4, which possesses only the polar flagellum, showed much faster attachment than the mutant YM18, which does not possess flagella, indicating that the polar flagellum plays an important role. The attachment of YM4 was dependent on Na⁺ concentration and was specifically inhibited by amiloride, an inhibitor of polar flagellum rotation. These results are quite similar to those for swimming speed obtained under the same conditions. Observations with other mutants showed that chemotaxis is not critical and that the flagellum does not act as an appendage for attachment. From these results, it is concluded that the attachment of V. alginolyticus to glass surfaces is dependent on swimming speed.     

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.     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium     

Polar Flagellar Motility of the Vibrionaceae

Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 μm/s. The sodium motive force powers rotation of the filament, which acts as a propeller. The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane. The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus. The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species. This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae. In addition to its propulsive role, the single polar flagellum of V. parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression. Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces. Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery.     

Vibrio harveyi Associated with Aglaophenia octodonta (Hydrozoa, Cnidaria)

A previously unknown association between a luminous bacterium, Vibrio harveyi, and a benthic hydrozoan, Aglaophenia octodonta, is described. Aglaophenia hydrocladia showed a clear fluorescence in the folds along the hydrocaulus and at the base of the hydrotheca, suggesting the presence of luminous bacteria. This hypothesis was confirmed by isolation of luminous bacteria from Aglaophenia homogenates. Phenotypic characterization of bacterial isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a single species: V. harveyi. The association between V. harveyi and A. octodonta has epidemiological as well as ecological significance. Therefore, A. octodonta may function as habitat “islands” providing a unique set of environmental conditions for luminous bacteria colonization, quite different from those already recorded from the plankton for other Vibrio species.