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.     

Regulation of polar flagellar number by the flhF and flhG genes in Vibrio alginolyticus

The number and location of bacterial flagella vary with the species. The Vibrio alginolyticus cell has a single polar flagellum, which is driven by sodium ions. We selected mutants on the basis of reduced swarming ability on soft agar plates. Among them, we found two mutants with multiple polar flagella, and named them KK148 and NMB155. In Pseudomonas species, it is known that FlhF and FleN, which are FtsY and MinD homologs, respectively, are involved in regulation of flagellar placement and number, respectively. We cloned homologous genes of V. alginolyticus, flhF and flhG. KK148 cells had a nonsense mutation in flhG; cells expressing transgenic flhG recovered the swarming ability and had a reduced number of polar flagella. NMB155 cells did not have a mutation in either flhF or flhG. In wild-type cells, expression of flhF increased the number of polar flagella; in contrast, expression of flhG reduced both the number of polar flagella and the swarming ability. These results suggest that FlhG negatively regulates the number of polar flagella in V. alginolyticus. KK148 cells expressing both flhF and flhG exhibited fewer polar flagella and better swarming ability than KK148 cells expressing flhG alone, suggesting that FlhG acts with FlhF.     

Characterization of the holdfast region of wild-type cells and holdfast mutants of Asticcacaulis biprosthecum

Mutants of Asticcacaulis biprosthecum lacking the ability to attach to various surfaces were selected by serial transfer in liquid media containing cheesecloth, to which wild-type cells attach but holdfast mutants do not. Congo red, incorporated into solid media, distinguishes between colonies of wild-type cells and those of holdfast mutants. Holdfast mutants were characterized and compared to wild-type cells according to their ability to swim, to attach to each other or to wild-type cells, for the presence on the cells of polar surface structures (holdfast, flagella, pili), and for sensitivity to phages. All holdfast mutants produced flagella, even though some mutants were nonmotile. Eighteen holdfast mutants fell into two groups: those apparently defective only in holdfast function and those defective in additional structures localized at the holdfast pole of the cell. None of these holdfast mutants was defective in prosthecal development. All holdfast mutants are capable of forming rosettes with wild-type cells, even though they are incapable of initiating attachment on their own, suggesting polymeric bridging as a likely mechanism for attachment.Abbreviation PYE peptone-yeast extract     

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.     

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.     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.     

Cohnella soli sp. nov. and Cohnella suwonensis sp. nov. Isolated from soil samples in Korea

Two bacterial isolates from soil samples taken in Korea, strains YM2-7^T and WD2-19^T, were characterized using a polyphasic approach. The cells were strictly aerobic, Gram-positive, motile with peritrichous flagella, and rod-shaped. Both strains formed ellipsoidal bulging positioned subterminal spores. Phylogenetic analysis of their 16S rRNA gene sequences revealed a clear affiliation with the Firmicutes. The 16S rRNA gene sequence similarity between YM2-7^T and WD2-19^T was 96.5%. Strains YM2-7^T and WD2-19^T showed 16S rRNA gene sequence similarities of 93.0–96.5% to type strains of recognized Cohnella species. The G+C contents of the DNA of strains YM2-7^T and WD2-19^T were 52.2 and 55.6 mol%, respectively. The major fatty acids of strains YM2-7^T and WD2-19^T were anteiso-C15:0 (44.4%), C16:0 (19.2%), and iso-C16:0 (16.8%) and anteiso-C15:0 (46.5%), iso-C16:0 (21.8%), and C16:0 (11.2%), respectively. Both strains contained menaquinone with seven isoprene units (MK-7) as the predominant quinone. Both strains had diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysophosphatidylglycerol as the major polar lipids. Comparative analysis of phenotypic and phylogenetic traits indicated that strains YM2-7^T and WD2-19^T represented two novel species of the genus Cohnella. The names Cohnella soli sp. nov. (type strain YM2-7^T =KACC 13346^T =NBRC 106486^T), and Cohnella suwonensis sp. nov. (type strain WD2-19T =KACC 13347^T =NBRC 106485^T) are proposed for these organisms.     

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.     

Polar flagellum biogenesis in Aeromonas hydrophila

Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.     

Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts

Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces.     

Viscous Dynamics of Lyme Disease and Syphilis Spirochetes Reveal Flagellar Torque and Drag

The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20–40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.     

Enhancement of the activity and pH-performance of chitosanase from Bacilluscereus strains by DNA shuffling

DNA shuffling was carried out with two chitosanase genes belonging to glycoside hydrolase family eight from Bacillus cereus KNUC51 and B. cereus KNUC55. The shuffled products, YM18 and YM20, which showed higher activity than the parents at 40°C, were selected for further studies. The 50 kDa chitosanases were purified using affinity chromatography with glutathione-Sepharose 4B. In general, the specific activity of YM18 is enhanced 250% and that of YM20 is 350% compared to the parents. YM20 exhibits a shift of the optimal pH level from 5.5 to 6.5. DNA sequence analysis revealed that YM18 and YM20 contained 2 amino acid substitutions (I13T and A87V for YM18; K66R and N352S for YM20). We presumed that these amino acid substitutions increase the specific activity and change the property of the two variants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spontaneous mutants of Microsporon gypseum resistant to griseofulvin

Summary 1) The spores of the microconidial mutant I–18 of the dermatophyteMicrosporon gypseum in agar medium with GF germinated and formed germ tubes deformated in a characteristic way. From 1µg GF/ml up with an increasing antibiotic concentration (expressed in logarithms) the munber of colonies grown (expressed in probits) decreased linearly.2) As a sensitivity measure of the spores the median efficient dose ED 50 was used which was determined by means of a graphic probit analysis. For the strain used this value was determined in the range between 1.35–1.95µg GF/ml in three independent experiments.3) From the smears of a thickened spore suspension (1.6–14.2 × 10⁷ viable spores) in medium containing a high GF concentration a very small, but as for the order a stable number of colonies grew, as found in eight independent experiments. On the medium containing 20µg GF/ml in average 61 colonies grew, on 40µg GF/ml 20 colonies, on 80µg GF/ml 3 colonies and on 160µg GF/ml 0.3 colony (expressed in 10⁷ viable spores tested).4) A part of these colonies were isolated and transferred 29 times on a medium without the antibiotic. Two isolates only show a permanently increased resistance to GF, viz. the strain D-29 which is 50 × more resistant and the strain N-53 which is 3.5 × more resistant than the wild strain I-18.     

Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus.

By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system. Pof+ Laf- cells could not swim in the high-viscosity environment (ca. 200 cP) in which Pof- Laf+ cells swam at 20 microns/s. The Pof- Laf+ cells swam at about 20 microns/s at normal viscosity (1 cP) without the viscous agent, and the speed increased to 40 microns/s at about 5 cP and then decreased gradually as the viscosity was increased further. These results show the functional difference between polar and lateral flagella in viscous environments.     

Galvanic Stimulation of Volvox globator II. Mechanism of Galvanic Response, an Effect of Calcium Displacement

Slight increases or decreases in calcium ions in solutions which supported the growth of Volvox globator colonies caused the colonies to fall to the bottoms of their containers. High speed cinematography (600 frames/sec) showed that the flagella beat normally (21/sec) in balanced electrolyte solutions which have calcium concentrations between 0.5 and 1.0 mM. When colonies were placed in 10.0 or 0.0 mM CaCl₂ solutions, flagellar beating disappeared within 1 hr. The cessation of flagellar beating was reversible when colonies were replaced in the balanced solution. The Volvox cell wall has been shown to be a fairly good cation-exchanger with calcium ions acting as the counterion to the fixed negative change. Colonies that were photopositive and gave a cathodal galvanotaxis responded to DC electrical potentials by producing solution patterns that were indicators of colony electronegativity. Colony resistance to electroosmotic flow was compared in potassium and calcium solutions. When colonies were placed in darkness for 24 hr and stimulated by DC electrical potentials, their cation-exchange properties became reduced and the cell walls appeared thinner. Application of a high DC electrical potential to dark-adapted colonies caused the colonies to shrink on their anode sides (anodal contraction). Other workers have found that the flagella on the anodal sides of dark-adapted colonies ceased beating during DC electrical stimulation. It is hypothesized that the electric current caused an increase of calcium ions on the anodal side of the colony that inhibited the flagellar mechanism of beating on that side. It is also hypothesized that the galvanotaxis associated with light-adapted (photopositive) colonies was due to calcium displacements in the colony cell walls that affected the flagellar beating on both sides of the colony.     

A new 'paralyzed' flagella mutant, OC-10, in Chlamydomonas reinhardtii (Volvocales, Chlorophyceae) that can be reactivated with adenosine triphosphate

A new ‘paralyzed’ mutant. OC–10, was isolated in Chlamydomonas reinhardtii Dangeard. OC-10 cannot swim and generally shows very little flagellar movement. However, when OC-10 was demembranated, axonemal motility was reactivated in the presence of adenosine triphosphate (ATP) or adenosine diphosphate (ADP). The beating form of the reactivated axonemes was almost the same as that of the wild-type axonemes. Flagellar regeneration of OC-10 was slower than that of the wild-type. Electron microscopic examination showed no abnormality in OC-10 flagella, but SDS/PAGE revealed that mobility of a flagellar membrane protein was changed and a few bands disappeared in OC-10 flagella, When the mutant was crossed to wild-type to form temporary dikaryon cells with 4 flagella, OC-10 flagella did not regain motility. Tetrad analysis of crosses between OC–10 and wild-type demonstrated a 1:1 segregation on the basis of flagellar motility. From these results, we suppose that OC-10 may be limited in ATP availability inside the flagella, or altered in flagellar membrane proteins important for motility.     

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.     

Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium

Background  

Many bacteria swim by rotating helical flagellar filaments [1]. Waterbury et al. [15] discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces [2,7]. The hypothesis that Synechococcus might swim using traveling surface waves [6,13] prompted this investigation.     

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.