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.     

A study of bacterial flagellar bundling

Certain bacteria, such as Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium), use multiple flagella often concentrated at one end of their bodies to induce locomotion. Each flagellum is formed in a left-handed helix and has a motor at the base that rotates the flagellum in a corkscrew motion.We present a computational model of the flagellar motion and their hydrodynamic interaction. The model is based on the equations of Stokes flow to describe the fluid motion. The elasticity of the flagella is modeled with a network of elastic springs while the motor is represented by a torque at the base of each flagellum. The fluid velocity due to the forces is described by regularized Stokeslets and the velocity due to the torques by the associated regularized rotlets. Their expressions are derived. The model is used to analyze the swimming motion of a single flagellum and of a group of three flagella in close proximity to one another. When all flagellar motors rotate counterclockwise, the hydrodynamic interaction can lead to bundling. We present an analysis of the flow surrounding the flagella. When at least one of the motors changes its direction of rotation, the same initial conditions lead to a tumbling behavior characterized by the separation of the flagella, changes in their orientation, and no net swimming motion. The analysis of the flow provides some intuition for these processes.     

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.     

Polar, peritrichous, and lateral flagella belong to three distinguishable flagellar families

The bacterial flagellum transforms its shape into several distinguishable helical shapes (polymorphs) under various environmental conditions. Polymorphs of each type of flagellum stay on a circle in the pitch-diameter (P versus πD) plot, indicating that they all belong to one family. Previously, we showed that the flagellar family of a marine bacterium Idiomarina loihiensis (Family II) differed from the conventional flagellar family of Salmonella typhimurium (Family I). The pitch and diameter of Family II flagella are half those of Family I flagella. We have suggested that Family I encompasses peritrichous flagella, while Family II forms a polar flagellum. In this study, we have surveyed the polymorphs of flagella from 18 other species and categorized their family types. Previous observations were confirmed; Family I form peritrichous flagella and Family II form polar flagella. Furthermore, we found that lateral flagella had helical parameters much smaller than those of the other two Families and thus belong to a new family (Family III).     

The bidirectional polar and unidirectional lateral flagellar motors of Vibrio alginolyticus are controlled by a single CheY species

The bacterial flagellar motor is an elaborate molecular machine that converts ion-motive force into mechanical force (rotation). One of its remarkable features is its swift switching of the rotational direction or speed upon binding of the response regulator phospho-CheY, which causes the changes in swimming that achieve chemotaxis. Vibrio alginolyticus has dual flagellar systems: the Na(+)-driven polar flagellum (Pof) and the H(+)-driven lateral flagella (Laf), which are used for swimming in liquid and swarming over surfaces respectively. Here we show that both swimming and surface-swarming of V. alginolyticus involve chemotaxis and are regulated by a single CheY species. Some of the substitutions of CheY residues conserved in various bacteria have different effects on the Pof and Laf motors, implying that CheY interacts with the two motors differently. Furthermore, analyses of tethered cells revealed that their switching modes are different: the Laf motor rotates exclusively counterclockwise and is slowed down by CheY, whereas the Pof motor turns both counterclockwise and clockwise, and CheY controls its rotational direction.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force.     

Analysis of the lateral flagellar gene system of Aeromonas hydrophila AH-3

Mesophilic Aeromonas strains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. Although Aeromonas lateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster of Aeromonas hydrophila AH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among the flgLL and lafA structural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. All Aeromonas lateral flagellar genes were located in a unique chromosomal region, in contrast to Vibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. In A. hydrophila mutations in flhAL, lafK, fliJL, flgNL, flgEL, and maf-5 resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced the A. hydrophila AH-3 alternative sigma factor sigma54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.     

Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni

Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar) flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that FlhG and polar flagella may function together in a broad range of bacteria to spatially regulate division.     

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.     

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 microm/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.     

A complete set of flagellar genes acquired by horizontal transfer coexists with the endogenous flagellar system in Rhodobacter sphaeroides

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a gamma-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other alpha-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster.     

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.     

The physics of flagellar motion of E. coli during chemotaxis

Flagellar motion has been an active area of study right from the discovery of bacterial chemotaxis in 1882. During chemotaxis, E. coli moves with the help of helical flagella in an aquatic environment. Helical flagella are rotated in clockwise or counterclockwise direction using reversible flagellar motors situated at the base of each flagellum. The swimming of E. coli is characterized by a low Reynolds number that is unique and time reversible. The random motion of E. coli is influenced by the viscosity of the fluid and the Brownian motion of molecules of fluid, chemoattractants, and chemorepellants. This paper reviews the literature about the physics involved in the propulsion mechanism of E. coli. Starting from the resistive-force theory, various theories on flagellar hydrodynamics are critically reviewed. Expressions for drag force, elastic force and velocity of flagellar elements are derived. By taking the elastic nature of flagella into account, linear and nonlinear equations of motions are derived and their solutions are presented.     

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.     

Peptidoglycan maturation enzymes affect flagellar functionality in bacteria

The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram‐negative pathogen Salmonella typhimurium and the Gram‐positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.     

MotX, the channel component of the sodium-type flagellar motor.

Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum. The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions. Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum. A gene, motX, encoding one component of the torque generator has been cloned and sequenced. The deduced protein sequence is 212 amino acids in length. MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor. Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli. The rate of lysis caused by induction of motX was proportional to the sodium ion concentration. Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis. Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride. The data suggest that MotX forms a sodium channel. The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains.