Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.     

Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources.

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V. parahaemolyticus.     

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.     

Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.     

Mutational analysis of flagellum-independent surface spreading of Serratia marcescens 274 on a low-agar medium.

In a previous study (J. O'Rear, L. Alberti, and R. M. Harshey, J. Bacteriol. 174:6125-6137, 1992) we reported the isolation of several transposon mutants of Serratia marcescens 274 that were defective either in swarming alone or in both swimming and swarming motility. All the nonflagellate (Fla-) mutants, while defective in both types of motility, were able to spread rapidly on the surface of low-agar (0.35%) media. We show here that some of the swarming-defective mutants are defective in the production of serrawettin W1, an extracellular cyclic lipopeptide produced by S. marcescens 274. When combined with a Fla defect, the serrawettin (Swt) mutants are deficient in spreading on low-agar media. The spreading deficiency can be overcome by serrawettin supplied extracellularly. Introduction of Fla defects into chemotaxis mutants does not affect this mode of surface translocation. These results suggest that spreading may be a passive form of translocation. We also report that swarming defects in all mutants showing a Dps phenotype (able to swarm within the inoculated area but unable to move outward) in the earlier study can be overcome by changing the commercial source of agar.     

3-Amino 1,8-naphthalimide,a structural analog of the anti-cholera drug virstatin inhibits chemically-biased swimming and swarming motility in vibrios

A screen for inhibitors of Vibrio cholerae motility identified the compound 3-amino 1,8-naphthalimide (3-A18NI), a structural analog of the cholera drug virstatin. Similar to virstatin, 3-A18NI diminished cholera toxin production. In contrast, 3-A18NI impeded swimming and/or swarming motility of V. cholerae and V. parahemolyticus suggesting that it could target the chemotaxis pathway shared by the polar and lateral flagellar system of vibrios. 3-A18NI did not inhibit the expression of V. cholerae major flagellin FlaA or the assembly of its polar flagellum. Finally, 3-A18NI enhanced V. cholerae colonization mimicking the phenotype of chemotaxis mutants that exhibit counterclockwise-biased flagellum rotation.     

Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function.

Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.     

Bacteria that express lateral flagella enable dissection of the multifunctional roles of flagella in pathogenesis

Flagella are much more than organelles of locomotion and have multiple roles that contribute to pathogenesis. Bacteria, such as Vibrio parahaemolyticus and Aeromonas spp., that possess two distinct flagellar systems (a polar flagellum for swimming in liquid and lateral flagella for swarming over surfaces) are relatively uncommon and provide ideal models for the independent investigation of the contributions of these different types of motility and other flagellar functions to virulence and how they are controlled. Studies with the above organisms have already increased our understanding of how bacteria sense and colonize surfaces forming biofilms that enable them to survive and persist in hostile environments. These insights are helping to identify possible new targets for novel antimicrobials that will both prevent or disrupt these processes and enhance the effectiveness of existing antibiotics. Aeromonas lateral flagella, in addition to mediating swarming motility, appear to be adhesins in their own right, contribute to microcolony formation and efficient biofilm formation on surfaces, and possibly facilitate host cell invasion. It is, therefore, likely that the ability to express lateral flagella is a significant virulence determinant for the Aeromonas strains able to cause persistent and dysenteric infections in the gastrointestinal tract, but further work is needed to establish this.     

Surface-induced swarmer cell differentiation of Vibrio parahaemoiyticus

Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface-induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria.     

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.     

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.     

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.     

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.     

Bacterial lateral flagella: an inducible flagella system

Flagella are complex surface organelles that allow bacteria to move towards favourable environments and that contribute to the virulence of pathogenic bacteria through adhesion and biofilm formation on host surfaces. There are a few bacteria that possess functional dual flagella systems, such as Vibrio parahaemolyticus, some mesophilic Aeromonas spp., Rhodospirillum centenum and Azospirillum brasilense. These bacteria are able to express both a constitutive polar flagellum required for swimming motility and a separate lateral flagella system that is induced in viscous media or on surfaces and is essential for swarming motility. As flagella synthesis and motility have a high metabolic cost for the bacterium, the expression of the inducible lateral flagella system is highly regulated by a number of environmental factors and regulators.     

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.