Linda L. McCarter

Polar flagellar motility of the Vibrionaceae.

SUMMARY 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.

Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus

Swarmer cell genes, laf, are induced when V. parahaemolyticus is grown on the surface of solidified media, embedded in solidified media, suspended in viscous media, or agglutinated with antibody in liquid media. These conditions have in common the constraint of the movement of the polar flagellum. To test the hypothesis that the polar flagellum functions as a sensor necessary for control of swarmer cell formation, we have constructed a variety of mutations in genes encoding components of the polar flagellum, fla. The consequence of such mutations is the constitutive expression of laf genes. So, the performance of the polar flagellum is coupled to the transcription of laf genes such that when function is perturbed, either physically or genetically, swarmer cell genes are induced. Because the polar flagellum appears to be capable of sensing external forces influencing its motion, we suggest it is acting as a dynamometer.

Dual flagellar systems enable motility under different circumstances.

Flagella are extremely effective organelles of locomotion used by a variety of bacteria and archaea. Some bacteria, including Aeromonas, Azospirillum, Rhodospirillum, and Vibrio species, possess dual flagellar systems that are suited for movement under different circumstances. Swimming in liquid is promoted by a single polar flagellum. Swarming over surfaces or in viscous environments is enabled by the production of numerous peritrichous, or lateral, flagella. The polar flagellum is produced continuously, while the lateral flagella are produced under conditions that disable polar flagellar function. Thus at times, two types of flagellar organelles are assembled simultaneously. This review focuses on the polar and lateral flagellar systems of Vibrio parahaemolyticus. Approximately 50 polar and 40 lateral flagellar genes have been identified encoding distinct structural, motor, export/assembly, and regulatory elements. The sodium motive force drives polar flagellar rotation, and the proton motive force powers lateral translocation. Polar genes are found exclusively on the large chromosome, and lateral genes reside entirely on the small chromosome of the organism. The timing of gene expression corresponds to the temporal demand for components during assembly of the organelle: RpoN and lateral- and polar-specific σ54-dependent transcription factors control early/intermediate gene transcription; lateral- and polar-specific σ28 factors direct late flagellar gene expression. Although a different gene set encodes each flagellar system, the constituents of a central navigation system (i.e., chemotaxis signal transduction) are shared.

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

Vibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose‐sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co‐regulated protein and an RTX toxin‐like membrane fusion transporter, as well as potential modifiers of cell surface molecules (nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H‐NS, a CsgD‐like repressor and an AraC‐like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.

Lateral Flagellar Gene System of Vibrio parahaemolyticus

Vibrio parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions in the structural and regulatory laf genes. A Tn5-based lux transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the laf regulon. Twenty-nine independent insertions were distributed within 16 laf genes. DNA sequence analysis identified 38 laf genes in two loci. Among the mutants isolated, 11 contained surface-induced lux fusions. A hierarchy of laf gene expression was established following characterization of the laf::lux transcriptional fusion strains and by mutational and primer extension analyses of the laf regulon. The laf system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however, laf regulation was different from the Salmonella-Escherichia coli paradigm. There is no apparent flhDC counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria. A potential sigma(54)-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific sigma(28) factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.

The sodium‐driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression

Certain marine Vibrio species swim in sea water, propelled by a polar flagellum, and swarm over surfaces using numerous lateral flagella. The polar and the lateral flagellar motors are powered by sodium‐ and proton‐motive forces, respectively. The lateral flagella are produced in media of high viscosity, and the relevant viscosity sensor is the polar flagellum. The cell might monitor either the rotation rate of the flagellar motor or the mechanical force applied against the flagellum. To test these possibilities, we examined the effects of amiloride and its derivatives, which inhibit the rotation of the sodium‐driven motor, on lateral flagellar gene (Iaf) expression in Vibrio parahaemolyticus. Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced Iaf transcription in media devoid of viscous agents in a dose‐dependent manner. The relationship between the average swimming speed and Iaf induction in the presence of various concentrations of phenamil was very similar to that observed when viscosity was changed. These results indicate that marine Vibrio sense a decrease in the rotation rate of (or the sodium influx through) the polar flagellar motor as a trigger for Iaf induction. Alternative mechanisms for Iaf induction are also discussed.

Relation of Capsular Polysaccharide Production and Colonial Cell Organization to Colony Morphology in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a ubiquitous, gram-negative marine bacterium that undergoes phase variation between opaque and translucent colony morphologies. The purpose of this study was to determine the factor(s) responsible for the opaque and translucent phenotypes and to examine cell organization within both colony types. Examination of thin sections of ruthenium red-stained bacterial cells by electron microscopy revealed a thick, electron-dense layer surrounding the opaque cells that was absent in preparations from translucent strains. Extracellular polysaccharide (EPS) material was extracted from both opaque and translucent strains, and the opaque strain was shown to produce abundant levels of polysaccharide, in contrast to the translucent strain. Compositional analysis of the EPS identified four major sugars: glucose, galactose, fucose, and N-acetylglucosamine. Confocal scanning laser microscopy was used to investigate cell organization within opaque and translucent colonies. Cells within both types of colonies exhibited striking organization; rod-shaped cells were aligned parallel to one another and perpendicular to the agar surface throughout the depth of the colony. Cells within translucent colonies appeared more tightly packed than cells in opaque colonies. In addition, a dramatic difference in the structural integrity of these two colony types was observed. When colonies were perturbed, the cell organization of the translucent colonies was completely disrupted while the organization of the opaque colonies was maintained. To our knowledge, this study represents the first description of how cells are organized in the interior of a viable bacterial colony. We propose that the copious amount of EPS produced by the opaque strain fills the intercellular space within the colony, resulting in increased structural integrity and the opaque phenotype.

Evolutionary Conservation of Methyl-Accepting Chemotaxis Protein Location in Bacteria and Archaea

The methyl-accepting chemotaxis proteins (MCPs) are concentrated at the cell poles in an evolutionarily diverse panel of bacteria and an archeon. In elongated cells, the MCPs are located both at the poles and at regions along the length of the cells. Together, these results suggest that MCP location is evolutionarily conserved.

Vibrio parahaemolyticus scrABC, a Novel Operon Affecting Swarming and Capsular Polysaccharide Regulation

Swarming is an adaptation of many bacteria to growth on surfaces. A search for genes controlling swarmer cell differentiation of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal-phosphate-dependent enzyme, an extracellular solute-binding protein, and a membrane-bound GGDEF- and EAL-motif sensory protein. The functions of these motifs, which are named after conserved amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling proteins. Studies with translational fusions supported the predicted localization of the gene products. When the operon was overexpressed, swarmer cell gene transcription was induced in liquid culture. Mutants with defects in any of the three genes exhibited decreased swarming and lateral flagellar (laf) gene expression. Complementation studies confirmed an operon organization and suggested that all three genes participated in laf regulation. The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of the operon inhibited transcription of the CPS gene cpsA. Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS.

Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence

Vibrio parahaemolyticus senses surfaces via impeded rotation of its polar flagellum. We have exploited this surface‐sensing mechanism to trick the organism into thinking it is on a surface when it is growing in liquid. This facilitated studies of global gene expression in a way that avoided many of the complications of surface‐to‐liquid comparisons, and illuminated ∼ 70 genes that respond to surface sensing per se. Almost all are surface‐induced (not repressed) and encode swarming motility proteins, virulence factors or sensory enzymes involved with chemoreception and c‐di‐GMP signalling. Follow‐up studies were performed to place the surface‐responsive genes in a regulatory hierarchy. Mapping the hierarchy revealed two surprises about LafK, a transcriptional activator that until now has been considered to be the master regulator for the lateral flagellar system. First, LafK controls a more diverse set of genes than previously appreciated. Second, some laf genes are not under LafK control, which means LafK is not the master regulator after all. Additional experiments motivated by the transcriptome analyses revealed that growth on a surface lowers c‐di‐GMP levels and enhances cytotoxicity. Thus, we demonstrate that V. parahaemolyticus can invoke a programme of gene control upon encountering a surface and the specific identities of the surface‐responsive genes are pertinent to colonization and pathogenesis.