Vibrio parahaemolyticus has dual flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum propels the swimmer cell in liquid environments. Numerous unsheathed lateral flagella move the swarmer cell over surfaces. The polar flagellum is produced continuously, whereas the synthesis of lateral flagella is induced under conditions that impede the function of the polar flagellum, e.g., in viscous environments or on surfaces. Thus, the organism possesses two large gene networks that orchestrate polar and lateral flagellar gene expression and assembly. In addition, the polar flagellum functions as a mechanosensor controlling lateral gene expression. In order to gain insight into the genetic circuitry controlling motility and surface sensing, we have sought to define the polar flagellar gene system. The hierarchy of regulation appears to be different from the polar system of Caulobacter crescentus or the peritrichous system of enteric bacteria but is pertinent to many Vibrio and Pseudomonas species. The gene identity and organization of 60 potential flagellar and chemotaxis genes are described. Conserved sequences are defined for two classes of polar flagellar promoters. Phenotypic and genotypic analysis of mutant strains with defects in swimming motility coupled with primer extension analysis of flagellar and chemotaxis transcription provides insight into the polar flagellar organelle, its assembly, and regulation of gene expression.Many bacterial species are motile by means of flagellar propulsion (reviewed in references 5, 32, and 33). Powered by a rotary motor, the flagellum acts as semirigid helical propeller, which is attached via a flexible coupling, known as the hook, to the basal body. The basal body consists of rings and rods that penetrate the membrane and peptidoglycan layers. Associating with the basal body and projecting into the cytoplasm is a structure termed the C ring, which contains the switch proteins and acts as the core, or rotating part, of the motor. Maintenance of a flagellar motility system is a sizable investment with respect to cellular economy in terms of the number of genes and the energy that must be committed to gene expression, protein synthesis, and flagellar rotation. As a result, flagellar systems are highly regulated. A hierarchy of regulation has been elucidated for peritrichously flagellated Escherichia coli and Salmonella enterica serovar Typhimurium (26,27,30). This scheme of control couples gene expression to assembly of the organelle. The pyramid of expression possesses three classes, or tiers, of genes. Genes in each class must be functional in order for expression of the subsequent class to occur. Class 1 genes, flhD and flhC, encode the master transcriptional activators of class 2 flagellar gene expression. The flhDC operon is controlled by a 70 promoter and a number of global regulatory factors (28). The majority of the class 2 flagellar genes encode components of the flagellar export system and the basal body (21). One clas...

In this work, we describe a new gene controlling lateral flagellar gene expression. The gene encodes ScrG, a protein containing GGDEF and EAL domains. This is the second GGDEF-EAL-encoding locus determined to be involved in the regulation of swarming: the first was previously characterized and named scrABC (for "swarming and capsular polysaccharide regulation"). GGDEF and EAL domain-containing proteins participate in the synthesis and degradation of the nucleotide signal cyclic di-GMP (c-di-GMP) in many bacteria. Overexpression of scrG was sufficient to induce lateral flagellar gene expression in liquid, decrease biofilm formation, decrease cps gene expression, and suppress the ⌬scrABC phenotype. Removal of its EAL domain reversed ScrG activity, converting ScrG to an inhibitor of swarming and activator of cps expression. Overexpression of scrG decreased the intensity of a 32 P-labeled nucleotide spot comigrating with c-di-GMP standard, whereas overexpression of scrG ⌬EAL enhanced the intensity of the spot. Mutants with defects in scrG showed altered swarming and lateral flagellin production and colony morphology (but not swimming motility); furthermore, mutation of two GGDEF-EAL-encoding loci (scrG and scrABC) produced cumulative effects on swarming, lateral flagellar gene expression, lateral flagellin production and colony morphology. Mutant analysis supports the assignment of the primary in vivo activity of ScrG to acting as a phosphodiesterase. The data are consistent with a model in which multiple GGDEF-EAL proteins can influence the cellular nucleotide pool: a low concentration of c-di-GMP favors surface mobility, whereas high levels of this nucleotide promote a more adhesive Vibrio parahaemolyticus cell type.

The bacterial flagellum is powered by a rotary motor capable of turning the helical flagellar propeller at very high speeds. Energy to drive rotation is derived from the transmembrane electrochemical potential of specific ions. Ions passing through a channel component are thought to generate the force to power rotation. Two kinds of motors, dependent on different coupling ions, have been described: proton-driven and sodium-driven motors. There are four known genes encoding components of the sodium-powered polar flagellar motor in Vibrio parahaemolyticus. Two, which are characterized here, are homologous to genes encoding constituents of the proton-type motor (motA and motB), and two encode components unique to the sodium-type motor (motX and motY). The sodium-channelblocking drugs phenamil and amiloride inhibit rotation of the polar flagellum and therefore can be used to probe the architecture of the motor. Mutants were isolated that could swim in the presence of phenamil or amiloride. The majority of the mutations conferring phenamil-resistant motility alter nucleotides in the motA or motB genes. The resultant amino acid changes localize to the cytoplasmic face of the torque generator and permit identification of potential sodium-interaction sites. Mutations that confer motility in the presence of amiloride do not alter any known component of the sodium-type flagellar motor. Thus, evidence supports the existence of more than one class of sodium-interaction site at which inhibitors can interfere with sodium-driven motility.Small but powerful rotary motors propel bacteria by turning semirigid helical propellers, the flagellar filaments (for recent reviews, see refs. 1-4). In Escherichia coli and Salmonella typhimurium, energy to power rotation derives from the proton motive force (5, 6). Somehow, the passage of protons through the torque generator is coupled to rotation of the flagellum (7). Although the molecular mechanism of coupling remains unsolved, the architecture of the proton-driven motor has been studied extensively. The stationary part of the torque generator consists of two cytoplasmic membrane proteins, MotA and MotB. MotA contains four transmembrane domains, and MotB possesses one transmembrane domain (8, 9). Together they form a proton channel (10-13). In addition, MotB contains a C-terminal domain, which is thought to anchor the MotA-MotB complex to the cell wall via an interaction with peptidoglycan (14,15).Torque is transmitted from the MotA͞B stator to the FliG protein, which acts as part of the rotor (16). FliG is found at the base of the flagellar basal body in a complex with FliM and FliN (17,18). This complex of interacting proteins, known as the ''switch complex,'' is essential for torque generation, flagellar assembly, and modulation of the direction of flagellar rotation (19)(20)(21)(22)(23)(24).Other bacteria, including alkalophilic Bacillus and marine Vibrio species, use the transmembrane electrochemical-potential gradient of Na ϩ to drive flagellar rotation (25). The sodiumdri...

Gene organization and hierarchical regulation of the polar flagellar genes of Vibrio parahaemolyticus, Vibrio cholerae, and Pseudomonas aeruginosa appear highly similar, with one puzzling difference. Two 54 -dependent regulators are required to direct different classes of intermediate flagellar gene expression in V. cholerae and P. aeruginosa, whereas the V. parahaemolyticus homolog of one of these regulators, FlaK, appears dispensable. Here we demonstrate that there is compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK.Acting as propellers driven by rotary motors, flagella are highly complex and efficient molecular machines. Flagellar assembly, which occurs via a flagellum-specific type three secretion system, initiates with the formation of a ring structure in the membrane. It proceeds to formation of a basal body hook and culminates with the polymerization of the extracytoplasmic propeller-like filament, subunits of which are secreted through the basal body hook structure (reviewed in reference 9). The assembly of this complex organelle requires the products of more than 35 genes. Flagellar gene expression is carefully regulated by a number of mechanisms such that there are sequential classes of gene expression coupled to morphogenesis of the organelle (reviewed in reference 1).Although the classes of temporally expressed flagellar genes are generally comparable (with a few variations), the specific regulators and sigma factors controlling early, middle, and late gene expression differ considerably. Flagellar gene organization and the specific transcriptional regulators of polar flagellar genes appear similar in many Vibrio and Pseudomonas species (reviewed in reference 14). In these organisms, 54 -dependent transcription factors regulate expression of intermediate genes, which encode components of the flagellar export-assembly complex and basal body hook structure. An alternate sigma factor ( 28 ) directs late polar gene expression, including transcription of propeller-encoding genes (e.g., flagellin genes). In Vibrio cholerae and Pseudomonas aeruginosa, experiments suggest that there are four classes in the temporal hierarchy of expression, with two subclasses of intermediate genes requiring There has been a puzzling discrepancy between observations of the phenotype of mutants of V. parahaemolyticus that is not in accordance with the regulatory scheme deciphered for other polarly flagellated Vibrio and Pseudomonas species. The flaK gene was discovered as an open reading frame encoding a potential 54 -dependent regulator located downstream of the flagellin chaperone-encoding gene fliS (formerly named flaJ). The gene was disrupted, but disappointingly it was not found to be important for swimming motility (21). Bacteria with flaK lesions have only slight swimming motility defects, an observation that is not consistent with FlaK being required for intermediate flagellar gene expression. Therefore, another regulatory scenario must be postulated for V. parahaemolyticus. In t...

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