SummaryVibrio 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.

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; rodshaped 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.Bacteria have evolved numerous adaptive mechanisms to facilitate their survival under changing environmental conditions. One such mechanism is phase variation, which occurs when the expression of a given factor is periodically altered such that it is either "on" or "off." These variations are generally spontaneous and reversible and occur at relatively high frequency (Ͼ10 Ϫ5 per generation) (13). Factors that are controlled by phase variation are often associated with the cell surface and include structures such as flagella, fimbriae, outer membrane proteins, and exo-and lipopolysaccharide (13). Variations in the expression of these structures often result in an observable phenotypic change, such as altered colony morphology.Vibrio parahaemolyticus is a ubiquitous, gram-negative marine bacterium that undergoes phase variation that results in different colony morphologies. This organism "switches" between a large, flat, translucent colony type (TR) and a smaller, mounded, opaque colony type (OP) (19,20). It is not clear whether environmental conditions influence OP-TR switching in V. parahaemolyticus or whether this variation is random. The factor(s) responsible for the...

purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement. We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6-phosphate [6-P] dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase). We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine.

Vibrio parahaemolyticus differentiates from a polarly flagellated, short, rod-shaped cell known as the swimmer to the elongated, hyperflagellated, and multinucleated swarmer cell type when it is grown on a surface. The swarmer is adapted to movement over and colonization of surfaces. To understand the signal transduction mechanism by which the bacterium recognizes surfaces and reprograms gene expression, we isolated a new class of mutants defective in surface sensing. These mutants were constitutive for swarmer cell gene expression, inappropriately expressing high levels of a swarmer cell gene fusion product when grown in liquid. They showed no defect in the swimming motility system, unlike all previously isolated constitutive mutants which have defects in the alternate, polar motility system. The lesions in the majority of the newly isolated mutants were found to be in a gene, lonS, which encodes a polypeptide exhibiting 81% sequence identity to the Escherichia coli Lon protein, an ATP-dependent protease. Upstream sequences preceding the lonS coding region resemble a heat shock promoter, and the homology extends to sequences flanking lonS. The gene order appears to be clpX lonS hupB, like the organization of the E. coli locus. V. parahaemolyticus lonS complemented E. coli lon mutants to restore UV resistance and capsular polysaccharide regulation to that of the wild type. Vibrio lonS mutants were UV sensitive. In addition, when grown in liquid and examined in a light microscope, lonS mutant cells were extremely long and thus resembled swarmer cells harvested from a surface.The bacterium Vibrio parahaemolyticus possesses two cell types, each appropriate for life under different circumstances (1, 29). The swimmer cell is adapted to life in a liquid environment. It is rod shaped and approximately 2 m long and has a single, sheathed polar flagellum. The flagellum is powered by the sodium motive force and can propel the cell at speeds as fast as 60 m/s (4). The swarmer cell is adapted to life on a solid surface or in a slime layer, i.e., conditions under which the polar flagellum is not functional or is poorly functional. This cell type is extremely long (approximately 30 m), multinucleate, and peritrichously flagellated. The peritrichous, or lateral, flagella are powered by the proton motive force and function to move the bacterium over surfaces or through viscous layers (4, 42). Swimming-negative mutants show no defect in swarming, and swarming-defective mutants swim as well as the wild type; therefore, the polar flagellar (Fla) and lateral flagellar (Laf) gene systems are distinct (27).The gene systems that encode the two motility systems are large, each composed of 40 or more genes. As is the case for all studied flagellar systems (23, 25), the polar and lateral flagellar systems seem to be carefully regulated in hierarchies of control whereby regulation of gene expression is coupled to morphogenesis of the organelles (32, 33). In addition, the two gene systems interact. Performance of the polar organelle is in ...

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme inSalmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.