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

Abstract

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...

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

“…We interpret the inclusion of the cys mutants in this class as the result of a requirement for activated sulphur in the biosynthesis of V. parahaemolyticus CPSA. Although the relative importance of CPSA in biofilm formation is consistent with observations in many organisms Danese et al, 2000;Sutherland, 2001), the fact that CPSA played a central role in both cell types was somewhat surprising in light of prior data demonstrating dramatic differences in the amount of CPSA produced by OP and TR strains (Enos-Berlage and McCarter, 2000). Perhaps TR strains produce sufficient CPSA to foster biofilm development and the additional CPSA produced by OP strains alters the properties and the architecture of the biofilm.…”

“…These mutants were confirmed as showing cysteine auxotrophy. In opaR + backgrounds, the cps and cys mutants did not form the characteristic sticky opaque c. All mutants with defects in genes described with 'CPSA biosynthesis' failed to produce extractable CPSA; detected as in Enos-Berlage and McCarter (2000). d. Minus pellicle phenotype of mutants with defects in CPSA biosynthesis was subtly different between OP and TR backgrounds.…”

“…The identification of mutants with transposon insertions in the nagAC operon suggests that a deficiency of UDP-N-acetylglucosamine may have altered the chemical composition of O-antigen and/or capsule structures. N-acetyl glucosamine accounts for 14% by weight of total CPSA (Enos-Berlage and McCarter, 2000).…”

“…obs.). OP colonies produce at least 10-fold more extracellular polysaccharide than TR colonies (Enos-Berlage and McCarter, 2000). By using the stain ruthenium red, the polysaccharide can be visualized in the electron microscope as a thick capsule of electron dense material surrounding the cells and was consequently described as capsular polysaccharide (denoted herein as CPSA) (Enos-Berlage and McCarter, 2000).…”

Persistence of Vibrio parahaemolyticus in the Pacific Oyster, Crassostrea gigas, Is a Multifactorial Process Involving Pili and Flagella but Not Type III Secretion Systems or Phase Variation

“…One possibility for oyster immune system evasion is phase variation in colony morphology from the opaque to the translucent phenotype, a result of altering surface polysaccharide production (26). In our analyses, we compared strains LM5431 (TR) and LM5432 (OP) to determine if phase variation affected persistence in the oyster.…”