Deletion of valR,a homolog of Vibrio harveyis´ luxR generates an intermediate colony phenotype between opaque/rugose and translucent/smooth in Vibrio alginolyticus

A previous study has shown that Vibrio alginolyticus ZJ-51 undergoes colony phase variation between opaque/rugose (Op) and translucent/smooth (Tr). The AI-2 quorum-sensing master regulator ValR, a homolog to V. harveyi LuxR, was suggested to be involved in the transition. To investigate the role of ValR in the variation and in biofilm formation, an in-frame deletion of valR in both Op and Tr backgrounds was carried out. The mutants in both backgrounds showed an intermediate colony morphotype, where the colonies were less opaque/rugose but not fully translucent/smooth either. They also showed an intermediate level of motility. However, biofilm formation was severely decreased in both mutants and polar flagella were depleted also. Quantitative PCR showed that most of the genes related to flagellar and polysaccharide biosynthesis were upregulated in the mutant of Op background (ΔvalR/Op) but downregulated in the mutant of Tr background (ΔvalR/Tr) compared with their parental wild-type strains. This suggests that ValR may control biofilm formation by regulating flagellar biosynthesis and affect the expression of the genes involved in colony phase variation in V. alginolyticus.     

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPS_C putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPS_C in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPS_C led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (eps^T) in EPS_C containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of eps^T resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPS_C that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.     

Capsular Polysaccharide Phase Variation in Vibrio vulnificus

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.     

Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.     

Capsular polysaccharide phase variation in Vibrio vulnificus

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.     

Genetic Analysis and Prevalence Studies of the brp Exopolysaccharide Locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus.     

Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca2(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca2(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca2(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca2(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca2(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.     

Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica)

Vibrio vulnificus infections are associated with raw oyster consumption, and disease reservoirs are determined by the ability of this bacterium to infect and persist in oysters. Surface structures, such as capsular polysaccharide (CPS), pili and flagella, function as virulence factors in mouse infection models. Furthermore, virulence is related to phase variation in colony morphology, which reflects CPS expression and includes opaque (encapsulated, virulent), translucent (reduced encapsulation, avirulent) and rugose (wrinkled, biofilm-enhanced) colony types. The role of these factors in environmental survival is unknown; therefore, mutational analysis and phase variation of V. vulnificus were examined in an oyster infection model. Oysters ( Crassostrea virginica ) were pre-treated with tetracycline to reduce background bacteria and subsequently inoculated via filter feeding with 10⁶ colony-forming units (cfu) ml⁻¹ of V. vulnificus wild-type strains and phase variants, as well as strains with deletion mutations in genes related to CPS (Δ wza ), pili (Δ pilA ), flagella (Δ flaCDE/ Δ flaFBA ) and motility (Δ motAB ). All mutants were significantly reduced in their dissemination to oyster haemolymph as compared with wild type; however, recovery of mutants from gills and intestinal tissue was generally similar to wild type. Translucent and rugose inocula showed induction of high-frequency phase variation to the opaque encapsulated phenotype (100% and 72% respectively) during oyster infections that did not occur in strains recovered from seawater. Thus, multiple bacterial factors determine uptake of V. vulnificus in oysters, and phase variation during oyster infection is a likely mechanism for environmental survival and for induction of the more virulent phenotype.     

A Confocal microscopic study on colony morphology and sporulation of Bacillus sp.

Hexavalent chromium Cr(VI) is one of the dominant oxidation states of chromium that exist in the environment and is highly toxic to all forms of life. In the present study, we employ a confocal laser scanning microscope (CLSM) and investigate the effect of Cr(VI) on colony morphology of a Bacillus sp. isolated from soil exposed to tannery effluent. The colonies grown at chromium concentrations, control and 100 ppm are found to be opaque and beyond 200 ppm the colonies were translucent thus exhibiting phase variation. CLSM studies on colonies grown on control plates showed significant increase in height and in biovolume as a function of time whereas, the translucent colonies showed very little change in height and biovolume corresponding to the colony growth. Exopolymeric substance (EPS) content of translucent colonies was lesser than that of opaque colonies, indicating that EPS also plays a role in the observed phenomenon of phase variation. Studies on the effect of Cr(VI) on spore formation showed that Cr(VI) concentrations up to 100 ppm favored spore formation, while concentrations beyond 100 ppm showed a steady decline in spore formation.     

The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the vps genes, which are organized into vps-I and vps-II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the vps intergenic region, termed rbmB to -F (rugosity and biofilm structure modulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of rbmB, which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the rbmC bap1 double deletion mutant and bap1 single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the rbmC bap1 double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of rbmDEF mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that vps intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.     

Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.     

Bacterial quorum sensing and cell surface electrokinetic properties

The hypothesis tested in this paper is that quorum sensing influences the microbial surface electrokinetic properties. Escherichia coli MG1655 and MG1655 LuxS- mutant (lacking quorum-sensing gene for Autoinducer synthase AI-2) were used for this study. AI-2 production (or lack of) in both strains was analyzed using the Vibrio harveyi bioassay. The levels of extracellular AI-2 with and without glucose in the growth medium were consistent with previously published work. The surface electrokinetic properties were determined for each strain of E. coli MG1655 by measuring the electrophoretic mobility using a phase amplitude light-scattering (PALS) Zeta potential analyser. The findings show that the surface charge of the cells is dependent upon the stage in the growth phase as well as the ability to participate in quorum sensing. In addition, significant differences in the electrophoretic mobility were observed between both strains of E. coli. These findings suggest that quorum sensing plays a significant role in the surface chemistry of bacteria during their growth.