Multiple Regulators Control Capsular Polysaccharide Production in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies cps genes and examines their regulation. Transposon insertions in the cps locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay. Reporter fusions to cpsA, the first gene in the locus, showed ϳ10-fold-enhanced transcription in the OP (opaR ؉ ) strain compared to a TR (⌬opaR) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA ⌬opaR strains. CpsR, which contains a conserved module found in members of the AAA؉ superfamily of ATP-interacting proteins, is homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor. Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in ⌻R strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in V. parahaemolyticus.Vibrio parahaemolyticus is a ubiquitous marine bacterium and human pathogen that can be isolated as a free-living bacterium and from a variety of animate and inanimate surfaces (24,25,29). V. parahaemolyticus strains retrieved from these natural sources display heterogeneity in colony morphology. The phase variation is slow and can be reversible, such that opaque (OP) and translucent (TR) clonal dimorphism is generally observed (30). The OP colony is small, mounded, and has a sticky character when touched with a toothpick. The TR colony is large, flat, and not sticky. Colony opacity correlates with increased capsular polysaccharide (CPS) production (18). One locus has been identified as controlling opacity (30). It encodes the transcriptional regulator, OpaR, which is homologous to Vibrio harveyi LuxR. Expression of opaR using an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible vector converts the TR colony to opaque, and mutation of opaR causes an OP strain to become translucent. Multiple mechanisms for this phase variation appear to exist. One mechanism involves spontaneous alterations in the opaR locus (30); many, but not all, TR strains can be converted to OP by using the opaR expression clone.In addition to the opaR locus, the scr locus is also known to aff...

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

“…Or, perhaps regulation of the cpsA locus gene expression in the context of biofilm development differs from our analysis of CPSA production in colonies on plates. (Guvener and McCarter, 2003).…”

“…without crystal violet) in microtiter wells for mutants that failed to produce a pellicle (Guvener and McCarter, 2003). CPSA mutants in the OP background were highly defective for pellicle formation and adhesion in the crystal violet assay.…”

“…Two mapped to a gene, cpsS (vpa1447), encoding a transcriptional regulator. The cpsS gene was also previously identified as encoding a negative regulator of CPSA on the basis of rough colony morphology, increased CPSA levels and cpsA gene transcription (Guvener and McCarter, 2003). In both OP and TR backgrounds, mutants with cpsS defects produced rough colonies, rough pellicles that were resistant to vortexing (LM6136 and LM6135 respectively; Fig.…”

“…Transposon mutagenesis identified an 11-gene cluster required for opacity and encoding CPSA. A transcriptional fusion to the first gene in the locus ( cpsA ) was shown to be differ-entially expressed in OP and TR strains (Guvener and McCarter, 2003).…”

“…Colonies can be opaque, sticky when touched with a toothpick and mounded (the OP phenotype), or translucent, non-sticky and flat (the TR phenotype). One contributing factor to the different colony morphotypes is variation in production of extracellular polysaccharide (although differences in cell type are multifactorial; Guvener and McCarter, 2003;L. McCarter and S. Jaques, unpubl.…”