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 approximately 10-fold-enhanced transcription in the OP (opaR+) strain compared to a TR (deltaopaR) 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 deltaopaR 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 TR strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in V. parahaemolyticus.

FIG. 1.

Physical map of the cpsA locus and location of transposons causing loss of opacity. Open reading frames and direction of transcription are depicted by arrows. Transposon insertions are indicated as balloon drawings and map at 1,946 bp (LM6296), 2,991 bp (LM6299), 5,337 bp (LM5959), 7,552 bp (LM6306), and 9,853 bp (LM6307). The 11,750 bp that were sequenced in this study (GenBank accession no. AY217749) are 98% identical with the sequenced V. parahaemolyticus strain RIMD2210633. Existence of cpsK is inferred from the genome database (GenBank accession no. AP005088).

FIG. 2.

SDS-PAGE analysis of CPS extracted from the OP, TR, and cps mutant strains. Gel was stained with Stains-All to visualize the polysaccharide. Lanes: 1, LM5312 (OP strain); 2, LM5674 (TR strain; ΔopaR); 3, LM6166 (cpsA2::Tn5); 4, LM6166/pLM3131 (cpsA2::Tn5/CPS cosmid); 5, LM6502 (cpsB1::Tn5); and 6, LM6502/pLM3131 (cpsB1::Tn5/CPS cosmid).

FIG. 3.

Pellicle and adherence phenotype of the cpsA mutant. (A) Pellicle formation by the LM5312 (OP) and LM5819 (cpsA1::lacZ-Kan) strains in polystyrene plastic microtiter wells. Cells inoculated in HI were statically grown for 16 h. Pictures show a view of the wells from above. (B) Crystal violet staining assay to quantitate attachment to a polystyrene plastic surface by LM5312 (OP), LM5819 (cpsA1::lacZ), LM5674 (TR; ΔopaR), and LM5818 (cpsA1::lacZ ΔopaR) strains after 12 h of growth at 30°C.

FIG. 4.

cpsA::lacZ expression in TR and OP strains. β-Galactosidase activities (solid lines) were monitored over time from cells grown on plates. Culture turbidity is shown with dotted lines. Open symbols represent LM5818 (TR; ΔopaR cpsA1::lacZ). Filled symbols represent LM5819 (OP; cpsA1::lacZ).

FIG. 5.

A cpsR lesion affects cpsA::lacZ expression in a scrA::Cam^r ΔopaR mutant but not in OP or TR (ΔopaR) strains. β-Galactosidase activities were measured from cells grown on plates for 24 h. Strains: LM5984 (TR; ΔopaR); LM6241 (scrA1::Cam^r ΔopaR); LM6133 (cpsR1::Tn5 ΔopaR); LM6243 (scrA1::Cam^r cpsR1::Tn5 ΔopaR); LM5985 (OP); LM6134 (cpsR1::Tn5).

FIG. 6.

The cpsS phenotype. (A) Pellicle; (B and C) colony morphologies. Strains: LM5674 (ΔopaR); LM6135 (cpsS1::Tn5 ΔopaR); LM6264 (cpsS1::Tn5 cpsR2::lacZ ΔopaR); LM6137 (cpsS1::Tn5 cpsA3::lacZ ΔopaR); LM5312 (OP); LM6136 (cpsS1::Tn5); LM6265 (cpsS1::Tn5 cpsR2::lacZ); LM6138 (cpsS1::Tn5 cpsA3::lacZ).

FIG. 7.

A lesion in cpsS increases cpsA::lacZ expression. β-Galactosidase activities were measured from cells grown on plates for 24 h. Strains containing cpsA1::lacZ were as follows: LM5984 (TR; ΔopaR); LM6137 (cpsS1::Tn5 ΔopaR); LM6369 (cpsS1::Tn5 cpsR3::Cam^r ΔopaR); LM6133 (cpsR1::Tn5 ΔopaR); LM6138 (cpsS1::Tn5); LM6654 (cpsS1::Tn5 cpsR3::Cam^r); LM6134 (cpsR1::Tn5); LM5985 (OP).

FIG. 8.

cpsR::lacZ expression. β-Galactosidase activities were measured from cells grown on plates for 24 h. Strains containing cpsR::lacZ were as follows: LM6261 (TR; ΔopaR); LM6263 (scrA1::Cam^r ΔopaR); LM6264 (cpsS1::Tn5 ΔopaR); LM6262 (OP).

FIG. 9.

Positive and negative modulators of CPS production in V. parahaemolyticus. Mutation of opaR converts the OP to the TR colony morphology and causes decreased CPS production (indicated by thickness of circle). Mutation of the scrA operon converts the opaR-defective TR strain to a slightly rough (or rugose) colony type as a result of production of an intermediate level of CPS (between levels of OP and TR strains). The positive regulator CpsR mediates enhanced transcription of the cpsA biosynthesis gene in the opaR scrA mutant but not the opaR⁺ scrA-deficient strain. Furthermore, introduction of the cpsR lesion has no effect on colony morphology or CPS production in the OP or the opaR-deficient TR strains. Introduction of a mutation in the negative regulatory gene cpsS causes derepression of cps biosynthetic gene expression and superrugose colonies, irrespective of the status of the opaR allele. In the opaR-deficient cpsS-deficient strain, derepression of cpsA gene expression is mediated through CpsR, whereas the significant portion of the cps derepression in the opaR⁺ cpsS-deficient strain is independent of CpsR. OpaR, CpsR, and CpsS may be global regulators of gene expression affecting other cell surface genes in addition to cps genes, because introduction of the cpsA lesion, which abolishes CPS production, does not completely reverse the mutant colony types (hence the “*” designation).