Identification of a c-di-GMP-regulated polysaccharide locus governing stress resistance and biofilm and rugose colony formation in Vibrio vulnificus

Abstract

As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. Its continued environmental persistence and transmission are bolstered by its ability to colonize shellfish, form biofilms on various marine biotic surfaces, and generate a morphologically and physiologically distinct rugose (R) variant that yields profuse biofilms. Here, we identify a c-di-GMP-regulated locus (brp, for biofilm and rugose polysaccharide) and two transcription factors (BrpR and BrpT) that regulate these physiological responses. Disruption of glycosyltransferases within the locus or either regulator abated the inducing effect of c-di-GMP on biofilm formation, rugosity, and stress resistance. The same lesions, or depletion of intracellular c-di-GMP levels, abrogated these phenotypes in the R variant. The parental and brp mutant strains formed only scant monolayers on glass surfaces and oyster shells, and although the R variant formed expansive biofilms, these were of limited depth. Dramatic vertical expansion of the biofilm structure was observed in the parental strain and R variant, but not the brp mutants, when intracellular c-di-GMP levels were elevated. Hence, the brp-encoded polysaccharide is important for surface colonization and stress resistance in V. vulnificus, and its expression may control how the bacteria switch from a planktonic lifestyle to colonizing shellfish to invading human tissue.

FIG. 1.

The vv21574-vv21582 (brp) polysaccharide gene cluster in the genome of V. vulnificus CMCP6. Open arrows represent the locations and directions of transcription of the respective open reading frames (ORFs). Small angled arrows indicate the positions of putative promoters, including a conserved ops (operon polarity suppressor) sequence (55), while the black hairpin structure following vv21582 denotes a putative Rho-independent transcriptional terminator. The open circles above vv21578 and vv21580 indicate the positions of targeted disruptions using the pSW23T suicide plasmid (15). Red, gray, green, and blue arrows denote glycosyltransferase, hypothetical proteins, export proteins, and EPS biosynthesis genes, respectively.

FIG. 2.

In vivo expression of the vv21574-vv21582 (brp) locus. The indicated strains were grown in HIN. RNA was isolated from mid-exponential (ME)- and stationary (S)-phase cells of the parental ATCC 27562 strain carrying pBAD24T (lane 1) or pBAD24T::dcpA (lane 2) and from an R variant, ATCC 27562R, with pBAD24T (lane 3). RT-PCR was used to determine if vv21574, vv21577, vv21578, and vv21580 were being transcribed. Expressions of the dcpA and the ribosomal rplT genes were used as positive controls. Primer sets were optimized for PCR using genomic DNA from V. vulnificus strain ATCC 27562, and PCR was performed on RNA samples to verify that they did not contain residual contaminating DNA. Target genes are indicated above the gels. M, 100-bp marker.

FIG. 3.

Lesions within the brp locus do not affect CPS production. The parental ATCC 27562 strain (A) appears opaque on solid medium, while the CPS biosynthesis mutant, Δwzy (B), appears translucent. The ΔbrpF (C) and ΔbrpI (D) mutant strains appear opaque. The strains were grown in HIN.

FIG. 4.

c-di-GMP-induced biofilm and aggregate formation is dependent on the vv21574-vv21582 (brp) locus. The indicated strains were grown in HIN. (A) Biofilm (top panel) and aggregate (middle panel) formation in culture tubes by the parental, mutant (Δvv21574 and Δvv21580), and complemented (Δvv21574/pSU38T::vv21574 and Δvv21580/pSU38T::vv21580) strains carrying the pBAD24T control vector or pBAD24T::dcpA. Control strains for complementation studies carried the empty pSU38T plasmid. Colony morphologies for the same strains are shown in the bottom panel. (B) Quantitative measurement of biofilm formation of the corresponding strains in panel A by CV staining. Results shown are representative of at least three independent experiments. Error bars represent standard deviations.

FIG. 5.

Expression of the brp locus is required for biofilm and rugose colony formation by the R variant. The indicated strains were grown in HIN. (A) Biofilm formation in culture tubes (top panel) and colony morphology (bottom panel) of the parental and ATCC 27562R strains and the ATCC 27562RΔbrpF and ATCC 27562RΔbrpI mutant strains carrying the pSU38T vector control. Complemented strains carry pSU38T::brpF or pSU38T::brpI, respectively. (B) Quantitative measurement of biofilm formation of the corresponding strains in panel A by CV staining. Results shown are representative of at least three independent experiments. Error bars represent standard deviations.

FIG. 6.

Sensitivity of the parental, ATCC 27562R, ATCC 27562RΔbrpF, and ATCC 27562RΔbrpI strains to chlorine and increasing salinity. The indicated strains were grown in LB containing 10⁰/₀₀ NaCl. (A) The strains were then incubated in PBS containing 3 ppm NaOCl for 5 min. The surviving bacteria were enumerated by plate counts. The number of viable bacteria relative to that obtained following treatment with PBS alone is plotted. (B) Strains were grown in LB containing 0⁰/₀₀ (white bars), 2⁰/₀₀ (dark gray bars), 10⁰/₀₀ (light gray bars), and 30⁰/₀₀ (black bars) NaCl. Bacterial growth (OD₅₉₅) is plotted. Results shown are representative of at least three independent experiments. Error bars represent standard deviations.

FIG. 7.

Scanning electron micrographs of biofilms formed by V. vulnificus. Biofilm formation by the parental and ATCC 27562R strains carrying pBAD24T::dcpA or the vector control on glass coverslips or oyster shells is shown. The indicated strains were grown in HIN. Images are in pairs of low (L) and high (H) magnification. Scale bars are indicated at the bottom of each image.

FIG. 8.

Depletion of intracellular c-di-GMP levels in the R variant prevents biofilm and rugose colony formation. The indicated strains were grown in HIN. (Top) ATCC 27562R carrying pME6041T or pME6041T::yhjH was grown in the presence (+) or absence (−) of l-arabinose. The arrow marks the position of the biofilm ring. (Bottom) Colony morphology of the strains above. Images shown are representative of at least three independent experiments.

FIG. 9.

BrpR and BrpT are required for biofilm and rugose colony formation by the R variant. The indicated strains were grown in HIN. (A) Biofilm formation in culture tubes (top panel) and colony morphology (bottom panel) of the parental strain, ATCC 27562R, and the ATCC 27562RΔbrpR and ATCC 27562RΔbrpT mutant strains carrying the pBAD24T vector control. Complemented strains carry pBAD24T::brpR or pBAD24T::brpT, respectively. (B) Quantitative measurement of biofilm formation of the corresponding strains in panel A by CV staining. Results shown are representative of at least three independent experiments. Error bars represent standard deviations.

FIG. 10.

Genomic comparison of the vps, cps, and brp loci. (A) Comparison of genes encoding the V. cholerae (Vch) vps, V. parahaemolyticus (Vpa) cps, and V. vulnificus (Vvu) brp loci. Open arrows represent the locations and directions of transcription of the respective genes. Genes depicted in gray are dissimilar. Homologous genes are shown in the same color and are connected by dotted lines. The rbmABCDEF genes, which modulate vps expression, are located between vpsI and vpsII. (B) Simplified schematic of the regulatory pathway of biofilm formation in V. cholerae, V. parahaemolyticus, and V. vulnificus. Activators are shown as circles, and repressors are shown as rectangles. Homologs are shown in the same color.