Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus

Abstract

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.

FIG. 1.

Domain architecture of DcpA. DcpA is a two-domain protein. The GAF (amino acids 66 to 200) and GGDEF (amino acids 215 to 370) domains are delimited by the gray rectangle and arrow, respectively. The corresponding amino acids of the GGEEF sequence and I site in DcpA are marked by dots below the GGDEF motif. The black bar represents the total length of the protein, at 379 amino acids.

FIG. 2.

Restoration of cellulose production in S. enterica serovar Typhimurium SL1344 by DcpA. S. enterica serovar Typhimurium strain SL1344 is deficient in cellulose production, and strain SL1344 carrying the empty pBAD24T vector does not fluoresce on calcofluor agar plates (A). The VCA0956 gene from V. cholerae N16961, previously shown to encode a DGC, was cloned into pBAD24T and transformed into strain SL1344 (B). The dcpA gene was also cloned into pBAD24T and transformed into strain SL1344 (C). Fluorescence is clearly visible in panels B and C.

FIG. 3.

Effects of DcpA and VCA0956 on the translucent phenotype of V. vulnificus CPS mutants. The CPS biosynthesis mutants wzy::Tn10 (A) and rmlC::Tn10 (B) and the CPS transport mutant Δwzc (C), which carried the pBAD24T plasmid, appear translucent on solid medium. The same strains carrying pBAD24T::dcpA (D to F) or pBAD24T::VCA0956 (G to I) appear opaque under inducing conditions.

FIG. 4.

Alcian blue staining and immunoblotting of V. vulnificus HMWP. HMWP extracted from the indicated strains was separated on a nondenaturing 40% polyacrylamide gel and detected by Alcian blue staining (A) or by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by immunoblotting with antiserum to formalin-killed whole cells of strain 27562 (B). The K band described previously (58) is indicated.

FIG. 5.

c-di-GMP induces biofilm formation by V. vulnificus and V. cholerae. (A) Biofilm formation in culture tubes by V. vulnificus and V. cholerae expressing dcpA or VCA0956. Cells carrying the empty pBAD24T vector or pBAD24T containing dcpA or VCA0956 were grown overnight under inducing conditions. (B) Quantification of biofilm formation by CV staining. The black bars indicate the level of biofilm formation for the various parental strains carrying the empty pBAD24T vector, while the adjacent gray bars indicated the level of biofilm formation in the same strain carrying pBAD24T that contains the indicated DGC. Microtiter plates were stained with CV 24 h after inoculation and static growth at 30°C. Results shown are representative of at least three independent experiments. Error bars represent standard deviations.

FIG. 6.

Colony morphologies of V. vulnificus 27562 variants expressing dcpA. All colonies were grown on LB agar supplemented with appropriate antibiotics for 72 h at 30°C. Panels: A, wt strain carrying pBAD24T; B, wt strain carrying pBAD24T::dcpA; C, wzy::Tn10 mutant strain carrying pBAD24T; D, wzy::Tn10 mutant strain carrying pBAD24T::wzy; E, wzy::Tn10 mutant strain carrying pBAD24T; F, wzy::Tn10 mutant strain carrying pBAD24T::dcpA; G, rmlC::Tn10 mutant strain carrying pBAD24T; H, rmlC::Tn10 mutant strain carrying pBAD24T::dcpA; I, strain 27562-T carrying pBAD24T; J, strain 27562-T carrying pBAD24T::dcpA. The colony images are representative of three biological replicates. Images were captured with an AxioCam MRc5 (Zeiss) digital camera attached to a dissecting microscope.

FIG. 7.

Colony morphologies of V. vulnificus 27562 variants and V. cholerae N16961 expressing VCA0956 or dcpA. All colonies were grown on LB agar supplemented with appropriate antibiotics for 72 h at 30°C. Panels: A, strain 27562 carrying pBAD24T; B, strain 27562 carrying pBAD24T::VCA0956; C, wzy::Tn10 mutant strain carrying pBAD24T; D, wzy::Tn10 mutant strain carrying pBAD24T::VCA0956; E, rmlC::Tn10 mutant strain carrying pBAD24T; F, rmlC::Tn10 mutant strain carrying pBAD24T::VCA0956; G, strain N16961 carrying pBAD24T; H, strain N16961 carrying pBAD24T::VCA0956; I, strain N16961 carrying pBAD24T; J, strain N16961 carrying pBAD24T::dcpA. The colony images are representative of three biological replicates. Images were captured with an AxioCam MRc5 (Zeiss) digital camera attached to a dissecting microscope.

FIG. 8.

Motility phenotypes of various V. vulnificus strains expressing dcpA or VCA0956. (A) Overnight cultures of the indicated strains carrying pBAD24T (top row) or pBAD24T::dcpA (bottom row) were adjusted to 10⁸ CFU/ml, and 1-μl aliquots were inoculated onto motility plates. Images were taken with a Bio-Rad Gel Doc 2000 imaging system following overnight incubation at 37°C. (B) Same as panel A but with strain 27562 carrying either pBAD24T (top) or pBAD24T::VCA0956 (bottom). (C) Quantitative measurement of the mobility of the same strains. The diameter of the net migration is shown. Results are representative of at least three independent experiments. Error bars represent standard deviations.