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

Abstract

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.

FIG. 1.

Genetic organization of the vps intergenic region. The genetic organization of rbmABCDEF and bap1 (open arrows) on the V. cholerae chromosome is depicted with locus annotations under each open reading frame. The vps-I and vps-II gene clusters, as well as the first and last genes of each cluster (vpsA and vpsK and vpsL and vpsQ) are marked and labeled. Unlinked chromosomal DNA regions are indicated (∥). Illustration is not to scale.

FIG. 2.

RbmB modulates rugose colony development and biofilm formation. (A) Colony morphologies of the rugose variant and RΔrbmB. (B) CLSM images of the top-down views (large panels) and orthogonal views (side panels) of biofilms formed by the rugose variant and RΔrbmB in a flow cell system after 24 h of incubation. Bars, 40 μm. (C) Quantitative analysis of biofilm formation by the rugose variant and RΔrbmB after 8 h of growth at 30°C under static conditions. Error bars represent standard deviations. (D) Expression of the vpsA::lacZ and vpsL::lacZ fusion genes in the rugose variant and RΔrbmB grown at 30°C to the mid-exponential growth phase. Error bars represent standard deviations.

FIG. 3.

SDS-PAGE analysis of VPS accumulation. Equal volumes (50 μl) of VPS samples were analyzed from the rugose variant, RΔrbmB, RΔrbmC, RΔrbmD, RΔrbmEF, RΔbap1, and RΔrbmC Δbap1. Cultures were normalized to the same cell density. The gel was stained with Stains-All, and the 5% polyacrylamide stacking portion of the gel is shown.

FIG. 4.

Overexpression of rbmB affects colony morphology and biofilm structures. (A) Colony morphologies of the rugose variant and RΔrbmB harboring the overexpression plasmid prbmB-myc or the vector pBAD/Myc-His B grown in the presence of arabinose. (B) CLSM images of the top-down views (large panels) and orthogonal views (side panels) of biofilms formed by RΔrbmB harboring prbmB-myc or the vector in a flow cell system after 24 h postinduction. Mature biofilms were allowed to form in the flow cell chambers in the absence of arabinose for 24 h, followed by overexpression of rbmB with the addition of 0.1% (wt/vol) arabinose to the growth medium. Bars, 40 μm.

FIG. 5.

RbmC and Bap1 are involved in rugose colony development. Colony morphologies are shown for the rugose variant, RΔrbmC, RΔbap1, RΔbap1 harboring prbmC, RΔbap1 harboring pbap1, RΔbap1 harboring the vector pACYC177, RΔrbmC Δbap1, RΔrbmC Δbap1 harboring prbmC, RΔrbmC Δbap1 harboring pbap1, and RΔrbmC Δbap1 harboring the vector pACYC177.

FIG. 6.

RbmC and Bap1 are involved in pellicle and biofilm formation. (A) Pellicle formation by the rugose variant, RΔrbmC, RΔbap1, RΔrbmC Δbap1, RΔrbmC Δbap1 harboring prbmC, RΔrbmC Δbap1 harboring pbap1, and RΔrbmC Δbap1 harboring the vector pACYC177 after 2 days of incubation at 30°C. Top and side views of the culture tubes are showed in the top and bottom panels, respectively. (B) Quantitative comparison of biofilm formation by the rugose variant, RΔrbmC, RΔbap1, and RΔrbmC Δbap1 after 8 h of growth at 30°C under static conditions. Error bars represent standard deviations. (C) Expression of the vpsA::lacZ and vpsL::lacZ fusion genes in the rugose variant, RΔrbmC, RΔbap1, and RΔrbmC Δbap1 grown at 30°C to the mid-exponential growth phase. Error bars represent standard deviations.

FIG. 7.

RbmC and Bap1 are secreted proteins and are involved in maintaining biofilm architecture. (A) CLSM images of the top-down views (large panels) and orthogonal views (side panels) of biofilms formed by the rugose variant, RΔrbmC, RΔbap1, and RΔrbmC Δbap1 in a flow cell system after 6 and 24 h of incubation. Bars, 40 μm. (B) Immunoblot analysis of proteins in the CS and WC fractions from arabinose-induced RΔrbmC (top) and RΔbap1 (bottom) harboring the overexpression plasmids prbmC-myc and pbap1-myc (lanes 1 and 3) as well as the vector pBAD/Myc-His B (lanes 2 and 4).

FIG. 8.

Overexpression of bap1 affects colony morphology and biofilm structure. (A) Colony morphologies of RΔrbmC Δbap1 harboring the overexpression plasmid pbap1-myc or the vector pBAD/Myc-His B. Strains were grown in the presence of 0.1% (wt/vol) arabinose. (B) CLSM images of the top-down views (large panels) and orthogonal views (side panels) of biofilms formed by RΔrbmC Δbap1 harboring pbap1-myc or the vector in a flow cell system after 24 h of growth in the presence of 0.1% (wt/vol) arabinose. Bars, 40 μm.

FIG. 9.

Colony morphology and biofilm formation phenotypes of the rbmDEF deletion mutants and the rugose variant are similar. (A) Colony morphology of the rugose variant, RΔrbmD, and RΔrbmEF. (B) Quantitative comparison of biofilm formation by the rugose variant, RΔrbmD, and RΔrbmEF after 8 h of growth at 30°C under static conditions. Error bars represent standard deviations. (C) Expression of the vpsA::lacZ and vpsL::lacZ fusion genes in the rugose variant, RΔrbmD, and RΔrbmEF grown at 30°C to the mid-exponential growth phase. Error bars represent standard deviations.

FIG. 10.

RbmD, RbmE, and RbmF are involved in biofilm structure formation and cell aggregation. (A) CLSM images of the top-down views (large panels) and orthogonal views (side panels) of biofilms formed by the rugose variant, RΔrbmD, and RΔrbmEF in a flow cell system after 6 and 24 h of incubation. (B) CLSM images of the top-down views of cell aggregates from overnight-grown cultures of the rugose variant, RΔrbmD, and RΔrbmEF. Bars, 40 μm.

FIG. 11.

Model of the biofilm developmental cycle in V. cholerae. Biofilm formation in the rugose variant begins with attachment of single or aggregated cells to surfaces. This is followed by the formation of microcolonies and mature biofilm structures, which require the presence of VPS as well as matrix proteins RbmA, RbmC, and Bap1, which are predicted to act as lectins. The biofilm developmental cycle is completed with detachment of bacteria from mature biofilms, which is likely to be mediated in part by RbmB, predicted to act as a VPS lyase.