Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae

Abstract

Phase variation between smooth and rugose colony variants of Vibrio cholerae is predicted to be important for the pathogen's survival in its natural aquatic ecosystems. The rugose variant forms corrugated colonies, exhibits increased levels of resistance to osmotic, acid, and oxidative stresses, and has an enhanced capacity to form biofilms. Many of these phenotypes are mediated in part by increased production of an exopolysaccharide termed VPS. In this study, we compared total protein profiles of the smooth and rugose variants using two-dimensional gel electrophoresis and identified one protein that is present at a higher level in the rugose variant. A mutation in the gene encoding this protein, which does not have any known homologs in the protein databases, causes cells to form biofilms that are more fragile and sensitive to sodium dodecyl sulfate than wild-type biofilms. The results indicate that the gene, termed rbmA (rugosity and biofilm structure modulator A), is required for rugose colony formation and biofilm structure integrity in V. cholerae. Transcription of rbmA is positively regulated by the response regulator VpsR but not VpsT.

FIG. 1.

Protein profiles of smooth and rugose variants are different. Two-dimensional gel electrophoretic analyses of proteins in whole-cell lysates from rugose and smooth variants of V. cholerae during exponential and stationary growth phases are shown. The arrowheads indicate the IEF internal standard, tropomyosin protein (33 kDa), whereas protein spots that were differentially produced between the two variants are circled and numbered. Protein spot 1 corresponds to the protein RbmA. The arrows in panel C indicate protein spots that are present in larger amounts in rugose whole-cell lysates than in those from the smooth variant during stationary growth phase. Sizes (in thousands) of molecular weight markers are indicated on the right side of each panel.

FIG. 2.

Amino acid sequence of protein spot 1 corresponds to that of VC0928. (A) Alignment of the 12-amino-acid sequence from protein spot 1 with a peptide sequence encoded by rbmA (locus VC0928), as obtained from TIGR. (B) The genetic organization of rbmA (open arrow) on the V. Cholerae chromosome is depicted with locus annotations under each ORF. The vpsI and vpsII gene clusters, as well as the first and last genes of each cluster (vpsA and vpsK; vpsL and vpsQ) are marked and labeled. Unlinked chromosomal DNA regions within the vps clusters are also indicated (∥). (Illustration is not to scale.)

FIG. 3.

RbmA is critical to rugose colony formation, and the expression of vpsA and vpsL is not altered in RΔrbmA. Panel A shows the colony morphologies of the rugose variant (A1), smooth variant (A2), RΔrbmA (A3), SΔrbmA (A4), RΔrbmA/pACYC177 (A5), and RΔrbmA/prbmA (A6). (B) Expression of the vpsA-lacZ and vpsL-lacZ fusion genes (carried in the pCC11 and pCC12 plasmids, respectively) in the wild-type rugose and RΔrbmA variants grown in LB at 30°C to mid-exponential growth phase (OD₆₀₀ = 0.4). Error bars represent standard deviations.

FIG. 4.

RbmA is required for biofilm structure. (A) Quantitative comparison of biofilm formation by the wild-type rugose variant, RΔrbmA, RΔrbmA/pACYC177, and RΔrbmA/prbmA after an 8-h incubation in LB medium at 30°C under static conditions. Error bars represent standard deviations. Crystal violet-stained biofilms formed in the wells of polyvinyl chloride microtiter plates, which were used in the quantitative biofilm assays, are shown in the inserts. (B) Pellicle formation by the wild-type rugose variant (B1 and B2) and RΔrbmA (B3 and B4) after 2 days of incubation in LB at 30°C. B2 and B4 show pellicles disturbed by shaking. (C) Confocal scanning laser microscopic images of horizontal (xy) and vertical (xz) projections of biofilm structures formed by the rugose variant and RΔrbmA in a flow cell system. Biofilm structures that developed after 12 and 24 h of incubation are shown in panels C1 to C4. Biofilm structure integrity studies using SDS are shown in C5 to C8, and the duration of SDS treatment is indicated on the left. In C1 and C2, bars represent 30 μm, whereas in C3 to C8, they represent 100 μm.

FIG. 5.

RbmA is a secreted protein. Immunoblot analysis of proteins in the CS and OM fractions from arabinose-induced (+) and uninduced (−) cultures of RΔrbmA harboring plasmid prbmA-myc. Mouse monoclonal antibody against the Myc epitope (α-Myc) and rabbit polyclonal antisera against V. cholerae OmpU (α-OmpU) were used to detect RbmA-Myc and OmpU, respectively.

FIG. 6.

VpsR is required for rbmA transcription. Expression of an rbmA-lacZ fusion gene (prbmA::lacZ) in the wild type and in vpsR, vpsT, and vpsR vpsT mutants of the rugose variant grown in LB at 30°C to mid-exponential growth phase (OD₆₀₀ = 0.4) is shown. Error bars represent standard deviations.