Identification and characterization of a Vibrio cholerae gene, mbaA, involved in maintenance of biofilm architecture

Abstract

The formation of biofilms is thought to play a key role in the environmental survival of the marine bacterium Vibrio cholerae. Although the factors involved in V. cholerae attachment to abiotic surfaces have been extensively studied, relatively little is known about the mechanisms involved in the subsequent maturation of the biofilms. Here we report the identification of a novel gene, which we have named mbaA (for maintenance of biofilm architecture), that plays a role in the formation and maintenance of the highly organized three-dimensional architecture of V. cholerae El Tor biofilms. We demonstrate that although the absence of mbaA does not significantly affect the initial attachment of cells onto the surface, it leads to the formation of biofilms that lack the typical structure, including the pillars of cells separated by fluid-filled channels that are evident in mature wild-type biofilms. Microscopic analysis indicates that the absence of mbaA leads to an increase in the amount of extracellular matrix material in the biofilms. The predicted mbaA product is a member of a family of regulatory proteins, containing GGDEF and EAL domains, suggesting that MbaA regulates the synthesis of some component of the biofilm matrix.

FIG. 1.

Disruption of a novel gene named mbaA in V. cholerae N16961 leads to increased biofilm formation. (A) Crystal violet-stained borosilicate tubes and quantification of the DMSO-solubilized dye are shown for the indicated strains after 30 h of growth in LB broth at room temperature. All assays were performed in triplicates. (B) Genetic organization of the mbaA region on the chromosome of V. cholerae N16961. The a, b, mbaA, c, and d open reading frames correspond to loci VC0705, VC0704, VC0703, VC0702, and VC0701, respectively, based on the DNA sequences obtained from The Institute for Genomic Research microbial genome database. Open reading frames a, mbaA, and c might constitute an operon. The Tn10 insertion in mbaA and the extent of the in-frame deletion created in mbaA are indicated.

FIG. 2.

Flagella and MSHA pili are required for biofilm formation in the absence of mbaA. Biofilm development of the indicated V. cholerae strains was followed in LB medium at room temperature over the course of 65 h. Cells that attached to borosilicate tubes after various periods of incubation were stained with crystal violet. The OD₅₇₀ was measured to quantify the amount of DMSO-solubilized dye. All assays were performed in triplicates. Shown are the biofilm developments of the WT (closed circles) and ΔmbaA (open circles) strains (A) and those of the ΔmshA (closed triangles) and ΔmbaA-ΔmshA (open triangles) mutant strains (B).

FIG. 3.

The production of EPS is required for biofilm formation in the absence of mbaA. WT, vsp59::pGp704, and ΔmbaA-vps59::pGp704 mutant cells of V. cholerae N16961 were incubated in LB broth at room temperature for 40 h. Phase contrast micrographs of biofilms formed on borosilicate coverslips are shown in the left panels. Crystal violet-stained borosilicate tubes are shown in the right panels.

FIG. 4.

The absence of mbaA leads to alterations in the biofilm architecture. WT and gfp-tagged ΔmbaA mutant cells of V. cholerae N16961 were incubated with borosilicate coverslips in LB broth at room temperature. Biofilm formation on the coverslips was observed at the indicated times. Shown are phase contrast micrographs of biofilms formed after 8 (A) and 40 h (B) and confocal scanning laser micrographs of biofilms formed after 65 h of incubation (C). Micrographs represent optical sections in the x-z plane, with the substratum located at the bottom. Bar, 5 μm.

FIG. 5.

The absence of mbaA leads to accumulation of a larger amount of biofilm matrix material. WT and ΔmbaA mutant strains of V. cholerae N16961 were incubated with borosilicate coverslips during 65 h in LB medium at room temperature. Biofilms were observed after fixation by SEM. The extracellular matrix of the biofilms appears on the micrographs as a white fibrous network among cells. Bar, 1 μm.