Environmental determinants of Vibrio cholerae biofilm development

Abstract

Vibrio cholerae is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean. Surface attachment is believed to be essential for colonization of all of these natural environments. Previous studies have demonstrated that the vps genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for V. cholerae biofilm development in Luria-Bertani broth. In this work, we showed that V. cholerae forms vps-dependent biofilms and vps-independent biofilms. The vps-dependent and -independent biofilms differ in their environmental activators and in architecture. Our results suggest that environmental activators of vps-dependent biofilm development are present in freshwater, while environmental activators of vps-independent biofilm development are present in seawater. The distinct environmental requirements for the two modes of biofilm development suggest that vps-dependent biofilm development and vps-independent biofilm development may play distinct roles in the natural environment.

FIG. 1.

Representative wild-type V. cholerae strain MO10 (WT) and ΔvpsL mutant PW328 biofilms formed over 18 h in 10 mM NaCl or commercial artificial seawater (ASW) supplemented with either YT or CAA. Biofilms were visualized by crystal violet staining.

FIG. 2.

Accumulation over time of wild-type V. cholerae strain MO10 (WT) and a ΔvpsL mutant (PW328) on a borosilicate surface in two types of media, 10 mM NaCl-YT (A) and commercial artificial seawater (ASW) supplemented with CAA (B).

FIG. 3.

Transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 biofilms formed in 10 mM NaCl-YT and commercial artificial seawater (ASW) supplemented with CAA. Transverse cross sections were obtained at the level of the substratum. Bars = 10 μm.

FIG. 4.

Architecture of V. cholerae biofilms formed in 10 mM NaCl supplemented with monosaccharides and/or CAA: transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and ΔvpsL mutant PW328 biofilms in 10 mM NaCl supplemented with CAA and 10 mM NaCl supplemented with CAA and monosaccharides (CRB). Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

FIG. 5.

Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in 10 mM NaCl-YT, 10 mM NaCl-CAA, and 10 mM NaCl supplemented with CAA and carbohydrates (CRB). This strain carries a chromosomal fusion of the vpsL promoter to lacZ.

FIG. 6.

Architecture of biofilms formed in DSW supplemented with CAA with and without Ca²⁺: transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and ΔvpsL mutant PW328 biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

FIG. 7.

Quantification of wild-type V. cholerae strain MO10 and ΔvpsL mutant PW328 surface-associated cells in DSW supplemented with CAA with and without Ca²⁺.

FIG. 8.

Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in commercial artificial seawater (ASW) supplemented with CAA and DSW supplemented with CAA. This strain carries a chromosomal fusion of the vpsL promoter to lacZ.

FIG. 9.

Architecture of wild-type V. cholerae and ΔvpsL mutant biofilms formed in water from the Charles River in Massachusetts (CRW) and in water from the Massachusetts coast (RSW) supplemented with CAA: transverse and vertical cross sections through DAPI-stained biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.