Steps in the development of a Vibrio cholerae El Tor biofilm - PubMed (original) (raw)

Steps in the development of a Vibrio cholerae El Tor biofilm

P I Watnick et al. Mol Microbiol. 1999 Nov.

Abstract

We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.

PubMed Disclaimer

Figures

Fig. 1

Confocal scanning laser micrograph through the xz-plane of a 5-day-old biofilm of V. cholerae. Biofilms were formed with V. cholerae that constitutively express GFP from a plasmid. Thus, bright areas represent biofilm-associated bacteria. The substratum is located at the bottom of the micrograph. Bar = 5 μm.

Fig. 2

Quantification of bacteria in biofilms formed by wild-type V. cholerae, an MSHA mutant (MSHA), an exopolysaccharide mutant (EPS) and a motility mutant (MOT). The biofilms were stained with crystal violet. Crystal violet was solubilized, and the optical density of the resultant solution was measured.

Fig. 3

Biofilm development over 72 h for V. cholerae El Tor and representative derivative mutants. OD570 quantifies the amount of crystal violet associated with the biofilm after staining.

Fig. 4

Phase-contrast micrographs comparing the attachment of wild-type V. cholerae and representative mutants after 15 min of incubation with a polystyrene surface. Bar = 5 μm.

Fig. 5

Phase-contrast micrographs comparing the attachment of wild-type V. cholerae and representative mutants after 24 h of incubation with a borosilicate coverslip. Bar = 2 μm.

Fig. 6

Confocal scanning laser micrographs comparing the attachment of wild-type V. cholerae and representative mutants after 72 h of incubation with a borosilicate coverslip. Micrographs represent optical sections in the xz-plane with the substratum located at the bottom of the micrograph. Bar = 5 μm.

Fig. 7

Proposed steps in the formation of a three-dimensional V. cholerae biofilm on an abiotic surface. Rods are V. cholerae with polar type IV MSHA pili and one polar flagellum. The bacteria swim towards the surface using flagellar motility. The bacteria attach to the surface using type IV pili, which then retract to draw the bacterium onto the surface. Flagella allow movement along the surface and microcolony formation, and EPS is important for the three-dimensional arrangement of bacteria.

References

1. Alexeyev MF, Shokolenko IN. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of Gram-negative bacteria. Gene. 1995;160:59–62. - PubMed
1. Anwar H, Strap JL, Costerton JW. Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob Agents Chemother. 1992;36:1347–1351. - PMC - PubMed
1. Blattner FR, Plunkett GI, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete genome sequence of Escherichia coli K12. Science. 1997;277:1453–1474. - PubMed
1. Bloemberg GV, O’Toole GA, Lugtenberg BJ, Kolter R. Green fluorescent protein as a marker for Pseudomonas spp. Appl Environ Microbiol. 1997;63:4543–4551. - PMC - PubMed
1. Colwell RR. Global climate and infectious disease: the cholera paradigm. Science. 1996;274:2025–2031. - PubMed

Steps in the development of a Vibrio cholerae El Tor biofilm - PubMed (original) (raw)