Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation

Abstract

The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.

Figure 1

The rugose colonial variant of V. cholerae O1 El Tor produces an extracellular glycocalyx. (A) Colonial morphology of the smooth and rugose variants grown on LB agar at 30°C for 72 hr. (B) Pellicle formation by the rugose variant grown in LB broth for 72 hr. The rugose colony type forms a floating membrane at the air-broth interface where it coats the glass surface of the culture tube. Scanning electron microscopy of the pellicle shows it to be composed of closely packed bacteria (Inset). The smooth variant grows mainly below the surface. (C) Ruthenium red-stained thin sections of smooth and rugose colonies. Electron micrographs demonstrate a stained matrix between rugose-type bacteria. (D) Immunogold electron microscopy of thin-sectioned smooth and rugose colonies. An EPS^ETr-specific antiserum localized the polysaccharide antigen between rugose-type bacteria. [Bars = 5 μm (B) and 1 μm (C and D).]

Figure 2

EPS^ETr-mediated survival in chlorine and biofilm formation by the rugose and smooth colonial variants. (A) Smooth-type bacteria were incubated for 5 min with 3 ppm chlorine (NaOCl) and the indicated concentrations of purified EPS^ETr. The surviving bacteria were enumerated by viable plate counts, and their numbers were compared with the number of surviving smooth bacteria that had not been incubated with NaOCl, denoted by C; this number was defined as 100% survival. Increasing survival of the smooth variant in chlorine was conferred by EPS^ETr concentrations of 250 and 500 μg/ml. Rugose and smooth bacteria, incubated with 3 ppm NaOCl in the absence of exogenous EPS^ETr, denoted by R and 0, respectively, revealed differences between the intrinsic resistances of the two colonial variants to the bactericidal activity of chlorine. (B) Preincubation of NaOCl (3 ppm) with the indicated concentrations of EPS^ETr for 5 min before the addition of smooth-type bacteria increased the protective effect of the polysaccharide. C denotes the survival of smooth-type bacteria in the absence of NaOCl and EPS^ETr. (C) Consumption of free chlorine by purified EPS^ETr. NaOCl (6 ppm) and the indicated concentrations of EPS^ETr were incubated in PBS for 1 min at 22°C, and the free chlorine concentration then was determined by the syringaldazine method. EPS^ETr caused rapid, concentration-dependent consumption of chlorine. (D) Quantitative analysis of biofilm formation by the smooth and rugose variants. Bacteria of each colony type (1–5 × 10⁶ colony-forming units) were incubated in separate wells of a poly(vinyl chloride) microtiter dish for the indicated time periods, the wells were emptied and washed, and the adherent bacteria were stained with a 1% solution of crystal violet. The dye was solubilized by the addition of 95% ethanol, and the absorbance was determined at 560 nm. Progressive biofilm formation by the rugose variant is indicated by an increase in A₅₆₀ during the experimental period.

Figure 3

Three-dimensional reconstructions of biofilms formed by the smooth and rugose colonial variants. Smooth and rugose type bacteria carrying a plasmid constituitively expressing the green fluorescent protein were incubated in chambers containing borosilicate coverglass bottoms. The wells were emptied at 1, 2, 4, 6, and 24 hr, washed, and examined with a scanning confocal laser microscope (MultiProbe 2010, Molecular Dynamics) using 488- and 510-nm excitation and emission wavelengths, respectively. (A and B) Horizontal (xy) projected images at low and high magnification, respectively, of biofilms formed by the two colony types after a 6-hr incubation period. Islands of adherent bacterial aggregates typify the rugose-type biofilm. (C) Image reconstruction led to a saggital (xz) view of the same biofilms and revealed dramatic differences between their heights. The relative intensity of the pseudo-colored images is shown at the lower right corner and correlates with cell density. [Bars 50 μM (A) and 10 μM (B and C)] ×18.

Figure 4

Identification of sequences on the V. cholerae O1, El Tor chromosome that are required for EPS^ETr production. (A) Physical map of cosmid-3 derived from restriction mapping and Southern analysis of insert DNA. E denotes EcoRI restriction sites. (B) Location of tagged DNA fragments within cosmid-3. Cosmid-3 was digested with EcoRI, the restriction fragments were separated by 0.8% agarose gel electrophoresis, transferred onto membranes, and hybridized with radiolabeled sequence tags from each of the indicated mutants (identified by the number above each autoradiograph and corresponding to the numbered designation in Table 2). The sizes of the hybridized fragments are indicated. (C) EPS^ETr production by the mutants harboring either the cloning vector alone or complemented with cosmid-3, as determined by ELISA on a nitrocellulose membrane using an EPS^ETr-specific antiserum. Supernatants of the smooth (S) and rugose (R) variants containing crude EPS^ETr were included as negative and positive controls, respectively. Purified EPS^ETr was used as an additional positive control. Cosmid-3 complemented EPS^ETr production by each of the mutants.