Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture

Abstract

Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of Escherichia coli K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms.

FIG. 1

Chemical structure of the colanic acid monomer. Fuc, l-fucose; Gal, d-galactose; GlcA, d-glucuronic acid; Glc, d-glucose; OAc, O-acetyl; Pyr, pyruvate. This figure was adapted from reference .

FIG. 2

Colanic acid is important for biofilm formation in E. coli K-12. The wild type {ZK2686 [W3110 Δ(argF-lac)U169]} and colanic acid mutant (ZK2687 [ZK2686 wcaF31::cam]) strains were grown in PVC microtiter dishes in LB at 30°C without shaking for 24 h and then subcultured (1:100) into PVC microtiter dishes containing LB. These cultures were then grown at 30°C in LB without shaking for the indicated times. (A) The dishes were then rinsed and stained with CV as previously described (16). (B) Quantification of CV staining. The amount of CV staining at each time point was determined as previously described (16). Note that the exponential growth rates of ZK2686 and ZK2687 were indistinguishable under the conditions tested (doubling times of ∼60 min).

FIG. 3

Overhead (xy) and sagittal (xz) images of wild-type and colanic acid-defective biofilms. Strains ZK2686 [W3110 Δ(argF-lac)U169] and ZK2687 [ZK2686 wcaF31::cam] were transformed with pND103 (which encodes the green fluorescent protein) and grown at 30°C in the presence of a borosilicate glass coverslip. Cultures were grown in LB with ampicillin (100 μg/ml) for 72 h without shaking. Saturated medium was replaced with fresh LB and ampicillin every 24 h. After 72 h, the coverslip was rinsed to remove nonattached cells, and the remaining attached cells were examined via epifluorescence microscopy using a 40× oil immersion objective (numerical aperture, 1.4) on an Olympus optical-sectioning microscope equipped with deconvolution software (Applied Precision). Sagittal images (c and d) were created from a collection of 130 consecutive Z-series scans of the wild-type and colanic acid-defective biofilms. The step size between each Z section was 0.2 μm. Sagittal images were generated using the Volume View program of the Deltavision software package (Applied Precision). Bar, 5 μm.