B-240. Biofilm Formation and Expression of Curli Fibres is Modulated by Cra in Escherichia coli

S. M. S. Reshamwala, S. B. Noronha;
Indian Inst. of Technology, Bombay, Mumbai, INDIA.

Background: Enteric bacteria attach to intestinal surfaces by means of various cell surface molecules, which may have potential roles as virulence factors. A number of regulatory proteins (e.g., Lrp, CRP, H-NS) activate expression of genes encoding virulence factors. Cra (catabolite repressor/activator) protein (formerly FruR) is a pleiotropic regulatory agent that influences, in a cAMP-independent manner, the relative activities of every major pathway concerned with carbon and energy metabolism in enteric bacteria. A computational search of the Escherichia coli K-12 genome using coliBASE identified putative Cra-binding sites upstream of genes coding for BarA, Crl, and an iron transport protein, all of which are implicated in biofilm formation and expression of curli fibres. Methods: Mutants were constructed using P1 bacteriophage transduction. Curli is well-characterized by its ability to bind the diazo dye Congo red. YESCA agar plates were incubated at 26 degC for 48 hours to allow visualization of curliated strains. A quantitative Congo red-binding assay was used to quantify dye-binding. Nomarski imaging and confocal microscopy was used to image bacteria attached to glass coverslips. E-SEM was used to study morphological differences at high magnification. A crystal violet-based spectroscopic assay was used to quantify biofilm formation. Results: E. coli MG1655 and BW25113 cra mutants formed colourless colonies on agar plates containing Congo red, while parental strains gave rise to red stained colonies. Congo red-binding was calculated to be decreased 6-8 times in the cra mutants. Biofilm formation on glass surfaces was decreased in the cra mutant by 60% as compared to the wild type. Morphological changes in cra mutants were evident in electron microscopy images. Conclusions: This is the first report that shows that Cra influences biofilm formation and expression of curli, which may affect colonization of host surfaces by pathogenic E. coli. Together with the general phenomenon of catabolite repression, Cra may exert its influence by regulating expression of genes that are required for initiation and development of a biofilm.