Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEG) forms thick biofilms on the intes tinal mucosa. Here we show that most EAEC strains form a biofilm on glass o r plastic surfaces when grown in cell culture medium with high sugar and os molarity. Biofilm-forming ability in two prototype EAEC strains required ag gregative adherence fimbriae (AAF), although many other EAEC strains that d o not express AAF also developed biofilms under these conditions. Ten thous and transposon mutants of EAEC strain 042 were isolated, and 100 were found to be deficient in biofilm formation. Of these, 93 were either deficient i n in vitro growth or mapped to genes known to be required for AAF/II expres sion. Of the seven remaining insertions, five mapped to one of two unsuspec ted loci. Two insertions involved the E. coli chromosomal fis gene, a DNA-b inding protein that is involved in growth phase-dependent regulation. Using reverse transcription-polymerase chain reaction (RT-PCR), we determined th at the effect of fis was at the level of transcription of the AAF/II activa tor aggR. Biofilm formation also required the product of the yafK gene, whi ch is predicted to encode a secreted 28 kDa protein. The yafK product is re quired for transcription of AAF/II-encoding genes. Our data do not suggest a role for type 1 fimbriae or motility in biofilm formation. EAEC appears t o form a novel biofilm, which may be mediated solely by AAF and may reflect its interactions with the intestinal mucosa.