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Novel biofilm formation by enteroaggregative Escherichia coli



Novel biofilm formation by enteroaggregative Escherichia coli



Abstracts of the General Meeting of the American Society for Microbiology 101: 442



Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrhea in adults and children. The organism has been observed to form thick biofilms of aggregating bacteria in in vivo models. To elucidate the requirements for biofilm formation, we developed an in vitro model for EAEC biofilm, characterized the EAEC biofilm, and screened for mutations that were deficient in biofilm-forming ability. Our results suggest that most EAEC strains are capable of forming a thick biofilm on glass or plastic in minimal essential medium with a high concentration of sugar. In prototype EAEC strain 042, biofilm-forming ability was dependent on the ability of the strain to express the fimbriae AAF/II, although many other EAEC strains that do not express AAF/II were also able to build similar biofilms. In contrast to previously described E. coli K12 biofilms, flagella and type 1 fimbriae were apparently not required for EAEC biofilm formation. We screened 10,000 transposon mutants and identified 100 that were deficient in biofilm formation. Of these, 93 were either deficient in in vitro growth or mapped to genes known to be required for AAF/II expression. Of the 7 remaining insertions, we identified two new loci that were required for biofilm formation. Two insertions mapped to the E. coli chromosomal fis gene, a DNA binding protein that is involved in growth phase dependent regulation. Complementation of the fis mutation resulted in restoration of wild type biofilm formation. Using RT-PCR we determined that the effect of fis was at the level of transcription of the AAF/II activator aggR. We also found that biofilm formation required the product of the yafK gene, which encodes a 28 kDa previously hypothetical protein that features a strongly predicted signal sequence. Complementation of the yafK mutations restored biofilm formation. The yafK product is required for expression of AAF/II at the post-transcriptional level. Our data suggest that EAEC forms a novel biofilm, which depends on growth phase-dependent expression of AAF fimbriae.

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