AGGR, A TRANSCRIPTIONAL ACTIVATOR OF AGGREGATIVE ADHERENCE FIMBRIA-I EXPRESSION IN ENTEROAGGREGATIVE ESCHERICHIA-COLI

Enteroaggregative Escherichia coli (EAggEC) has been associated with p ersistent pediatric diarrhea in the developing world, yet the pathogen etic mechanisms of EAggEC infection are unknown. Our previous data hav e suggested that aggregative adherence of some EAggEC strains to HEp-2 cells is mediated by flexible, bundle-forming fimbriae, which we have termed aggregative adherence fimbriae I (AAF/I). Genes sufficient to confer expression of AAF/I are located on the 60-MDa plasmid of EAggEC 17-2; AAF/I genes are present as two unlinked plasmid regions (region s 1 and 2), separated by 9 kb of DNA. Here we report the complete DNA sequencing of region 2 and the identification of an open reading frame which is involved in the expression of AAF/I. One open reading frame of 794 bp encodes a protein (designated AggR) with a predicted molecul ar size of 29.4 kDa, which shows a high degree of amino acid sequence identity to CfaR and other members of the AraC class of gene regulator s. The cloned aggR gene (or, alternatively, a cloned cfaR gene) was su fficient to complement a region 1 done to confer AAF/I expression. To further substantiate the role of aggR in the regulation of AAF/I, we c onstructed a 289-bp in-frame aggR deletion and replaced the native gen e in 17-2 by allelic exchange, using the temperature-sensitive vector pIB307. The resulting aggR deletions were negative for AAF/I expressio n, but expression was restored when the aggR gene (cloned into pBluesc ript II SK) was reintroduced into the aggR mutant. RNA slot blot exper iments using a probe for the putative AAF/I pilin subunit (aggA) revea led that aggR operates as a transcriptional activator of aggA expressi on. aggA::phoA fusions were constructed in 17-2 and in 17-2 Delta aggR . AggR was found to promote expression of the aggA gene under a variet y of conditions of temperature, osmolarity, oxygen tension, and medium . At acid pH, aggA expression was maximal and was regulated by both Ag gR-dependent and AggR-independent mechanisms.