Expression of the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is regulated at the transcriptional level by growth phase, temperature, calcium and ammonium. Genes required for the transcriptional activation of bfpA were localized to a 1.8 kb fragment of the enteroadherent factor (EAF) plasmid of EPEC that is separated from the bfp operon by 6 kb. Within this fragment three identically oriented and closely spaced open reading frames (ORFs) were identified and designated bfpT, bfpV and bfpW. bfpT is predicted to encode a 31.8 kDa protein that shares homology with the AraC family of transcriptional regulators, including the presence of a conserved C-terminal DNA-binding helix-turn-helix motif. Insertional inactivation of bfpT led to the loss of bfpA transcription, BfpA protein production and the localized adherence (LA) phenotype; this mutant phenotype could be complemented by introduction of bfpTVW and, on separate plasmids, bfpT + bfpW. However, introduction of bfpT + bfpV, bfpV alone, bfpW alone, or bfpV + bfpW did not enable recovery of the wild-type phenotype. Maximal efficiency of bfpA transcription required all three genes, but bfpV and bfpW each enhanced transcription providing bfpT was also present. A series of deletions of the bfpA upstream promoter region was prepared; with respect to the bfpA transcription start site, sequence between nucleotides -94 and -55 was found to bind bfpT. BfpT also bound a DNA fragment containing the eaeA promoter region on the EPEC chromosome. From these results we conclude that bfpTV W causes transcriptional activation of bfpA, and possibly eaeA, by a trans-acting mechanism that may co-ordinately regulate the expression of EPEC virulence determinants.

Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundleforming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.

bfp, the structural gene of the major repeating bundle-forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (O111:NM). The bfp open reading frame encoded a 193-amino-acid protein; comparison of this sequence with the biochemically determined N-terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13-amino-acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21,000 dalton protein that co-migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonella serotypes. Fifty per cent of these bfp probe-sensitive salmonellae exhibited the localized-adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid-containing EPEC strains. Scanning electron micrographs of a bfp probe-sensitive, LA-positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP-like fibres form inter-bacterial linkages. For EAF plasmid-containing EPEC strains and for several Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.