A CLUSTER OF 14 GENES FROM ENTEROPATHOGENIC ESCHERICHIA-COLI IS SUFFICIENT FOR THE BIOGENESIS OF A TYPE-IV PILUS

Enteropathogenic Escherichia coil (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA), LA is depen dent upon the presence of 50-70 MDa plasmids, termed EPEC adherence fa ctor (EAF) plasmids, Expression of an EAF plasmid-encoded type IV fimb ria, the bundle-forming pilus (BFP), is associated with the LA phenoty pe. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be c omplemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10 kb of downstream sequence, sug gesting that such mutations have polar effects on downstream genes, Th e identification and characterization of a cluster of 13 genes immedia tely downstream of bfpA are described. The introduction into a laborat ory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragmen t derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred, This work also pr ovides evidence to show that the fragment cloned in pJPN14 encodes a f actor(s) which results in increased levels of the pilin protein, Final ly, it is shown that expression of the 14 genes in the bfp cluster fro m an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. coil strain, but is insufficient for LA. This is the first report demonstrating the recons titution of a type IV pilus in a laboratory E. coil strain with a defi ned set of genes. The BFP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.