The chromosomal iroBCDEN gene cluster first described for Salmonella enterica is involved in the uptake of catecholate-type siderophore compounds. An orthologous gene cluster has recently been detected in Escherichia coli strains which cause extraintestinal disease. This E. coli iroBCDEN gene cluster has an impact on virulence and has been reported to be located in a pathogenicity island on the chromosome. In this study we characterized an iro gene cluster of a uropathogenic E. coli isolate which is located on a transmissible plasmid related to the R64 plasmid of S. enterica. This cluster is highly homologous to the chromosomal iro cluster of E. coli. When introduced into an E. coli fepA cir fiu aroB mutant, IroN, but not IroBCDE, mediated the utilization of structurally related catecholate siderophores, including 2,3-dihydroxybenzoyl-L-serine, 2,3-dihydroxybenzoyl-D-ornithine, 2,3-dihydroxybenzoic acid, and enterochelin. This study supports the idea of an ongoing horizontal transfer of putative virulence factors and the mobilization of single virulence gene clusters, which lead to a modular assembly of virulence determinants such as pathogenicity islands.