The complete nucleotide sequence of the 8.3 kilobase operon of the enterobacterial virulence plasmid pColV-K30, which encodes a high-affinity iron transport system mediated by the hydroxamate siderophore aerobactin, has been determined. The region includes five open reading frames which correspond to the genes iucA, iucB, iucC and iucD, encoding the enzymes of the biosynthetic pathway for aerobactin, and iutA for the outer membrane receptor of ferri-aerobactin complexes. The sequences of the iucABCD genes are tightly coupled without any intervening non-coding sequence. The predicted secondary mRNA structures at the gene junctions within the iucABCD cluster, along with their codon usage, may account for the differential expression of each of the protein products, as observed in vivo with minicells. The genes iucA and iucC, which determine the two subunits of the aerobactin synthetase complex, showed a considerable homology within three stretches of their amino acid sequence. A potential operator sequence (iron box) for the binding of the iron(II)-responsive Fur repressor protein was found within the iucA coding region, suggesting that the operon is subjected to an additional level of transcriptional repression by iron (II).