Molecular genetic analysis of aerobactin receptors.

Some Enterobacteriaceae produce a low molecular weight compound, aerobactin, with a high affinity for ferric iron. The genes encoding the aerobactin system have been identified in plasmid or chromosomal DNA; they are arranged in an operon consisting of five genes; four genes encode enzymes responsible for the biosynthesis of aerobactin and the fifth encodes an outer membrane receptor protein specific for ferric aerobactin. The aerobactin receptor protein appears to be different in size (molecular weight) in different species. Escherichia coli (ColV-K30) express a 74 kDa protein, while Shigella spp. express a 76 kDa protein. The aerobactin receptor protein also acts as a receptor for the bacteriocin cloacin DF13. Shigella spp. expressing the aerobactin receptor protein were less sensitive to cloacin DF13 than E. coli (ColV-K30). The aerobactin receptor genes of several Shigella spp. isolate have been cloned and the aerobactin receptor gene from Shigella flexneri ser.6 {lcub}iutAsh) has been sequenced. The restriction maps of iutAsh and iutA (aerobactin receptor gene from ColV-K30) were similar. The main difference was the existence of a BamHI site in the middle of iutAsh but not in iutA. The predicted protein product of iutAsh consists of 732 amino acid residues, the same as the aerobactin receptor protein from ColV-K30 (iutA), and with 93% similarity. Construction of iutA::iutAsh hybrid genes demonstrated that although verious parts of iutA were able to increase the cloacin sensitivity function of iutAsh, the main function was located in the 3'-terminus of iutA. Constructs with the 3'-terminus of iutA expressed a 74 kDa, while constructs with 3'-terminus of iutAsh expressed a 76 kDa outer membrane protein. The aerobactin uptake of E. coli strains carrying constructs with the 3'-terminus of iutAsh was higher than that of E. coli strains carrying constructs with the 3'-terminus of the iutA. The highest aerobactin uptake was showed by E. coli strain carrying construct 2, which consist of the iutA fragment upstream of the ClaI site and the iutAsh fragment downstream of the ClaI site. Bacteriophage B74K was capable of using the aerobactin receptor protein, but it was also capable of using other outer membrane proteins as receptors, and so could not be used for aerobactin receptor protein identification. In addition, the presence of aerobactin receptor protein increased the sensitivity of E. coli strains to phage BF23.