Aerobactin, a hydroxamate iron transport compound, is synthesized by some, but not all, Shigella species. Conjugation and hybridization studies indicated that the genes for the synthesis and transport of aerobactin are linked and are found on the chromosome of Shigellaflexneri, S. boydii, and S. sonnei. The genes were not found in S. dysenteriae. A number of aerobactin synthesis mutants and transport mutants have been isolated. The most common mutations are deletions of the biosynthesis or biosynthesis and transport genes. The Shigella aerobactin genes share considerable homology with the E. coli CoIV aerobactin genes. On the CoIV plasmid and in the Shigella chromosome, the aerobactin genes are associated with a repetitive sequence which has been identified as IS]. Shigellai species synthesize both phenolate and hydroxamate siderophores for acquisition of iron. Sliigell(a soniiei utilizes enterobactin (enterochelin) (25), a phenolate siderophore common to many enteric species (19). and some strains produce an additional hydroxamate siderophore (25). Similarly, S. boydii has been reported to produce both phenolate and hydroxamate siderophores (25). S. flexvneri strains normally synthesize only aerobactin (22), a secondary hydroxamate originally isolated from cultures of Aerobacter aerogenes (9) and more recently from Esclhrichia coli ColV strains (29). Rare isolates and laboratory variants of S. flexneri produce enterobactin in addition to aerobactin (23). The genetics of iron transport in enteric bacteria has been studied primarily in E. coli and Sailm)ioniellai typhiminriumi. The genes for enterobactin synthesis and transport are linked and are carried on the chromosome in both species (12-14, 26). Aerobactin genes, however, are encoded by the CoIV plasmid in E. (/li (29) and by a large plasmid in A. aerogenies (18). In other species, the location of the aerobactin genes is unclear. Since differences were observed in the types of iron transport systems expressed by different Shliie/lla species (22, 23, 25) and even by members of the same species, it was of interest to examine the genetics of siderophore synthesis and transport in Shige/lla spp. and to compare it to the CoIV system. MATERIALS AND METHODS Strains and media. Bacterial strains, plasmids, a phage, and their sources are listed in Table 1. All other Shliige/la

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.

Mutants of Shigellaflexneri defective in aerobactin-mediated iron transport were assayed for virulence in several model systems. A TnS insertion mutant was invasive in HeLa cells, lethal in the chicken embryo, and produced keratoconjunctivitis in the guinea pig, indicating little or no loss of ability to invade and multiply intracellularly. Although the mutant failed to grow in low-iron medium in vitro, growth equivalent to that of the wild type was observed in HeLa cell lysates. Thus, there appears to be sufficient available iron inside the HeLa cell to allow growth in the absence of siderophore synthesis. Possible host iron sources were tested, and both the mutant and wild type utilized hemin or hematin as a sole source of iron. Only the wild-type, aerobactin-producing strain could remove iron from transferrin or lactoferrin. Two deletion mutants were also assayed for virulence and were found to be avirulent for the chicken embryo. These deletions encompass flanking sequences as well as the aerobactin genes; therefore, adjacent genes may be required for virulence.

Shigella flexneri strains were assayed for the ability to synthesize and utilize phenolate and hydroxamate siderophores. The hydroxamate aerobactin was synthesized by all isolates tested, whereas phenolates were only rarely produced. Expression of aerobactin was accompanied by production of a single ironregulated outer membrane protein (Mr = 74,000). This protein was not produced by a mutant defective in aerobactin utilization and may serve as the aerobactin receptor. Phenolate (enterobactin)-producing strains synthesized three additional outer membrane proteins (Mr = 74,000, 81,000, and 83,000) in response to iron starvation. These proteins are the same apparent size as those produced by Escherichia coli K-12 strains. Ent sequences are apparently present in strains which do not synthesize this compound. Although normally silent, ent genes can be activated in Ent-strains to produce Ent' variants. These laboratory variants are phenotypically indistinguishable from clinical Ent' isolates.

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