Iron is essential for bacterial growth but is of limited availability in animal tissues. Therefore, bacteria have evolved several strategies for iron uptake, most of which have been explained in molecular terms (15). The socalled aerobactin operon (24) encodes IucABCD, four enzymes for biosynthesis of the hydroxamate-type siderophore aerobactin and IutA, the outer membrane receptor specific to ferric aerobactin. This operon is present not only on plasmids including pColV-K30 (41), F1me (7), and a 180-kb plasmid (25) in certain strains of Escherichia coli, Salmonella spp., and Klebsiella pneumoniae, respectively, but also in chromosomal pathogenicity islands of Shigella spp. (29,39) and other E. coli isolates (37). The wide distribution of this iron acquisition system has been hypothesized to depend on the mobility of the aerobactin gene complex between plasmids and chromosome and between bacterial species via conjugative plasmids (39). In addition, several chromosomally located genes, tonB exbBD and fhuBCD are required for transport of ferric aerobactin into cell cytosol (4). The energy for transport of ferric aerobactin across the outer membrane is provided by the TonB-ExbBD complex, which transduces energy from the inner membrane (3). Its transport through the periplasm and across the inner membrane is facilitated by an ATP-binding cassette (ABC) transport system which is encoded by fhuBCD, and this system also serves to utilize other hydroxamate siderophores such as ferrichrome, rhodotorulic acid, and coprogen (4). In E. coli, the fhuBCD genes and the fhuA gene, which codes for the ferrichrome receptor, constitute a single operon (4). Thus, two operons, iucABCD iutA and fhuABCD, exist independently in the enteric bacterial species described above and are located far from each other. Moreover, the contribution of aerobactin to virulence has been extensively assessed for enterobacterial pathogens (11).Vibrio mimicus was first described by Davis et al. (9) for a group of biochemically atypical strains of V. cholerae, and has been reported as an agent for enteric and extraintestinal infections (9, 34). It has been proposed that the production of multiple toxins or toxic substances is characteristic of this species (6,30,35). We previously reported that V. mimicus also produces aerobactin as a major siderophore in response to iron star- Identification and Characterization of Two Contiguous Operons Required for Aerobactin Transport and Biosynthesis in Vibrio mimicusYong-Hwa Moon, Tomotaka Tanabe, Tatsuya Funahashi, Kei-ichi Shiuchi, Hiroshi Nakao, and Shigeo Yamamoto* Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Okayama 700-8530, Japan Received January 6, 2004. Accepted February 6, 2004 Abstract: In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron-regulated operons. One contains ...

The present authors have previously reported that Vibrio mimicus expresses 77-kDa and 80-kDa outer membrane proteins in response to iron-limited conditions, and that the 77-kDa protein serves as the receptor for ferriaerobactin. In this study, it was found that V. mimicus can use heme and hemoglobin as iron sources. FURTA was then applied to V. mimicus 7PT to obtain candidate gene fragments involved in utilization of heme and hemoglobin. One FURTA-positive clone was shown to contain a partial gene, whose predicted amino acid sequence correlated with the N-terminal amino acid sequence determined for the 80-kDa outer membrane protein and also shared homology with heme/hemoglobin receptors of Gram-negative bacteria. Based on this information, the entire gene (named mhuA),andageneupstreamof mhuA (named mhuB) encoding a LysR family of transcriptional activator, were cloned and analyzed. RNA analysis indicated that mhuA and mhuB are each transcribed from individual Fur-regulated promoters. Moreover, RNA analysis of an mhuB deletion mutant and a promoter reporter assay coupled with β-galactosidase suggested that MhuB could function as an activator for mhuA transcription. Finally, the role of MhuA as the heme/hemoglobin receptor was confirmed by construction of an mhuA deletion mutant and its complemented strain followed by growth assay.