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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 ...

In response to iron starvation, many bacteria produce siderophores which mediate high-affinity iron transport enabling iron to be scavenged from their surroundings. [1][2][3] In addition to their native siderophores, some bacteria are endowed with the uptake systems for heterologous siderophores produced by other bacterial and fungal species. In gram-negative bacteria, these siderophores loaded with ferric ion are transported via an energy dependent system across the membrane into the cytoplasm. The components of such systems include a receptor protein located in the outer membrane, a periplasmic binding protein and an ATP-binding cassette (ABC) type transporter located in the inner membrane. 2,4)The energy required for transport via the outer membrane receptor is provided by interaction of the receptor with the TonB-ExbB-ExbD complex located in the inner membrane, which couples the proton motive force created by the inner membrane for energy transduction. In general, siderophores display higher receptor specificity than proteins constituting the ABC transport systems. 4) Furthermore, expression of genes responsible for siderophore biosynthesis and uptake of its ferric complex are controlled by concentration of iron in the environment through a global iron-binding repressor called ferric utilization regulation (Fur) protein, with increased expression occurring under iron-limiting conditions. 2,5)Vibrio parahaemolyticus is a gram-negative halophilic bacterium that is found in estuarine waters worldwide and is recognized as a leading cause of food-borne gastroenteritis. 6)V. parahaemolyticus secretes the siderophore vibrioferrin in response to iron starvation.7) The pvsABCDE genes are involved in production 8) and secretion 9) of vibrioferrin, and uptake of its ferric complex occurs via the outer membrane receptor PvuA 10) and the ABC transporter PvuBCDE. have been annotated in the genomic sequences of V. parahaemolyticus RIMD2210633, 15) but none of these genes have been experimentally characterized to date. We report here that the V. parahaemolyticus fhuACDB orthologs are responsible for ferrichrome utilization and that the fhuCDB genes are also needed for transport of ferric aerobactin across the inner membrane. MATERIALS AND METHODSStrains, Plasmids, and Media Bacterial strains and plasmids used in this study are listed in Table 1. Unless otherwise noted, bacterial cells were routinely grown with shaking at 37°C in Luria-Bertani (LB) broth (pH 7.5, 1% tryptone peptone, 0.5% yeast extract) or grown on LB agar plates (1.5% agar) containing 3% (V. parahaemolyticus) and 0.5% (E. coli) NaCl. (LB) media with and without addition of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA) (Sigma) at 25 mM were used for growth under ironlimiting (ϪFe) and iron-replete (ϩFe) conditions, respectively. The media for E. coli were supplemented with ampicillin (50 mg/ml) or chloramphenicol (10 mg/ml), when required.Growth Assay V. parahaemolyticus TNB6 which carries a deletion in pvsA of strain AQ3354, result...

A pvsB-vctA-irgA triple deletion mutant of Vibrio parahaemolyticus can utilize enterobactin under iron-limiting conditions by inducing a previously undescribed receptor, PeuA (VPA0150), in response to extracellular alkaline pH and enterobactin. In silico analyses revealed the existence of a two-component regulatory system operon, peuRS, immediately upstream of peuA, which constitutes an operon with the TonB2 system genes. Both the peuRS and peuA-tonB2 operons were found to be upregulated under iron-limiting conditions in a ferric uptake regulator (Fur)-dependent manner. The involvement of peuA and peuRS in enterobactin utilization was analyzed by complementation experiments using deletion mutants. Primer extension analysis indicated that, under iron-limiting conditions, the transcription of peuA was initiated from the +1 site at pH 7.0 and from both the +1 and +39 sites at pH 8.0 in the presence of enterobactin. The +39 transcript was absent from the peuRS deletion mutant. Secondary structure prediction of their 5′-untranslated regions suggested that translation initiation is blocked in the +1 transcript, but not in the +39 transcript. Consistent with this, in vitro translation analysis demonstrated that production of PeuA was determined only by the +39 transcript. These studies establish a novel gene regulation mechanism in which the two-component regulatory system PeuRS enhances expression of the alternative +39 transcript that possesses non-inhibitory structure, allowing the peuA expression to be regulated at the translation stage.

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