In order to complement

the null mhuA allele, the full-length mhuA gene was amplified by PCR with the primer pair A1 and A4, and the resulting amplicon was ligated into the XbaI site of a broad-host-range plasmid, pRK415. The resulting hybrid plasmid, pRK415-mhuA, was transformed into E. coliβ2155, and crossed with the ΔiucDΔmhuA strain. Tetracycline-resistant colonies were selected, and plasmid transfer confirmed by PCR and restriction enzyme analysis of the extracted plasmid. Two kinds of DNA fragments containing upstream regions of the mhuA gene were amplified by PCR with primer pairs Xba-I-P (5′-ctagtctagaACGGAACCGCAGACATGGTGTTG-3′), or Xba-I-P2 (5′-ctagtctagaTTTGATAACTCAAGGAGCTAGGAGC-3′) and Sph-I-P (5′-acatgcatgcTACAACAATTGCACTAGCGAGC-3′) Selleckchem Metformin Proteasome assay (the small italic letter sequences in Xba-I-P and Xba-I-P2, and Sph-I-P are polylinkers with XbaI and SphI sites, respectively). PCR fragments digested with

SphI-XbaI were ligated into the same enzyme sites of pAA224 (17), and the resulting promoter-lacZ reporter plasmids, termed pVMB2 and pVMB3, were then individually transformed into E. coli WAM131 (15). The degree of expression of the promoter-fused lacZ gene in E. coli WAM131 cells harboring the respective reporter plasmids was estimated by β-galactosidase activity measured by the method of Miller (22), after they had been grown at 37oC in +Fe or −Fe (with DPD) medium for 20 hr and 12 hr, respectively. Nucleotide sequence data for the V. mimicus mhuA and mhuB genes have been deposited in the EMBL/GenBank/DDBJ databases under the accession number AB048382. In order to eliminate background growth resulting from aerobactin-mediated iron uptake in the −Fe medium, a ΔiucD mutant incapable of synthesizing aerobactin was first constructed from V. mimicus 7PT and then Amino acid used to assess whether this species can utilize heme and hemoglobin as iron sources. The deletion

in the iucD gene was confirmed by PCR analysis of the ΔiucD chromosomal DNA with the primer pair D5 and D6, which revealed an amplicon of the expected size (ca. 2.8-kb) (Fig. 1a). In the growth assay, the ΔiucD strain showed no growth in the −Fe medium, while the addition of hemin at 10 μM or hemoglobin at 2.5 μM to the same medium restored growth to a degree comparable to that found in the +Fe medium (Fig. 1b) These data clearly indicate that V. mimicus can utilize heme and hemoglobin as iron sources. The ORF in the 5121-bp cloned region together with relevant inserts in the constructed plasmids are shown in Figure 2. Fur-box-containing gene fragments from V. mimicus 7PT (10, 21) were isolated through application of FURTA system (14). One of the FURTA-positive clones, termed pVM3, possessed two partial ORF, whose deduced amino acid sequences were significantly homologous to the V. cholerae HutA and VCA0575 proteins involved in heme and hemoglobin utilization (11, 23). To clone surrounding regions of these partial ORF, V.