BamHI-BamHI fragments hybridized check details with the probe D were ligated into the same site of pUC19, and the resulting plasmids transformed in E. coli H1717. Positive clones were selected by colony blot hybridization with the same probe, and one of the recombinant plasmids, termed pVMB1, was extracted (Fig. 2). The nucleotide sequence of the 5121-bp fragment from pVMB1 was determined by primer
walking. Two entire ORF located divergently were identified; these were named mhuAB (V. mimicus heme utilization). The two other partial genes (orf1 and orf4) were not relevant to iron acquisition or iron-regulated gene expression. As shown in Figure 3a, each of the mhuA and mhuB genes possesses the predicted RBS and promoter elements (−35 and −10). Potential Fur boxes sharing 15/19 and 12/19 base matches with the E. coli consensus Fur box (24) are located in the upstream regions of mhuA and mhuB, respectively, overlapping with the −35 elements. Although the normal initiation codon (AUG) was missing at the predicted start position of mhuB transcript, an alternative initiation codon, UUG (25), was found in seven bases downstream of the RBS. V. mimicus has been reported to produce 77-kDa (IutA) and 80-kDa IROMP, whose N-terminal amino acid sequences have been determined to be EEQTLFDEMV and EQQSQFNEVV,
respectively (9, 10). An amino acid sequence compatible with the latter Selleckchem Nutlin 3a was found in the N-terminal portion of the deduced amino acid sequence of MhuA. To gain better separation of the IROMP, SDS-PAGE was carried out under the conditions shown in Figure 3b. As a result, the IROMP were separated into five protein bands, and the N-terminal amino acid sequences of the smallest band and a second large-molecular weight band corresponded with those of 77-kDa IutA (Fig. 3b, lane 1, open arrowhead) and 80-kDa MhuA (Fig. 3b, lane 1, solid arrowhead), respectively. The functions of the three other IROMP are at present unknown.
The protein product of mhuA shared homology with the heme/hemoglobin receptors of Vibrio species (11, 12, 26), ranging from 33% to 62% identity and from 52% to 80% similarity (Table 2). Selected proteins were aligned with MhuA (Fig. HAS1 4). A probable TonB box (28NEVVVTA34) present in MhuA, which is thought to interact physically with TonB protein, was similar in amino acid sequence to those in the heme/hemoglobin receptors of other Vibrio species (1, 27). Furthermore, MhuA possesses FRTP and NPNL amino acid boxes characteristic of the bacterial heme/hemoglobin receptors (28). However, the conserved histidine residue between FRTP and NPNL boxes (corresponding to His-461 in the Yersinia enterocolitica HemR, a receptor for heme/heme-containing proteins) (28) was not found in MhuA.