FIG. 3.
Effects of substituting segments of ArcB with MalF on the expressions of Φ(cydA′-lacZ) and Φ(lldP′-lacZ). To construct Φ(arcB1-22-malF17-35-arcB42-778), PCR was performed with pIBW as a template and BPH-N and BMF-22 as primers. The purified PCR product and BMF-35 were used as primers for the second PCR with pDHB32 (6) as a template. The PCR product and B3NRU were used as primers for a third PCR with the pABW as a template. The product was digested with NdeI and NruI and cloned between the corresponding sites of the pIBW, resulting in pIBM1. To construct Φ(arcB1-57-malF40-58- arcB78-778), PCR was performed with pIBW as a template and BPH-N and BMF-40 as primers. The purified PCR product and BMF-58 were used as primers for the second PCR with pDHB32 as a template. The PCR product and B3NRU were used as primers for a third PCR with the pABW as a template. The product was digested with NdeI and NruI and cloned between the corresponding sites of the pIBW, resulting in pIBM2. To construct a Φ(arcB1-22-malF17-39-arcB58-778), PCR was performed with pIBW as a template and BPH-N and BMF-22 as primers. The purified PCR product and BMF-39 were used as primers for the second PCR with pDHB32 as a template. The PCR product and B3NRU were used as primers for a third PCR with pABW as a template. The product was digested with NdeI and NruI and cloned between the corresponding sites of the pIBW, resulting in pIBM3. Plasmids pIBM1, pIBM2, and pIBM3 were used to integrate the modified arcB sequences into the chromosome of the reporter-bearing strains by the gene replacement techniques, as described in the legend of Fig. 2. For the β-galactosidase activity assay, the Φ(cydA′-lacZ)-bearing strains were cultured in buffered Luria-Bertani broth containing 0.1 M MOPS (morpholinepropanesulfonic acid) (pH 7.4) and 20 mM d-xylose. For the growth of Φ(lldP′-lacZ)-bearing strains, the above medium was supplemented with 20 mM l-lactate as an inducer (9). The data are averages of four experiments and the standard deviations are indicated. The different alleles of arcB are shown as follows: 1, arcB+; 2, Φ(arcB1-22-malF17-35-arcB42-778); 3, Φ(arcB1-57-malF40-58-arcB78-778); 4, Φ(arcB1-22-malF17-39-arcB58-778); 5, ΔarcB. The topology of the chimeric proteins is illustrated at the bottom: solid segments represent ArcB sequences, and hatched segments represent MalF sequences. Solid bars, aerobically grown cells; hatched bars, anaerobically grown cells.