Bacterial Strains, Plasmids, and Growth Conditions. The bacterial strains and plasmids used in this study are listed in Table 4.

Cloning ncbA into Mammalian Expression Vector. To clone ncbA into the mammalian expression vector pTracer-CMV3 (Invitrogen), PCR was carried out with primer sets SC-CMV-K1 and SC-CMV-ER1 (Table 5). SC-CMV-K1 contains the artificial Kozak sequence for expression in CHO-K1 cells. The PCR product was digested with EcoRI and KpnI. The purified fragment was ligated into EcoRI- and KpnI-double digested pTracer-CMV, yielding pCMV-SC7.

Sequencing of Bacillus pseudofirmus OF4 Na_VBP-Encoding Gene ncbA. PCR was carried out by using primer set BH-SC1 and BH-SC2 (Table 5), which were designed from a conserved region of the NaChBac-encoding genes of Bacillus halodurans C-125 and Magnetococcus sp. MC-1. Genomic DNA from B. pseudofirmus OF4 was used as the template. A PCR product of » 450 bp was amplified and was ligated into SmaI-digested pMW118. The insert was sequenced in both directions. Two inverse PCRs were then used to amplify the rest of the region containing the Na_VBP-encoding gene, designated ncbA. The first two reactions used the primer set OF4-SC4 and OF4-SC6 and primer set OF4-SC1 and OF4-SC3 to amplify from SauIIIAI-cleaved chromosomal DNA, which had been religated. The resulting 1.3- and 0.7-kb fragments amplified by these respective reactions were sequenced. To obtain sequence further upstream of ncbA to facilitate gene disruption, an additional inverse PCR was conducted by using primer set OF4-SC9 and OF4-SC14 and mspI-cleaved chromosomal DNA that had been religated. After sequencing, a total 2,542 bp of a region containing the apparently monocistronic ncbA gene was deposited in GenBank (accession no. AY376071).

Disruption of the ncbA Gene in B. pseudofirmus OF4 and Restoration of the Gene. Two independent PCRs were performed on a wild-type DNA template with the primer sets OF4-SC14 and OF4-SC-MR-Sm1 plus OF4-SC12 and OF4-SC-MR-Sm1. The two purified PCR products were used as templates for a second PCR with primers OF4-SC14 and OF4-SC12. The purified product of this reaction was cloned into SmaI-digested pMW118, yielding pMWD ncbA::Spc^R. The plasmid was digested with HindIII and KpnI, and then the D ncbA::Spc^R fragment was cloned into HindIII- and KpnI-digested pG⁺host4, yielding pGD ncbA::Spc^R. The plasmid was used to transform B. pseudofirmus OF4-811M protoplasts and the protocol for isolation of single and then double crossover candidates was followed as described (1). The deletion was confirmed by PCR and Southern analyses. The D ncbA mutant was designated strain SC34 and an up-motile version selected by repeated transfers from the motile edge of SC34 colonies on soft agar motility plates (see below) was designated SC34-M.

Restoration of a functional ncbA gene to B. pseudofirmus OF4 ncbA channel mutants, under control of its own promoter, was achieved either by introduction of the gene on a low copy plasmid or by its restoration to the chromosome. For construction of a plasmid carrying intact ncbA, PCR was performed on wild-type DNA with primer set OF4-SC14 and OF4-SC12. The 2.53-kb purified PCR product was digested with EcoRI and XbaI, and the 1.52-kb DNA fragment containing ncbA was then cloned into EcoRI- and XbaI-digested pYM1, yielding pSC. A recombinant plasmid carrying ncbA ₁₉₁LESWAS_{196® 191}LDDWAD₁₉₆, which functions as a Ca²⁺ channel, two independent PCRs were performed on wild-type DNA with the primer sets OF4-SC15 and Sea-R and OF4-SC12 and Sca-F. The two purified PCR products were used as templates for a second PCR with primers OF4-SC15 and OF4-SC12. The purified product of this reaction was cloned into SmaI-digested pGEM7zf(+), yielding pGEMD ncbA. The plasmid was digested with EcoRI and XbaI, and the resulting 1.52-kb DNA fragment containing ncbA-Ca was cloned into EcoRI- and XbaI-digested pYM1, yielding pSC-Ca. The inserts of pSC and pSC-Ca were completely sequenced.

Wild-type ncbA was restored to the chromosome of ncbA mutants SC34 and SC34-M under its own promoter, resulting respectively in strains SC34-R and -MR, by replacement of the region containing the D ncbA::Spc^R with nucleotides 477-2,267 of the deposited sequence. A plasmid containing this region was constructed with a single silent mutation in E244 of the coding sequence that introduced a SacI site. The two PCRs used were performed on wild-type DNA: (i) using primer sets OF4SC-KF and OF4SC-SR, producing a 1.2-kb fragment corresponding to the 5' part of the region to be cloned, and containing a 5' KpnI site and 3' SacI site; and (ii) OF4SC-SF and OF4SC-BR, producing a 0.7-kb fragment corresponding to the 3' part if the region and containing a 5' SacI site and 3' BamHI site. After digestion of the purified fragments with the appropriate restriction enzymes, the two PCR products were ligated at the SacI sites and inserted into KpnI- and BamHI-digested pGEM3-zf(+), producing pGSCR11. The insert of this plasmid was completely sequenced and verified and was then released and cloned into KpnI- and BamHI-digested pG⁺Host4, producing pG⁺Host4-SCR11. Transformation of protoplasts of SC34 and SC34-M with pG⁺Host4-SCR11 and subsequent selection of single and double crossovers that completed the ncbA gene restoration were carried out as described (14), using the restoration of Sp-sensitivity as a screen. The sequence of the ncbA locus of the restored SC34R and SC34-MR strains was confirmed by sequencing.

1. Ito, M., Guffanti, A. A., Zemsky, J., Ivey, D. M. & Krulwich, T. A. (1997) J. Bacteriol. 179, 3851-3857.