Complementation of the Mg²⁺ phenotype of S. typhimurium MM281 by atpZ, atpI, and atpZI. S. typhimurium MM281 transformants were grown for 6.5 h in LB/Ap medium, supplemented with the indicated concentrations of MgSO₄ as described in Materials and Methods. The averages and SD of five independent growth experiments, each done in duplicate, are shown.

Demonstration of a promoter upstream of atpZ and of an AtpZ protein. (A) RT-PCR was carried out by using primer 3378R for the RT reaction. The reaction was subjected to PCR by using 3378R, with either primer 2568F (set 1) or 2619F (set 2; see Fig. 1B for precise locations of these primers). The reverse transcriptase enzyme was left out (-) or included (+) in the RT reaction as indicated. The control lanes show the product generated from PCR with the appropriate primer pairs, with chromosomal DNA as the template. The standards are labeled in base pairs. Below the gel is a diagram showing the location of the primers, with the reverse primer in atpB, and the forward primers either just after the proposed start site (set 1) or before the start site (set 2), as indicated by the numbered arrows. (B) Western blot of atpZ expression in E. coli and B. pseudofirmus OF4. In E. coli, everted membrane vesicles were isolated from induced cultures of strains with the empty vector, or with the cloned atpZ. In B. pseudofirmus OF4, everted membrane vesicles were prepared from the wild-type carrying pYH56 (empty vector), or ΔatpZ carrying the empty vector, or with cloned atpZ. Lane 1, E. coli with empty vector (0.25 μg); lane 2, E. coli with atpZ (0.25 μg); lane 3, B. pseudofirmus OF4 wild-type with empty vector (100 μg); lane 4, ΔatpZ with empty vector (100 μg); lane 5, ΔatpZ with atpZ (100 μg). The AtpZ band is marked on the left, and the other bands represent nonspecific reactions. Molecular weight markers are indicated on the left. (C) Alignment of AtpZ homologues. Boxed residues are charged amino acids shared by three of four of the candidate proteins. Other residues shared by three or four proteins are shaded. The two predicted transmembrane helices (TMS) are overlined. Bps, B. pseudofirmus OF4; Bh, Bacillus halodurans C-125; Bm, Bacillus megaterium; Ba, Bacillus anthracis. According to the National Center for Biotechnology Information ORF finder, the B. anthracis candidate has an additional 10 residues at the N terminus for which no strong ribosome binding site (RBS) can be identified; there is a good RBS for the leucine codon that lines up with the first amino acid of B. pseudofirmus OF4 AtpZ. The B. megaterium candidate shown is an incomplete ORF with at least 14 more amino acids at the N terminus. In this alignment, these extended segments have not been included.

atp operon and upstream region with proposed promoter elements of B. pseudofirmus OF4. (A) atp operon structure showing an upstream gene, vpr (an incomplete ORF), and the atpBEFHAGDC structural genes, as well as the Z and I genes that are proposed to be part of the atp transcriptional unit. The solid arrow indicates the proposed promoter from this article, the dotted arrow is the previously described promoter (17), and the upward arrowhead indicates the site of insertion of a spectinomycin-resistance cassette in a preliminary experiment that led to a malate-negative phenotype, and hence to this article. (Upper) The bar is marked in kb. Below the operon is an expansion of the region from ≈2 to 5 kb that shows the regions deleted in the mutants constructed for this article. (B) The sequence from bases 2541-3052. The primers used in the transcriptional start mapping (3024R and 2700R) are shown. The start site from the earlier study is shown with a downward dotted arrow (base 2925), and the new start site is indicated with a downward solid arrow (base 2613). The proposed -35 promoter and -10 promoter elements are bold. The primers used in conjunction with primer 3378R in the RT-PCR of Fig. 2B are also shown (2568F and 2619F). The one-letter amino acid designations are shown above (atpZ) or below (atpI) their codons.

Effect of [MgSO₄] on the growth of deletion strains of B. pseudofirmus OF4. (A) The MgSO₄ concentration was varied for overnight growth in the pH 7.5 QA-Mops-defined media described in Materials and Methods. (B) Growth of the indicated transformants of wild-type B. pseudofirmus OF4 and the atpZ mutant was monitored as in A. The enhanced response of the wild-type with the control vector to low [MgSO₄] was due to a carryover of Mg²⁺ from the richer medium used for the pregrowth of transformed alkaliphile strains, as described in Materials and Methods. The results are from two independent experiments, each carried out in duplicate.