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Proc Natl Acad Sci U S A. Sep 2, 2003; 100(18): 10213–10218.
Published online Aug 13, 2003. doi:  10.1073/pnas.1832982100
PMCID: PMC193541
Biochemistry

A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake

Abstract

The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2+ for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg2+-sensitive 45Ca2+ uptake by vesicles of an Escherichia coli mutant that is defective in Ca2+ and Na+ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg2+ transporter, Ca2+ transporter, or channel proteins. Such proteins could provide Mg2+, which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation.

Prokaryotic atp operons encode the cell membrane F-type ATPase (ATP synthase) that couples the energy of an electrochemical H+ gradient (or sometimes Na+), to the synthesis of ATP, from ADP and Pi. In the reverse reaction, the ATPase hydrolyzes ATP concomitant with H+ (or Na+) efflux, thereby contributing to cytoplasmic pH regulation and/or generation of a transmembrane electrochemical gradient under fermentative conditions (1-4). Most atp operons, like that of Escherichia coli, contain the eight structural genes for the ATPase, atpBEFHAGDC, which are preceded by atpI (5). The Escherichia coli atpI is expressed, and its product associates with the membrane, as predicted from its deduced sequence (6-9). Whereas there is no demonstrated effect of AtpI on expression or assembly of the ATPase, an atpI deletion strain of E. coli has been reported to have a reduced growth yield (7). There is no function established for this “mysterious ninth gene” (10) that accounts for such an effect. We report here the finding of another gene, encoding a membrane protein, that is upstream of the atpI gene, and within the atp operon of alkaliphilic Bacillus pseudofirmus OF4. This gene, designated atpZ, was discovered during attempts to introduce site-directed changes in alkaliphile-specific motifs of the membrane-embedded F-ATPase subunits of B. pseudofirmus OF4 (11). A cassette introduced just upstream of the putative atp promoter abolished atp expression. This finding led us to reexamine the location of the atp operon promoter, to the inclusion of atpZ in the extended operon, and then to an exploration of the effects of deleting atpI as well as atpZ. The results suggest a cation translocation function for AtpI and AtpZ, which could be widely relevant to the diverse prokaryotes that possess homologues of one or both of these proteins.

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions. Alkaliphilic Bacillus pseudofirmus OF4 strain 811M, a methionine auxotroph of B. pseudofirmus OF4 (12), was the parent strain for deletion mutants in atpZ, atpI, and atpZI. Routine cloning was performed in Escherichia coli DH5α, except for pG+host4 plasmids (Appligene, Pleasanton, CA), which were cloned in E. coli XL-1 Blue (Promega). The phenotypic effects of atpZ, atpI, and atpZI were examined in a Na+/H+ and Ca2+/H+ antiporter mutant of E. coli, KNabc (ΔnhaAΔnhaBΔchaA) (13), and in the Mg2+ uptake mutant of Salmonella typhimurium MM281 (corA45::MudJ mgtA21::MudJ mgtB10::MudJ) (14). Inducible gene expression was carried out in E. coli C43(DE3) (15). All plasmid inserts and PCR products were completely sequenced at the Utah State Biotechnology Center (Logan, UT), or at the DNA Core at the Mount Sinai School of Medicine. Ampicillin (Ap) was used at 100 μg·ml-1 in E. coli and S. typhimurium, and erythromycin (Em) selection was conducted at 250 μg·ml-1 in E. coli. For B. pseudofirmus OF4811M in complex or defined media, Em was used at 0.6 μg·ml-1, and in protoplast regeneration DM3 plates (16), 0.1-0.3 μg·ml-1 Em was used.

Complementation of the Mg2+-uptake defect of S. typhimurium MM281 was determined by passing 100-μl portions of overnight cultures (grown in LB/Ap medium plus 100 mM MgSO4) that were pelleted and resuspended in LB medium (with no MgSO4 or Ap) into 2 ml of test medium (LB/Ap with different concentrations of MgSO4). The tubes were shaken at 37°C for 6.5 h, and the absorbance at 600 nm was determined. Alkaliphile strains were grown in semidefined (16) or defined media at 30°C, with glucose or malate as the carbon source. The media were buffered with 0.1 M Mops-NaOH at pH 7.5, and 0.1 M Na2CO3/NaHCO3 at pH 10.5. The [Na+] of the pH 7.5 buffer was raised to 0.2 M Na+ by the addition of NaCl. In the defined media, QA, the yeast extract was omitted, and 0.1% glutamine and 0.1% alanine, 1 μg/ml thiamine and biotin, and 10 μg/ml methionine were added. For growth experiments with transformed alkaliphile strains, overnight cultures grown in a rich medium at pH 7.5 (16), containing 50 μg/ml kanamycin (Km), were diluted 200-fold into defined medium to start the experiment. The richer medium overcame deleterious effects of the plasmids on growth. Carryover from this medium resulted in the faster growth of transformed, but not of untransformed strains, in complementation experiments (Fig. 4 B vs. A).

Fig. 4.
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 ...

Transcriptional Start Site Mapping. RNA was isolated as described (17) from late-logarithmic cultures of B. pseudofirmus OF4 grown in semidefined media at pH 10.5. Reverse transcriptase reactions were carried out by using the SUPERSCRIPT preamplification system (GIBCO/BRL, Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. RNA (50 μg) and 50 pmol of a primer that had been end-labeled with [32P]ATP by using T4 polynucleotide kinase were used in each reaction. Two primers were used: 2700R in atpZ, and 3024R in atpI (see Fig. 1B). The latter primer corresponds to that used in the earlier study (17) that mapped the start site to base 2925 (all numbering is based on the deposited sequence in the GenBank database, accession number AF330160). The reverse transcriptase reactions were resolved on an 8 M urea/6% polyacrylamide denaturing gel (12 × 16 cm), along with 32P-labeled 100-bp markers. They were further analyzed on DNA sequencing gels, by using a Bio-Rad apparatus, along with a sequencing ladder generated by using primer 2700R or primer 3024R, and a template consisting of the region from 2389 to 3108, which was obtained by PCR, and cloned into pGEM7Zf(+). RT-PCRs were carried out by annealing primer 3378R, corresponding to the 5′ end of atpB, to 1 μg of RNA. The reverse transcription (RT) reaction (0.5-2 μl of 20 μl) was then used as the template for PCR, by using the HotStarTaq master mix kit (Qiagen, Valencia, CA).

Fig. 1.
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 ...

Construction of Mutants. Mutant constructs were made in pG+host4 carrying a temperature-sensitive replicon, and recombinant plasmids were introduced into the alkaliphile by protoplast transformation. The general strategy for making the mutant construction was the one described by Biswas et al. (18), as applied to alkaliphiles (16, 19, 20).

For construction of in-frame ΔatpZ, ΔatpI, and ΔatpZI mutations, two sets of PCRs for each mutant were performed on wild-type chromosomal DNA to amplify and introduce appropriate restriction sites into segments upstream and downstream of the region to be deleted: nucleotides 2740-2889 for ΔatpZ, nucleotides 2924-3292 for ΔatpI, and nucleotides 2740-3292 for ΔatpZI. Digests of the upstream and downstream pair of PCR products were prepared, and then were ligated to the appropriately digested low-copy Gram-negative vector pMW118 (Nippon Gene, Toyama, Japan). EcoRI digests of each of these plasmids were ligated with EcoRI-digested pG+host4 to produce a chimeric plasmid that was used to create each of the deletions in the alkaliphile. A growth temperature of 40°C was used during manipulations in E. coli, so that maintenance of the plasmid depended on the low-copy replicon of pMW118, thus minimizing potential toxicity of the hydrophobic gene products.

Cloning of atpZ, atpI, and atpZI. A PCR product generated from B. pseudofirmus OF4 chromosomal DNA with designed BamHI and EcoRI sites was cloned into pMW118. The product contained atpZ and 1.3 kb of upstream region. atpZI was cloned in a similar way. For the cloning of atpI, the same strategy was followed, except that the template for the PCR was chromosomal DNA from the ΔatpZ mutant. Expression of atpI in this construct would be under the control of the native promoter of the operon. For overexpression of atpZ, the gene was cloned into the vector pET-3a (Novagen), by using designed NdeI and XhoI sites at the 5′ and 3′ end of atpZ, respectively. Everted membrane vesicles were isolated from IPTG-induced cultures of E. coli C43(DE3) harboring the empty plasmid or pET-3a-atpZ. For the expression of atpZ in B. pseudofirmus OF4, a PCR product (using Vent DNA polymerase, New England Biolabs) that contained atpZ and 1.3 kb of upstream sequence was blunt-end-cloned into the shuttle vector pYH56 (16).

ATPase and Protein Assays. Everted membrane vesicles were prepared from strains grown to mid-logarithmic stage in defined media at pH 7.5 and 10.5 with malate as the carbon source, and were assayed for octyl glucoside-stimulated ATPase activity (21). Protein was measured by the Lowry method (22).

Assays of 45Ca2+ Accumulation and Uptake by E. coli Transformants. E. coli KNabc transformed with pMW118, or the recombinant plasmid with atpZ, atpI, or atpZI were grown overnight in LBK (LB medium containing KCl instead of NaCl) (23) and Ap. The cells were washed once and were resuspended in 50 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane buffer, pH 7.5. Three milliliters of cell suspension (≈1 mg of protein per ml) were shaken in 15-ml conical tubes at 37°C with 100 μM 45CaCl2 (0.1 μCi/mmol; 1 Ci = 37 GBq). At the end of a 1-h incubation, with 1 mM glucose where indicated, 1 ml was removed and vacuum filtered by using 25-mm GF/F glass microfiber filters (Whatman). The air-dried filters were counted by liquid scintillation counting. Experimental values were corrected for binding. Binding was assessed in the presence of 5% butanol, which released internalized solute. For 45Ca2+ uptake assays, right-side-out membrane vesicles were prepared from the same transformants of E. coli KNabc by the method of Kaback (24) in 10 mM Bis-Tris propane, pH 7.5. 45Ca2+ uptake was measured by adding 100 μM 45CaCl2 to a 1-ml suspension containing 0.25 mg vesicle protein. At 5-sec intervals during incubation at 37°C, the vesicle suspension was rapidly filtered through 0.45-μm HAWP filters (Millipore) and washed with 2.5 ml of the Bis-Tris propane buffer. Radioactivity was measured by scintillation counting.

Western Analysis. Everted membrane vesicles were prepared from E. coli transformants grown on LB/Ap medium, and the indicated alkaliphile strains grown on pH 7.5 QA media, with malate and 50 μg/ml kanamycin. The vesicles, denatured in SDS sample buffer, were resolved on mini-SDS/12% polyacrylamide gels (25). After transfer to nitrocellulose membranes, Western blots were developed by a chemiluminescence protocol (Amersham Pharmacia Biosciences, Piscataway, NJ), according to manufacturer's recommendations. A synthetic peptide was prepared that corresponded to the last 15 amino acids of AtpZ, with an additional cysteine at the N terminus. The peptide, conjugated to keyhole limpet hemocyanin, was injected in rabbits to raise polyclonal antisera (Covance Research Products, Denver, PA), from which the IgG fraction was purified (26), and used for these analyses.

Results

Reexamination of the Transcriptional Start and Probable Promoter of the atp Operon. Introduction of a cassette in the chromosomal region just upstream of atpI, in the position within atpZ indicated by the arrowhead in Fig. 1 A, resulted in a malate-minus growth phenotype, and loss of ATPase expression in B. pseudofirmus OF4, even though the promoter had earlier been predicted to be downstream of the cassette-insertion site, just upstream of atpI (17). To reexamine that promoter identification, primer extension was carried out with the same primer, 3024R, that was used in the earlier work (17). This primer sequence is in atpI, as shown in Fig. 1B. The product of the RT reaction was electrophoresed on a polyacrylamide/urea gel that enabled approximate sizing of the product from ≈50 to 500-600 nucleotides, a broader range than would have been detected in the earlier experiments (17). Two products of the reaction were visualized: one that corresponded approximately to the size originally determined, which was 85 nucleotides, and a second product of ≈400 nucleotides. To precisely size the larger product by primer extension, it was desirable to use a primer upstream of 3024R, i.e., 2700R. The resulting smaller reverse transcriptase product was determined to be 67 or 68 nucleotides on a 6% polyacrylamide/urea sequencing gel (data not shown). This result indicated that the transcriptional start site is base 2613 or 2614 of the deposited sequence, which is 293 (292) nucleotides upstream of the predicted start codon of atpI. The smaller product observed with the 3024R primer presumably resulted from either premature termination of the RT reaction, because of RNA secondary structure, or from a processed atp transcript fragment.

Additional support for an atp transcriptional start near base 2613 was obtained by RT-PCR. The reverse transcriptase reaction was carried out by using a primer corresponding to a sequence from the 5′ end of atpB (3378R), which was the first ATPase structural gene in the operon. The RT product was then subjected to PCR, by using two sets of primers. For the first set, 3378R was used with 2619F, whose 5′ end is six nucleotides downstream of the proposed start site. In the second set, 3378R was used with 2568F, whose 3′ end is 25 nucleotides upstream of the start site (Fig. 1B). As shown in Fig. 2A, a product of the right size, 760 base pairs, was observed for the wild-type with the set 1 primers, whereas no product was generated from primer set 2. The set 1 product was absent when the RT enzyme was omitted from the RT reaction, indicating that the observed product was not due to DNA contamination of the RNA preparation. RT-PCR was also carried out on RNA from the ΔatpI mutant, and similar results were obtained, except that the size of the set 1 product was approximately the predicted size of 392 base pairs, taking into account the deleted region.

Fig. 2.
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. ...

The suggested promoter sequence shown in Fig. 1B has the consensus sequence for the Bacillus subtilis -35 element, TTGACA, and a -10 element, TACGAT, which is an infrequently used element in B. subtilis (27). The transcriptional start site is four nucleotides upstream of the predicted start codon of atpZ (National Center for Biotechnology Information ORF finder), a putative ORF upstream of atpI that had not earlier been considered part of the operon. A more likely start for AtpZ is that shown in Fig. 1B, which is based on the presence of a potential ribosome-binding site, and the size of the product detected in Western blots by antibodies raised against a synthetic peptide corresponding to the C-terminal sequence of AtpZ (see Fig. 2B). An alignment of several possible homologues of AtpZ from Bacillus species is shown in Fig. 2C, and additional homologues are found in the database entries for genomes of Clostridium difficile and Desulfitobacterium hafniense.

Detection of AtpZ. Initial assessments of whether atpZ actually produces a protein product were conducted by Western blot analysis of membranes from wild-type B. pseudofirmus OF4 grown in semidefined malate medium at pH 7.5 and 10.5, but no AtpZ was detected. Apparently the antibody could not detect the low levels of AtpZ found in such cells. Therefore, atpZ was cloned into the plasmid pET-3a behind an IPTG-inducible promoter, by using the start codon deduced from this article. As shown in Fig. 2B, AtpZ was expressed at high levels in E. coli C43(DE3) (lane 2); the negative control lacked a reactive product of the same size. atpZ was also expressed in the alkaliphile ΔatpZ mutant transformed with recombinant pYH56 containing the atpZ coding sequence, and 1 kb of upstream sequence. A band was observed that was the same size as that seen in E. coli (lane 5), supporting the conclusion that the actual start site corresponds to that shown in Fig. 1B. No AtpZ signal was detected from either the ΔatpZ, or from the wild-type strains harboring the empty plasmid (lanes 3 and 4).

Phenotype of Deletion Mutants. The ΔatpZ and ΔatpI mutant strains of B. pseudofirmus OF4 grew like the wild-type strain on semidefined media, with either malate or glucose as the carbon source. The specific ATPase activities in membrane vesicles from ΔatpZ and ΔatpI were similar to the wild-type (data not shown), which was consistent with the nonfermentative growth on malate. This result confirmed the nonpolar nature of the deletions. Little growth deficit was observed on defined medium at pH 10.5, ruling out a specific role of atpZ or atpI in alkaliphily. However, the mutant strains showed a pronounced growth deficit when grown in a defined medium at pH 7.5. Recent studies (28-31) suggest a role in Mg2+ transport for several putative 2 TMS proteins. This finding led us to consider the possibility that one or both of the first atp operon genes was involved in Mg2+ acquisition. Because of the poor solubility of Mg2+ in high-pH settings, the alkaliphile is likely to require special Mg2+ acquisition mechanisms for growth at pH 10.5, but might have a lower overall capacity for this important function at pH 7.5. As shown in Fig. 3A, all three deletion strains exhibited a greatly increased requirement for Mg2+ in malate-containing QA-Mops media at pH 7.5. Although not shown, a growth deficit was also observed when glucose was the primary carbon source instead of malate. As shown in Fig. 3B, transformation of the ΔatpZ strain with a plasmid-bearing atpZ resulted in nearly wild-type levels of growth over a broad range of MgSO4 concentrations. There were differences in pregrowth conditions and Mg2+ carryover (see Materials and Methods), which were necessitated by effects of plasmids on growth. This finding led to the apparent ability of the wild-type transformant and complemented mutant to grow at lower Mg2+ concentrations than the untransformed wild-type shown in Fig. 3A.

Fig. 3.
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 ...

Effects of atpZ, atpI, or atpZI Expression on the Phenotypes of Heterologous Transport Mutants. S. typhimurium MM281 is deficient in Mg2+ uptake by virtue of disruptions in the genes for three different transport systems, CorA, MgtA, and MgtB (14); complementation of this strain has proved useful in identifying and developing information about new, heterologous Mg2+ transporters (32, 33). As shown in Fig. 4, low-copy plasmids expressing atpZ, atpI, or atpZI complemented the growth phenotype of S. typhimurium MM281. The AtpZI combination supported significantly greater growth of the mutant on contaminating and low added concentrations of Mg2+ than did either AtpI or AtpZ alone.

Because of the lack of availability of radioactive isotopes of Mg2+, assays of Mg2+ transport are usually performed as the Mg2+-dependent inhibition of transport of other divalent cations. This method was used to characterize the MgtE Mg2+ transporter of B. pseudofirmus OF4 (32), which exhibited Mg2+ -inhibitable uptake of 57Co2+ in S. typhimurium MM281 transformants. However, no evidence for AtpZ-, AtpI-, or AtpZI-dependent Co2+ uptake was obtained, although such activity was conferred by the positive control plasmid in which the alkaliphile mgtE gene was cloned (data not shown). Because crossinhibition occurs between Mg2+ and Ca2+ with some transporters (34), the possibility that AtpZ and AtpI might confer Mg2+-inhibitable 45Ca2+ accumulation was assessed in E. coli KNabc, which is deficient in Ca2+ efflux (13). As shown in Table 1 for a 1-h accumulation experiment, the control transformant (cells harboring the empty vector) exhibited little Mg2+-inhibitable 45Ca2+ accumulation. The transformant expressing atpI exhibited a significant increase in activity only when glucose was also added, but not as much as the transformants expressing atpZ and atpZI, both of which exhibited enhanced accumulation even in the absence of glucose. In followup experiments, right-side-out membrane vesicles from the same transformants were assayed for 45Ca2+ uptake in the absence of any intravesicular solute. Initial experiments showed that uptake was extremely rapid, such that the highest uptake was observed at the shortest time points that could be taken (5 sec). Subsequent time points indicated a loss of 45Ca2+ from the vesicles, as would be expected under unenergized conditions. Addition of an electron donor, D-lactate, did not cause more sustained uptake, but instead inhibited uptake overall. This result was presumed to reflect energization of the remaining Ca2+ efflux pathway(s), which were expected to be present in the chaA deletion background of E. coli KNabc (35). Subsequent vesicle experiments were conducted in the absence of added electron donors at 5-sec points. As shown in Table 2, AtpZ-, AtpI- and AtpZI-dependent 45Ca2+ uptake was observed. This uptake, after subtracting the background binding and uptake in the control transformant, was completely inhibited by addition of either 1 mM MgCl2 or 200 μM of the general cation channel inhibitor GdCl3 (36) to the extravesicular space, just before the addition of 45Ca2+ to start the reaction.

Table 1.
AtpZ-, AtpI-, and AtpZI-enhanced 45Ca2+ accumulation by transformants of Escherichia coli KNabc
Table 2.
AtpZ-, AtpI-, and AtpZI-dependent 45Ca2+ uptake in right-side-out membrane vesicles of Escherichia coli KNabc

Discussion

The results indicate that a tenth gene, designated atpZ, is part of the B. pseudofirmus OF4 atp operon. Because atpZ homologues are found in a comparable position in atp operons of several nonalkaliphilic Bacillus species, as well as C. difficile and D. hafniense, the indications that AtpZ and AtpI have a role in divalent cation translocation may have broad applicability. In addition to the phenotypes of the atpZ, atpI, and atpZI mutants of the alkaliphile, the enhancement of Mg2+ acquisition in the Mg2+ uptake-deficient mutant strain, S. typhimurium MM281, and of Mg2+-inhibitable 45Ca2+ uptake in E. coli KNabc, are further evidence that AtpZ and AtpI support inward Mg2+ transport. In the whole-cell experiments in the heterologous systems, the relative effectiveness of the alkaliphile genes was atpZI > atpZ > atpI in enhancing both growth of the Mg2+ uptake mutant of S. typhimurium and the Mg2+-sensitive accumulation of Ca2+ in E. coli. Only in the vesicle uptake experiments did atpI seem to confer higher activity than the other genes, with atpZI now conferring the lowest. The likely explanation for this deviation from the whole-cell experiments is that the measurements in vesicles were too slow to capture the peak points of the fastest transporters. The faster the initial transport, the further the 5-sec assay point was down the slope of the subsequent loss of internalized 45Ca2+. Such a loss would reflect an expected reequilibration of the Ca2+ in the absence of an imposed potential.

Different properties conferred by AtpZ, AtpI, and AtpZI suggest that these molecules constitute the translocation pathway themselves, rather than acting through an indirect mechanism, by affecting some independent transporter in the cells. AtpZ and AtpI would be likely to function as homooligomers and, perhaps, AtpZ and AtpI can also form heterooligomers. Homooligomeric assemblies of CorA (37), and the 2 TMS ALR1 and Mrs2 transporters of yeast (30, 38), have been reported, and may function as channels. Although no conclusion can yet be drawn about the transport mechanism of AtpZ and AtpI, the properties observed thus far raise the possibility of a cation channel.

The unusual and diverse nature of the proteins to which a Mg2+ transport function has been ascribed is notable (39). The current findings add to that diversity. Neither AtpZ nor AtpI has the conserved motif of the CorA family of Mg2+ transporters (39) and the 2-TMS Mrs2-type transporters (28-31), nor do they show sequence similarity to other putative Mg2+ transporters such as MgtE, which was first isolated from an alkaliphilic B. pseudofirmus DNA library (32). The CorA family possesses a putative magnesium transporter signature, Y/FGMNF, which is critical for activity (30, 31, 37). There is no evident counterpart in AtpZ or AtpI. We note, however, that the AtpZ proteins of two alkaliphilic Bacillus species possess three contiguous acidic residues at their C terminus. A discontinuous DDE motif has been shown to function in divalent metal binding (40), and contiguous acidic residues have been associated with Ca2+ binding and translocating proteins (34, 41).

Coexpression of a cation translocation pathway with the F-ATPase from a single operon is a functionally satisfying combination. The nucleotide substrates for the enzyme are usually complexed with Mg2+, and the cation has been suggested to play a role in establishing the asymmetry of the catalytic sites (42). In addition, when the ATPase is functioning hydrolytically to lower the cytoplasmic pH (43), a capacity of AtpZI, AtpZ, or AtpI to support cation flux could allow charge-compensating, inward movement of cations that would maximize the proton efflux. AtpI gene products from different organisms do not exhibit strong sequence conservation, and are less conserved than are deduced AtpZ sequences. Perhaps inactivation, loss, or modifications in AtpZ and AtpI function will ultimately be found to relate to different physiological needs of diverse organisms.

It is expected that extremely alkaliphilic bacteria require well adapted mechanisms for acquiring needed concentrations of ions such as Fe2+ and Mg2+, when growing at pH values as high as 10.5. The transporter complement encoded in the genome of alkaliphilic Bacillus halodurans C-125 is predicted to include four distinct MgtE-like proteins (www.membranetransport.org). The finding that the phenotype of the atpZI deletion is more pronounced at near-neutral pH than at pH 10.5, suggests that the major Mg2+ uptake systems of B. pseudofirmus OF4 are better adapted for function at pH 10.5 than at pH 7.5. Although a B. pseudofirmus OF4 MgtE conferred significant Mg2+ transport capacity at pH 7.0 in a heterologous system (32), pH optima of MgtE proteins may be higher in the native alkaliphile membrane. Several examples of such hard-wired adaptations to extreme alkaliphily have been found to disadvantage B. pseudofirmus OF4 at near-neutral pH (20).

Acknowledgments

This work was supported by National Institute of General Medical Sciences Grant GM28454 (to T.A.K.).

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Ap, ampicillin; RT, reverse transcription.

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