Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a-and c-subunits of ATP synthase. Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk ⌬p that are characteristic of alkaliphiles. The aLys 180 and aGly 212 residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pHdependent restriction of proton flux through the ATP synthase to and from the bulk phase. The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH. The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for nonfermentative growth and OXPHOS at high pH.Aerobic organisms maximize catabolic energy conservation by carrying out OXPHOS.1 Energy stored in NADH or FADH 2 during catabolism is used to produce a bulk ⌬p, acid and positive out, across the mitochondrial or bacterial cell membrane by respiration-dependent proton extrusion. Inward proton flux through the proton-coupled ATP synthase, energized by the ⌬p, then leads to ATP production (1-4). Among the important unresolved issues is whether protons are always captured from the bulk medium as in Mitchell's (1) chemiosmotic model or whether they can be sequestered as they emerge from the respiratory chain (2, 5-9). Robust H ϩ -coupled OXPHOS by extremely alkaliphilic Bacillus strains growing on non-fermentable carbon sources at external pH values Ն10.5 poses one of the most striking challenges to the strictly bulk energization model (10 -13). At such pH values, maintenance of a cytoplasmic pH that is much lower than the external pH, i.e. a ⌬pH that is acid in, lowers the total chemiosmotic driving force, and yet OXPHOS proceeds optimally (10, 13).A variety of solutions to the energetic conundrum of alkaliphile OXPHOS have been proposed (for reviews, see Refs. 10 and 14). We have hypothesized that special properties of the alkaliphile ATP synthase are needed for OXPHOS at high pH that depend upon the presence of specific amino acid residues or stretches of amino acids in functionally important regions of the membrane-embedded a-and c-subunits of the enzyme (10, 15). Other hypotheses have suggested global features of the alkaliphile membrane, membrane surface, or cell wall-associated polymers (see Refs. 10 and 14) that contribute to the resolution of the alkaliphile energetic problem. It has recently been proposed that a single global feature of this kind, i.e. a sufficiently low pH near the membrane surface, can completely account for alkaliphile OXPHOS (14), ...

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 Mg 2؉ 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 Mg 2؉ uptake. atpZ and atpI, separately and together, increased the Mg 2؉ -sensitive 45 Ca 2؉ uptake by vesicles of an Escherichia coli mutant that is defective in Ca 2؉ and Na ؉ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg 2؉ transporter, Ca 2؉ transporter, or channel proteins. Such proteins could provide Mg 2؉ , 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. P rokaryotic atp operons encode the cell membrane F-type ATPase (ATP synthase) that couples the energy of an electrochemical H ϩ gradient (or sometimes Na ϩ ), to the synthesis of ATP, from ADP and P i . In the reverse reaction, the ATPase hydrolyzes ATP concomitant with H ϩ (or Na ϩ ) efflux, thereby contributing to cytoplasmic pH regulation and͞or 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 alkaliphilespecific 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 ...