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Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary [Na.sup.+] ([Li.sup.+])/[H.sup.+] antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited [Na.sup.+]([Li.sup.+])/[H.sup.+] antiport. A second paralogue of S. aureus (Mnh2) did not. [K.sup.+], [Ca.sup.2+], and [Mg.sup.2+] did not support significant antiport by any of the test antiporters. All three [Na.sup.+] ([Li.sup.+])/[H.sup.+] Mrp antiporters had alkaline pH optima and apparent [K.sub.m] values for [Na.sup.+] that are among the lowest reported for bacterial [Na.sup.+]/[H.sup.+] antiporters. Using a fluorescent probe of the transmembrane electrical potential ([DELTA][PSI]), Mrp [Na.sup.+]/[H.sup.+] antiport was shown to be [DELTA][PSI] consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher [DELTA][PSI] levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, [DELTA][PSI]-consuming antiporters can elicit increased capacity for [DELTA][PSI] generation in a bacterial host.

In extreme alkaliphiles, [Na.sup.+]/[H.sup.+] antiporters play a central role in the [Na.sup.+] cycle that supports pH homeostasis, [Na.sup.+] resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a [Na.sup.+]([Li.sup.+])/[H.sup.+] antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and [Na.sup.+] concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 ([DELTA]nhaA [DeLTA]nhaB), the pH optimum for NhaD-dependent [Na.sup.+]([Li.sup.+])/[H.sup.+] antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH [less than or equal to]8.5. NhaD supported low [Na.sup.+]/[H.sup.+] antiport activity at pH 9.5 that was detectable over a range of [Na.sup.+] concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the [Li.sup.+] sensitivity of the triple mutant E. coli strain KNabc ([DELTA]nhaA [DELTA]nhaB [DELTA]chaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine ([GlyL.sup.327])[right arrow]serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at [Na.sup.+] concentrations up to 200 mM but lower activity at 400 to 600 mM [Na.sup.+], with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.

Mrp catalyzes secondary [Na.sup.+]/[H.sup.+] antiport and was hypothesized to have an additional primary energization mode. Mrp-dependent complementation of nonfermentative growth of an Escherichia coli respiratory mutant supported this hypothesis but is shown here to be related to increased expression of host malate:quinone oxidoreductase, not to catalytic activity of Mrp.

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 atpl 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 atpl. 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, atpl, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of [Mg.sup.2+] for growth at pH 7.5. Either atpZ, atpl, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in [Mg.sup.2+] uptake, atpZ and atpl, separately and together, increased the [Mg.sup.2+]-sensitive [sup.45][Ca.sup.2+] uptake by vesicles of an Escherichia coli mutant that is defective in [Ca.sup.2+] and [Na.sup.+] efflux. We hypothesize that AtpZ and Atpl, as homooligomers, and perhaps as heterooligomers, are [Mg.sup.2+] transporter, [Ca.sup.2+] transporter, or channel proteins. Such proteins could provide [Mg.sup.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.

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both [Na.sup.+]- and [K.sup.+]-dependent pH homeostasis and in [Na.sup.+] resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)-hexahistidine proteoliposomes exhibit high activities of tetracycline-cobalt/[H.sup.+], [Na.sup.+]/[H.sup.+], and [K.sup.+]/[H.sup.+] antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline-cobalt/[H.sup.+] and [Na.sup.+]/[H.sup.+] antiports is presented. [K.sup.+] and [Li.sup.+] inhibit [Na.sup.+] uptake, but there is little cross-inhibition between [Na.sup.+] and tetracycline-cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.

Use of primer extension and Northern and Western blot methods in the biochemical and molecular analysis of the F1F0-ATP synthase of Bacillus firmus OF4 reveals the presence of excessive levels of membrane F1F0-ATPase and atp gene transcripts. The excessive levels appear when the bacterial strains are grown at a pH of 7.5 and 10.5, with the enzyme from the atp operon exhibiting similar subunit and stoichiometric properties as the c-subunit/holoenzyme. The transcriptional start site can be detected in the RNAs from the strains grown at a pH of 7.5 and 10.5.

The energy-linked activities of F1F0 ATP synthases from wild type Bacillus subtilis do not differ from uncoupler-resistant strains. There is no change in the HT/ATP stoichiometry in B. subtilis, mediated by the mutant lipids. This leads to protonophore carbon cyanide m-chlorophenylhydrazone (CCCP) inhibition of energy-linked functions of the enzyme. The CCCP resistance results from an interaction that warrants a suitable electron-transfer complex and its natural electron donor than ATP synthase alone.

The experimental details and results of differential scanning calorimetry and saturation transfer electron paramagnetic resonance studies with cytochrome ca[a.sub.3] and ATP synthase embedded in phospholipid vesicle is reported. The result demonstrate a physical interaction between the cytochrome ca[a.sub.3] and [F.sub.1][F.sub.0]-ATP synthase from Bacillis pseudofirmus O[F.sub.4] in a reconstituted system, which suggest that sequestered proton transfers during alkaliphile oxidative phosphorylation at high pH.

Focuses on a study that investigated the bonding of silicon to silicon by solid-phase epitaxy. Discussion of a silicon-to-silicon bonds; Techniques in which silicon-to-silicon bonds can be made; Information on solid-phase epitaxy.