Alkaliphilic Bacillus Pseudofirmus OF4 Research Articles

Mutational loss of carotenoids in alkaliphilic Bacillus pseudofirmus OF4 results in sensitivity to oxidative stress and growth at high pH.

Alkaliphilic Bacillus pseudofirmus OF4, which has a broad pH growth range of 7.5 to above 10.5, is yellow-pigmented due to carotenoids. Carotenoids contribute to membrane rigidity and can alleviate cellular oxidative stress. This study was undertaken to gain insight into the roles carotenoids play in alkaliphile physiology. Carotenoid content was high in stationary phase and in cells grown nonfermentatively at pH 10.5 A colourless mutant was isolated by the in-frame deletion of a key carotenogenic gene, crtM. In cells grown to stationary phase in a pH 10.5 medium with a suboptimal concentration of Na+, the ∆crtM strain exhibited lower resistance to paraquat and hydrogen peroxide. Preincubation of the mutant in a nutrient-free pH 10.5 buffer revealed a pronounced sensitivity to hydrogen peroxide in growth at pH 7.5. In growth curves in media with optimal or suboptimal nutrient concentrations conducted at 37°, the mutant grew identically to the wild-type at pH 7.5 but its lag time was longer than the wild-type at pH 10.5 and growth was slower when the carbon source, malate, was limiting. When the temperature of the growth curves was lowered to 25°, the mutant no longer had a pH 10.5 phenotype, implicating the effect of carotenoids on membrane rigidity for the pH 10.5 growth phenotype. These results suggest that carotenoids in B. pseudofirmus OF4 play a role in managing oxidative stress when cells are adapting to other stressful conditions such as nutrient limitation while also helping to maintain membrane fluidity/rigidity balance for membrane-linked functions.

A Hydrophobic Small Protein, BpOF4_01690, Is Critical for Alkaliphily of Alkaliphilic Bacillus pseudofirmus OF4.

A monocistronic small protein, BpOF4_01690, was annotated in alkaliphilic Bacillus pseudofirmus OF4. It comprises 59 amino acids and is hydrophobic. Importantly, homologs of this protein were identified only in alkaliphiles. In this study, a mutant with a BpOF4_01690 gene deletion (designated Δ01690) exhibited weaker growth than that of the wild type in both malate-based defined and glucose-based defined media under low-sodium conditions at pH 10.5. Additionally, the enzymatic activity of the respiratory chain of Δ01690 was much lower than that of the wild type. These phenotypes were similar to those of a ctaD deletion mutant and an atpB-F deletion mutant. Therefore, we hypothesize that BpOF4_01690 plays a critical role in oxidative phosphorylation under highly alkaline conditions.

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

It is well known that the Na+ cycle and the cell wall are essential for alkaline adaptation of Na+-dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na+ concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na+ concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

Cardiolipin Is Dispensable for Oxidative Phosphorylation and Non-fermentative Growth of Alkaliphilic Bacillus pseudofirmus OF4

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration-dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from (32)P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH 7.5 during log phase. The % CL was higher (28-33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration-dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5.

Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter

Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).

Crystallization and preliminary X-ray study of alanine dehydrogenase fromBacillus pseudofirmusOF4

Alanine dehydrogenase (OF4Ald) from the alkaliphilic Bacillus pseudofirmus OF4 was expressed and purified with a His6 tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K using a solution consisting of 0.1 M Tris-HCl pH 8.0, 0.2 M LiSO4, 22%(w/v) PEG 3350. X-ray diffraction data were collected to 2.8 Å resolution. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 88.04, b = 105.59, c = 120.53 Å, α = 88.37, β = 78.77, γ = 82.65°.

Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

AtpI, a membrane protein encoded by many bacterial atp operons, is reported to be necessary for c-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream of atpI that has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilic Bacillus pseudofirmus OF4. A strain with a chromosomal deletion of atpI grew nonfermentatively, and its purified ATP synthase had a c-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion of atpI, but not atpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F(1) domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. Both spoIIIJ and yqjG, but not atpI or atpZ, complemented a YidC-depleted Escherichia coli strain. Consistent with such overlapping functions, single deletions of spoIIIJ or yqjG in the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression of yqjG was elevated at pH 7.5 relative to that at pH 10.5 and in ΔspoIIIJ strains, but it was lower than constitutive spoIIIJ expression. Deletion of atpZ caused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.

Characterization and purification of the multi subunit type Na+/H+ antiporter from alkaliphilic Bacillus pseudofirmus OF4

Characterization and purification of the multi subunit type Na+/H+ antiporter from alkaliphilic Bacillus pseudofirmus OF4

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant: MUTATIONS IN THE CRITICAL ALKALIPHILE-SPECIFIC RESIDUE LYS-180 AND OTHER RESIDUES THAT SUPPORT ALKALIPHILE OXIDATIVE PHOSPHORYLATION

A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and β-subunit levels at least as high as wild-type. Purified mutant F(1)F(0)-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R mutant of a thermoalkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395-17404). The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found in the thermoalkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness.

Adaptations of the ATP synthase a-subunit to support synthesis at low protonmotive force at both pH 7.5 and 10.5 may underpin the more stringent requirement for lysine-180 in TMH-4 by alkaliphilic Bacillus pseudofirmus OF4 than by more modestly alkaliphilic thermoalkaliphile Bacillus sp. TA2.A1

Adaptations of the ATP synthase a-subunit to support synthesis at low protonmotive force at both pH 7.5 and 10.5 may underpin the more stringent requirement for lysine-180 in TMH-4 by alkaliphilic Bacillus pseudofirmus OF4 than by more modestly alkaliphilic thermoalkaliphile Bacillus sp. TA2.A1

Identification and characterization of functional residues in a multi-subunit type Na+/H+ antiporter Mrp complex from alkaliphilic Bacillus pseudofirmus OF4

Identification and characterization of functional residues in a multi-subunit type Na+/H+ antiporter Mrp complex from alkaliphilic Bacillus pseudofirmus OF4

Single, double and triple alanine to glycine replacements in the AxAxAxA motif of alkaliphilic Bacillus pseudofirmus OF4 c-subunits affect c-ring stability, change both monomer and c-ring mobility in SDS-PAGE and lead to deficits in ATP synthesis

Single, double and triple alanine to glycine replacements in the AxAxAxA motif of alkaliphilic Bacillus pseudofirmus OF4 c-subunits affect c-ring stability, change both monomer and c-ring mobility in SDS-PAGE and lead to deficits in ATP synthesis

Characterization of the Functionally Critical AXAXAXA and PXXEXXP Motifs of the ATP Synthase c-Subunit from an Alkaliphilic Bacillus

The membrane-embedded rotor in the F(0) sector of proton-translocating ATP synthases is formed from hairpin-like c-subunits that are protonated and deprotonated during energization of ATP synthesis. This study focuses on two c-subunit motifs that are unique to synthases of extremely alkaliphilic Bacillus species. One motif is the AXAXAXA sequence found in the N-terminal helix-1 instead of the GXGXGXG of non-alkaliphiles. Quadruple A-->G chromosomal mutants of alkaliphilic Bacillus pseudofirmus OF4 retain 50% of the wild-type hydrolytic activity (ATPase) but <18% of the ATP synthase capacity at high pH. Consistent with a structural impact of the four alanine replacements, the mutant ATPase activity showed enhanced inhibition by dicyclohexylcarbodiimide, which blocks the helix-2 carboxylate. Single, double, or triple A-->G mutants exhibited more modest defects, as monitored by malate growth. The key carboxylate is in the second motif, which is P(51)XXE(54)XXP in extreme alkaliphiles instead of the (A/G)XX(E/D)XXP found elsewhere. Mutation of Pro(51) to alanine had been shown to severely reduce malate growth and ATP synthesis at high pH. Here, two Pro(51) to glycine mutants of different severities retained ATP synthase capacity but exhibited growth deficits and proton leakiness. A Glu(54) to Asp(54) change increased proton leakiness and reduced malate growth 79-90%. The Pro(51) and the Glu(54) mutants were both more dicyclohexylcarbodiimide-sensitive than wild type. The results highlight the requirement for c-subunit adaptations to achieve alkaliphile ATP synthesis with minimal cytoplasmic proton loss and suggest partial suppression of some mutations by changes outside the atp operon.

Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4

An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadX(OF4)), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadX(OF4) was purified to homogeneity by His(6)-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 degrees C and the optimal pH was 10.5. The kinetic parameters K(m) and V(max) at 40 degrees C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively.

Crystallization and preliminary X-ray study of alkaline alanine racemase from Bacillus pseudofirmus OF4.

Alanine racemase (DadX(OF4)), a dimeric endogenous PLP-dependent alkaline enzyme from alkaliphilic Bacillus pseudofirmus OF4, was expressed in Escherichia coli and purified with a His(6) tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 291 K using a solution containing 1.4 M sodium/potassium phosphate pH 8.2. The protein crystallized in space group P2(1)2(1)2(1), with two protein molecules in the asymmetric unit.

Single Gene Deletions of mrpA to mrpG and mrpE Point Mutations Affect Activity of the Mrp Na + /H + Antiporter of Alkaliphilic Bacillus and Formation of Hetero-Oligomeric Mrp Complexes

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na +/H + antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na +-resistance on an antiporter-deficient E. coli strain, nor did they confer Na +/H + antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.

The voltage-gated Na+ channel NaVBP co-localizes with methyl-accepting chemotaxis protein at cell poles of alkaliphilic Bacillus pseudofirmus OF4

Na(V)BP, found in alkaliphilic Bacillus pseudofirmus OF4, is a member of the bacterial voltage-gated Na(+) channel superfamily. The alkaliphile requires Na(V)BP for normal chemotaxis responses and for optimal pH homeostasis during a shift to alkaline conditions at suboptimally low Na(+) concentrations. We hypothesized that interaction of Na(V)BP with one or more other proteins in vivo, specifically methyl-accepting chemotaxis proteins (MCPs), is involved in activation of the channel under the pH conditions that exist in the extremophile and could underpin its role in chemotaxis; MCPs transduce chemotactic signals and generally localize to cell poles of rod-shaped cells. Here, immunofluorescence microscopy and fluorescent protein fusion studies showed that an alkaliphile protein (designated McpX) that cross-reacts with antibodies raised against Bacillus subtilis McpB co-localizes with Na(V)BP at the cell poles of B. pseudofirmus OF4. In a mutant in which Na(V)BP-encoding ncbA is deleted, the content of McpX was close to the wild-type level but McpX was significantly delocalized. A mutant of B. pseudofirmus OF4 was constructed in which cheAW expression was disrupted to assess whether this mutation impaired polar localization of McpX, as expected from studies in Escherichia coli and Salmonella, and, if so, whether Na(V)BP would be similarly affected. Polar localization of both McpX and Na(V)BP was decreased in the cheAW mutant. The results suggest interactions between McpX and Na(V)BP that affect their co-localization. The inverse chemotaxis phenotype of ncbA mutants may result in part from MCP delocalization.

Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

Properties of four two-component bacterial transport systems of the cation/proton antiporter-2 (CPA2) family led to suggestions that this CPA2 subset may use a channel rather than an antiport mechanism [see Booth IR, Edwards MD, Gunasekera B, Li C, Miller S (2005) in Bacterial Ion Channels, eds Kubalski A, Martinac B (Am Soc Microbiol, Washington, DC), pp 21-40]. The transporter subset includes the intensively studied glutathione-gated K(+) efflux systems from Escherichia coli, KefGB, and KefFC. KefG and KefF are ancillary proteins. They are peripheral membrane proteins that are encoded in operons with the respective transporter proteins, KefB and KefC, and are required for optimal efflux activity. The other two-component CPA2 transporters of the subset are AmhMT, an NH(4)(+) (K(+)) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K(+) efflux system from Bacillus subtilis. Here a K(+)/H(+) antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent K(m) for K(+) was in the low mM range. The peripheral protein was required for YhaU- and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb(+) approximately K(+)>Li(+)>Na(+). Glutathione significantly inhibited KefFC-mediated K(+)/H(+) antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H(+) uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH(4)(+), the results also imply that AmhMT catalyzes NH(4)(+)/H(+) antiport, which would prevent net cytoplasmic H(+) loss during NH(4)(+) efflux.