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.

Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis.

Staphylococcus aureus has three types of cation/proton antiporters. The type 3 family includes two multisubunit Na+/H+ (Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane-bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 in S. aureus Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter-deficient KNabc Escherichia coli strain. The catalytic properties of the antiporters were measured in everted (inside out) vesicles. The Mnh1 antiporter exhibited a significant exchange of Na+/H+ cations at pH 7.5. Mnh2 showed a significant exchange of both Na+/H+ and K+/H+ cations, especially at pH 8.5. Under elevated salt conditions, deletion of the mnhA1 gene resulted in a significant reduction in the growth rate of S. aureus in the range of pH 7.5 to 9. Deletion of mnhA2 had similar effects but mainly in the range of pH 8.5 to 9.5. Double deletion of mnhA1 and mnhA2 led to a severe reduction in the S. aureus growth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters in S. aureus were also assessed via their support of virulence in a mouse in vivo infection model. Deletion of the mnhA1 gene led to a major loss of S. aureus virulence in mice, while deletion of mnh2 led to no change in virulence.IMPORTANCE This study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters in S. aureus and their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+ antiport, and it plays a significant role in maintaining halotolerance at pH 7.5 while the Mnh2 antiporter has catalytic antiporter activities for Na+/H+ and K+/H+ that have roles in both osmotolerance and halotolerance in S. aureus Study of S. aureus with a single deletion of either mnhA1 or mnhA2 was assessed in an infection model of mice. The result shows that mnhA1, but not mnhA2, plays a major role in S. aureus virulence.

The Lysine 299 Residue Endows the Multisubunit Mrp1 Antiporter with Dominant Roles in Na+ Resistance and pH Homeostasis in Corynebacterium glutamicum.

Corynebacterium glutamicum is generally regarded as a moderately salt- and alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+ resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the Δmrp1 Δmrp2 double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+ antiporters of C. glutamicum were differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrp-type antiporters. When the major Mrp1 antiporter was overwhelmed, C. glutamicum might employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+ resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136 Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+ level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation during C. glutamicum stress acclimatization.IMPORTANCE The capacity to adapt to harsh environments is crucial for bacterial survival and product yields, including industrially useful Corynebacterium glutamicum Although C. glutamicum exhibits a marked resistance to salt-alkaline stress, the possible mechanism for these adaptations is still unclear. Here, we present the physiological functions and expression patterns of C. glutamicum putative Na+/H+ antiporters and conserved residues of Mrp1 subunits, which respond to different salt and alkaline stresses. We found that the Mrp-type antiporters, particularly the Mrp1 antiporter, played a predominant role in maintaining intracellular nontoxic Na+ levels and alkaline pH homeostasis. Loss of the major Mrp1 antiporter had a profound effect on gene expression of other antiporters under salt or alkaline conditions. The lysine 299 residue may play its essential roles in conferring salt and alkaline tolerance by affecting the ion translocation channel of the Mrp1A subunit. These findings will contribute to a better understanding of Na+/H+ antiporters in sodium antiport and pH regulation.

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4.

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.

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.

The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus†OF4 is adapted to alkaliphilic lifestyle.

In the c-ring rotor of ATP synthases ions are shuttled across the membrane during ATP synthesis by a unique rotary mechanism. We investigated characteristics of the c-ring from the alkaliphile Bacillus pseudofirmus OF4 with respect to evolutionary adaptations to operate with protons at high environmental pH. The X-ray structures of the wild-type c13 ring at pH 9.0 and a 'neutralophile-like' mutant (P51A) at pH 4.4, at 2.4 and 2.8 Šresolution, respectively, reveal a dependency of the conformation and protonation state of the proton-binding glutamate (E(54) ) on environmental hydrophobicity. Faster labelling kinetics with the inhibitor dicyclohexylcarbodiimide (DCCD) demonstrate a greater flexibility of E(54) in the mutant due to reduced water occupancy within the H(+) binding site. A second 'neutralophile-like' mutant (V21N) shows reduced growth at high pH, which is explained by restricted conformational freedom of the mutant's E(54) carboxylate. The study directly connects subtle structural adaptations of the c-ring ion binding site to in vivo effects of alkaliphile cell physiology.

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.

A new type of proton coordination in an F(1)F(o)-ATP synthase rotor ring.

We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 A, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle.

Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and P(i)-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS-PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the intact c-rings, only Ala16-to-Gly mutants exhibited significantly increased mobility relative to that of the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested, but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The A16/20G double mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to that of the WT c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.

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 ß-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.

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.

F1F0-ATP synthases of alkaliphilic bacteria: lessons from their adaptations.

This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values>10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na+- instead of H+-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H+ transfers to ATP synthases via membrane-associated microcircuits between the H+ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.

Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

Mutations alter the sodium versus proton use of a Bacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors.

Bacterial flagella contain membrane-embedded stators, Mot complexes, that harness the energy of either transmembrane proton or sodium ion gradients to power motility. Use of sodium ion gradients is associated with elevated pH and sodium concentrations. The Mot complexes studied to date contain channels that use either protons or sodium ions, with some bacteria having only one type and others having two distinct Mot types with different ion-coupling. Here, alkaliphilic Bacillus clausii KSM-K16 was shown to be motile in a pH range from 7 to 11 although its genome encodes only one Mot (BCl-MotAB). Assays of swimming as a function of pH, sodium concentration, and ion-selective motility inhibitors showed that BCl-MotAB couples motility to sodium at the high end of its pH range but uses protons at lower pH. This pattern was confirmed in swimming assays of a statorless Bacillus subtilis mutant expressing either BCl-MotAB or one of the two B. subtilis stators, sodium-coupled Bs-MotPS or proton-coupled Bs-MotAB. Pairs of mutations in BCl-MotB were identified that converted the naturally bifunctional BCl-MotAB to stators that preferentially use either protons or sodium ions across the full pH range. We then identified trios of mutations that added a capacity for dual-ion coupling on the distinct B. subtilis Bs-MotAB and Bs-MotPS motors. Determinants that alter the specificity of bifunctional and single-coupled flagellar stators add to insights from studies of other ion-translocating transporters that use both protons and sodium ions.

Cation/proton antiporter complements of bacteria: why so large and diverse?

Most bacterial genomes have five to nine distinct genes predicted to encode transporters that exchange cytoplasmic Na(+) and/or K(+) for H(+) from outside the cell, i.e. monovalent cation/proton antiporters. By contrast, pathogens that live primarily inside host cells usually possess zero to one such antiporter while other stress-exposed bacteria exhibit even higher numbers. The monovalent cation/proton antiporters encoded by these diverse genes fall into at least eight different transporter protein families based on sequence similarity. They enable bacteria to meet challenges of high or fluctuating pH, salt, temperature or osmolarity, but we lack explanations for why so many antiporters are needed and for the value added by specific antiporter types in specific settings. In this issue of Molecular Microbiology, analyses of the pH dependence of cytoplasmic [Na(+)], [K(+)], pH and transmembrane electrical potential in the 'poly extremophile'Natranaerobius thermophilus are the context for assessment of the catalytic properties of 12 predicted monovalent cation/proton antiporters in the genome of this thermophilic haloalkaliphile. The results provide a profile of adaptations of the poly extremophilic anaerobe, including a proposed role of cytoplasmic buffering capacity. They also provide new perspectives on two large monovalent cation/proton antiporter families, the NhaC and the cation/proton antiporter-3 antiporter families.

ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry.

Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F(1)F(o)-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F(1)F(o)-ATP synthases. Under selected buffer conditions the mass of the intact F(1)F(o)-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases.

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.

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.

Do physiological roles foster persistence of drug/multidrug-efflux transporters? A case study.

Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.

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.