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 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.

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.

Mrp Antiporters Have Important Roles in Diverse Bacteria and Archaea.

Mrp (Multiple resistance and pH) antiporter was identified as a gene complementing an alkaline-sensitive mutant strain of alkaliphilic Bacillus halodurans C-125 in 1990. At that time, there was no example of a multi-subunit type Na+/H+ antiporter comprising six or seven hydrophobic proteins, and it was newly designated as the monovalent cation: proton antiporter-3 (CPA3) family in the classification of transporters. The Mrp antiporter is broadly distributed among bacteria and archaea, not only in alkaliphiles. Generally, all Mrp subunits, mrpA-G, are required for enzymatic activity. Two exceptions are Mrp from the archaea Methanosarcina acetivorans and the eubacteria Natranaerobius thermophilus, which are reported to sustain Na+/H+ antiport activity with the MrpA subunit alone. Two large subunits of the Mrp antiporter, MrpA and MrpD, are homologous to membrane-embedded subunits of the respiratory chain complex I, NuoL, NuoM, and NuoN, and the small subunit MrpC has homology with NuoK. The functions of the Mrp antiporter include sodium tolerance and pH homeostasis in an alkaline environment, nitrogen fixation in Schizolobium meliloti, bile salt tolerance in Bacillus subtilis and Vibrio cholerae, arsenic oxidation in Agrobacterium tumefaciens, pathogenesis in Pseudomonas aeruginosa and Staphylococcus aureus, and the conversion of energy involved in metabolism and hydrogen production in archaea. In addition, some Mrp antiporters transport K+ and Ca2+ instead of Na+, depending on the environmental conditions. Recently, the molecular structure of the respiratory chain complex I has been elucidated by others, and details of the mechanism by which it transports protons are being clarified. Based on this, several hypotheses concerning the substrate transport mechanism in the Mrp antiporter have been proposed. The MrpA and MrpD subunits, which are homologous to the proton transport subunit of complex I, are involved in the transport of protons and their coupling cations. Herein, we outline other recent findings on the Mrp antiporter.

Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

Draft Genome Sequence of Bacillus alcalophilus AV1934, a Classic Alkaliphile Isolated from Human Feces in 1934.

Bacillus alcalophilus AV1934, isolated from human feces, was described in 1934 before microbiome studies and recent indications of novel potassium ion coupling to motility in this extremophile. Here, we report draft sequences that will facilitate an examination of whether that coupling is part of a larger cycle of potassium ion-coupled transporters.

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.

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.

Transcriptional profiling of Staphylococcus aureus during growth in 2 M NaCl leads to clarification of physiological roles for Kdp and Ktr K+ uptake systems.

UNLABELLED: Staphylococcus aureus exhibits an unusually high level of osmotolerance and Na(+) tolerance, properties that support survival in various host niches and in preserved foods. The genetic basis of these traits is not well understood. We compared the transcriptional profiles of S. aureus grown in complex medium with and without 2 M NaCl. The stimulon for growth in high-osmolality media and Na(+) included genes involved in uptake of K(+), other compatible solutes, sialic acid, and sugars; capsule biosynthesis; and amino acid and central metabolism. Quantitative PCR analysis revealed that the loci responded differently from each other to high osmolality imposed by elevated NaCl versus sucrose. High-affinity K(+) uptake (kdp) genes and capsule biosynthesis (cap5) genes required the two-component system KdpDE for full induction by osmotic stress, with kdpA induced more by NaCl and cap5B induced more by sucrose. Focusing on K(+) importers, we identified three S. aureus genes belonging to the lower-affinity Trk/Ktr family that encode two membrane proteins (KtrB and KtrD) and one accessory protein (KtrC). In the absence of osmotic stress, the ktr gene transcripts were much more abundant than the kdpA transcript. Disruption of S. aureus kdpA caused a growth defect under low-K(+) conditions, disruption of ktrC resulted in a significant defect in 2 M NaCl, and a ΔktrC ΔkdpA double mutant exhibited both phenotypes. Protective effects of S. aureus Ktr transporters at elevated NaCl are consistent with previous indications that both Na(+) and osmolality challenges are mitigated by the maintenance of a high cytoplasmic K(+) concentration. IMPORTANCE: There is general agreement that the osmotolerance and Na(+) tolerance of Staphylococcus aureus are unusually high for a nonhalophile and support its capacity for human colonization, pathogenesis, and growth in food. Nonetheless, the molecular basis for these properties is not well defined. The genome-wide response of S. aureus to a high concentration, 2 M, of NaCl revealed the upregulation of expected genes, such as those for transporters of compatible solutes that are widely implicated in supporting osmotolerance. A high-affinity potassium uptake system, KdpFABC, was upregulated, although it generally plays a physiological role under very low K(+) conditions. At higher K(+) concentrations, a lower-affinity and more highly expressed type of K(+) transporter system, Ktr transporters, was shown to play a significant role in high Na(+) tolerance. This study illustrates the importance of the K(+) status of the cell for tolerance of Na(+) by S. aureus and underscores the importance of monovalent cation cycles in this pathogen.

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.

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.

Role of institutional climate in fostering diversity in biomedical research workforce: a case study.

This article reviews the barriers to diversity in biomedical research and describes the evolution of efforts to address climate issues to enhance the ability to attract, retain, and develop underrepresented minorities, whose underrepresentation is found both in science and medicine, in the graduate-school biomedical research doctoral programs (PhD and MD/PhD) at Mount Sinai School of Medicine. We also describe the potential beneficial impact of having a climate that supports diversity and inclusion in the biomedical research workforce. The Mount Sinai School of Medicine diversity-climate efforts are discussed as part of a comprehensive plan to increase diversity in all institutional programs: PhD, MD/PhD, and MD, and at the residency, postdoctoral fellow, and faculty levels. Lessons learned from 4 decades of targeted programs and activities at the Mount Sinai School of Medicine may be of value to other institutions interested in improving diversity in the biomedical science and academic medicine workforce.

Diversity in the biomedical research workforce: developing talent.

Much has been written about the need for and barriers to achievement of greater diversity in the biomedical workforce from the perspectives of gender, race, and ethnicity; this is not a new topic. These discussions often center around a "pipeline" metaphor that imagines students flowing through a series of experiences to eventually arrive at a science career. Here we argue that diversity will only be achieved if the primary focus is on (1) what is happening within the pipeline, not just counting individuals entering and leaving it; (2) de-emphasizing the achievement of academic milestones by typical ages; and (3) adopting approaches that most effectively develop talent. Students may develop skills at different rates based on factors such as earlier access to educational resources, exposure to science (especially research experiences), and competing demands for time and attention during high school and college. Therefore, there is wide variety among students at any point along the pipeline. Taking this view requires letting go of imagining the pipeline as a sequence of age-dependent steps in favor of milestones of skill and talent development decoupled from age or educational stage. Emphasizing talent development opens up many new approaches for science training outside of traditional degree programs. This article provides examples of such approaches, including interventions at the postbaccalaureate and PhD levels, as well as a novel coaching model that incorporates well-established social science theories and complements traditional mentoring. These approaches could significantly impact diversity by developing scientific talent, especially among currently underrepresented minorities.

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 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.

Molecular aspects of bacterial pH sensing and homeostasis.

Diverse mechanisms for pH sensing and cytoplasmic pH homeostasis enable most bacteria to tolerate or grow at external pH values that are outside the cytoplasmic pH range they must maintain for growth. The most extreme cases are exemplified by the extremophiles that inhabit environments with a pH of below 3 or above 11. Here, we describe how recent insights into the structure and function of key molecules and their regulators reveal novel strategies of bacterial pH homeostasis. These insights may help us to target certain pathogens more accurately and to harness the capacities of environmental bacteria more efficiently.

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.

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.