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Biochimica et Biophysica Acta 1797 (2010) 1362–1377

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Biochimica et Biophysica Acta

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Review

F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations David B. Hicks a, Jun Liu a, Makoto Fujisawa b, Terry A. Krulwich a,⁎

a Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA b Faculty of Food Life Sciences, Toyo University, Ora-gun, Gunma 374-0193, Japan

article info abstract

Article history: This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully Received 19 January 2010 overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values Received in revised form 22 February 2010 N10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for Accepted 23 February 2010 ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a Available online 1 March 2010 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 Keywords: ATPase ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory + + Oxidative phosphorylation alkaliphilic bacteria do not use Na - instead of H -coupled ATP synthases. Nor do they offset the adverse pH c-ring gradient with a compensatory increase in the transmembrane electrical potential component of the Bacillus pseudofirmus OF4 protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve Bacillus TA2.A1 the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Spirulina platensis 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. © 2010 Elsevier B.V. All rights reserved.

1. Introduction reports of ectopic F1F0-ATP synthases in caveolae/lipid rafts on the surface of a variety of mammalian cell types, including cancer cells [2–5]. ATP is the dominant chemical energy currency of living cells. A The homologous but structurally simpler bacterial ATP synthases are minority of cells, such as mature red blood cells and some non- found in the cytoplasmic membrane or thylakoid membranes [6–8]. respiratory microbial cells, generate ATP exclusively through substrate ATP synthesis by ATP synthases is energized by electrochemical level phosphorylation. By contrast, the majority of ATP synthesized by gradients of H+ or Na+ across the membrane. ATP synthesis is coupled animal, plant and microbial cells is obtained at higher energy yield from to downhill movement of ions fostered by those gradients across catabolic substrates or light via the use of oxidative phosphorylation mitochondrial, thylakoid and bacterial cell membranes [6,8].ATP (OXPHOS) or photo-phosphorylation, which depend upon the catalytic synthases are reversible, i.e., they can function as ATPases that hydrolyze

activity of F1F0-ATP synthases (which will be called ATP synthases in this ATP in concert with ion pumping (energetically uphill) in the opposite article). These synthases have been part of the physiological landscape direction of ion uptake during synthesis. For many fermentative through much of the evolutionary path of contemporary organisms, bacteria, ATPase activity is the only physiological activity of the enzyme. perhaps as far back as LUCA, the putative “last universal common By contrast, the hydrolytic activity of ATP synthases in most other ancestor” of living organisms [1]. ATP synthases of animals and plants settings is carefully controlled, or even latent, in order to protect the

are embedded by one of their domains, the F0 domain, in the inner cytoplasmic ATP pool [9–11]. Structure–function information that membranes of mitochondria or in chloroplast thylakoid membranes. An emerged over the past decade and a half supports a rotary mechanism interesting exception to these classic organelle localizations are recent for ATP synthesis that involves sequential conformational changes in the three catalytic sites of the synthase. Experiments on single synthase complexes have captured steps and sub-steps of the rotation. An ⁎ Corresponding author. Department of Pharmacology and Systems Therapeutics, extensive collection of high resolution structures of major portions of Box 1603, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY ATP synthases has also emerged. Although not yet representing the 10029-6500, USA. Tel.: +1 212 241 7280; fax: +1 212 996 7214. entire structure, the structural information has enormously fostered E-mail addresses: [email protected] (D.B. Hicks), [email protected] (J. Liu), [email protected] (M. Fujisawa), [email protected] hypothesis-building and experimental work aimed at greater mecha- (T.A. Krulwich). nistic understanding [12–24].

0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2010.02.028 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 1363

The importance of resolving the mechanistic details of ATP the F0 and the catalytic domain of the F1 that contains the three α- and synthase function arises from the enzyme's central role in cell phys- β-subunit pairs [50–54]. The rotor element found in the F0 (highlight- iology. The importance of further insights is underscored by the ed in green in Fig. 1) is a homo-oligomer of hairpin-like c-subunits serious consequences of genetic defects in ATP synthase structural in most ATP synthases. The cytoplasmic loops that are exposed on the genes, regulatory genes or assembly factors [25,26]. Dysfunctions in N-side of the membrane (the electrically negative side) interact with respiration-dependent ATP synthesis, i.e. oxidative phosphorylation the γε sub-complex which forms the central stalk, with the γ-subunit

(OXPHOS), are further correlated with complex disorders whose extending asymmetrically into the F1 catalytic domain [55] (see frequency increases with ageing, such as Type 2 diabetes [27]. The Section 2.3). mechanistic details of ATP synthase function will also enhance our The number of c-subunits observed per c-rotor varies from 10 to 15, ability to inactivate the ATP synthase of bacterial pathogens in which depending upon the organism [18,20,56–65]. The two c-subunit the enzyme plays an essential role in part of their pathogenic life stoichiometries so far determined for eukaryotic c-rotors are at opposite cycle. This is the promising basis for the use of diarylquinolines that ends of those ranges with the yeast stoichiometry at 10 [66] and the target the ATP synthase of pathogenic Mycobacterium tuberculosis and spinach chloroplast stoichiometry at 14 [67–69]. By contrast to the its multi-drug resistant forms [28–31]. The resulting inhibition of ATP typical homo-oligomeric rotor, the Na+-coupled ATP synthase of the synthase reduces viability of the pathogens under hypoxic conditions anaerobic, acetogenic bacterium Acetobacterium woodii instead has a in which they otherwise persist in the host [32]. Analogously, growing hetero-oligomeric rotor with two different c-subunits. The A. woodii c- insights into the mechanisms of natural ATP synthase modulators, and rotor has 10 c-subunits/rotor; 9 of them are the products of two inhibitors of its hydrolytic mode, could lead to rational design of identical genes in the atp operon that have the usual 2-helix hairpin therapeutic strategies for human disorders that are linked to ATP structure of other F-type ATP synthase c-subunits. The remaining rotor synthase and to strategies for inhibiting the ATP synthases that are subunit is encoded by a third c-subunit gene and is a 4-transmembrane ectopically expressed on cancer cell surfaces [10,33,34]. helix (TMH) “c”-subunit of the type found in V-type ATP synthases/ There is another gateway to progress on the structure–function ATPases [70]. The V-type ATPase from Enterococcus hirae has 10 of the V- underpinnings of ATP synthase activity and its contributions to phys- type subunit in its K-rotor, the V-type ATPase equivalent of the c-rotor iology. This is the growing awareness that ATP synthases from higher [71]. The larger V-type subunit has only one ion-binding site, like that of organisms, as well as the cytochrome oxidases that participate in ATP the smaller ones. Its presence in the A. woodii rotor is expected to impact synthase-dependent OXPHOS, have multiple isoforms of important the functional properties of the enzyme. Perhaps the V-type subunit subunits [35–38]. Some of these have been shown to relate to tissue- enhances the propensity for ATP hydrolysis in a manner that relates to specific functional difference [39–41]. Among bacteria, the pace of its specialized physiological circumstances [70,72]. evolutionary adaptations and the extraordinary range of environ- ments in which ATP synthases function have yielded a wealth of 2.2. atp operons adaptations and variants in the OXPHOS machinery [42–44]. Microbial ATP synthases exhibit variations that support their adap- Bacterial ATP synthases are most often encoded in single operons tation to very distinct conditions of pH, temperature, oxygen levels, that have the eight structural genes in the order atp (or unc) BEFHAGDC, salt etc. [11,31,43,45,46]. These variations have structural and func- respectively encoding the a, c, b, δ, α, γ, β, ε subunits (as shown for tional impacts on the ATP synthase that presumably contribute Bacillus subtilis in Fig. 2). Some bacteria, including Rhodosprillum rubrum adaptive value under particular conditions. The recognition of these and the cyanobacterium Synechococcus, have distinct operons for genes variations has challenged and expanded our understanding of how encoding F0 proteins and those encoding the F1 proteins, consistent with ATP synthases work. In this article, the discussion will center on the the hypothesis that the genes for the two domains evolved as separate ATP synthase of alkaliphilic bacteria that are highly adapted to grow modules [73,74]. In these particular bacteria there are two homologous well at pH values as high as 11 [45,47–49]. The adaptive strategies by genes encoding the two b-subunits [74]. In other bacterial operons, which alkaliphiles overcome the energetic challenges to ATP synthesis there are different gene orders, e.g. atpEBFHAGDC in Streptococcus at high pH started as an esoteric puzzle and grew into a model system mutans and Streptococcus sanguis [75].Inmostbacterialatp operons, a with implications for ATP synthases in general. Before focusing on ninth gene precedes the eight structural genes for the ATP synthase these bioenergetic challenges and adaptive strategies of alkaliphiles, itself. This is an atpI gene that encodes a putative hydrophobic protein we will present an overview of structural features of bacterial ATP predicted to have 4 TMH. The AtpI protein sequence is not highly synthases, of the operons that encode them and of the process of ATP conserved across species [76]. In some instances atpI is under the control synthase-mediated ATP synthesis. Variations that have been observed of a separate promoter from the other atp genes [76], in some instances in subunit structure of diverse non-alkaliphile ATP synthases and in atpI is absent entirely [77] and in some instances atpI is absent but a the organization of atp operons will be noted as part of this overview. different gene of unknown function takes its usual place [78]. Gay and Walker suggested a chaperone/assembly type of function for the AtpI 2. Overview of bacterial ATP synthase structure–function proteinwhentheyfirst described the atpI gene [79]. Partial homology of AtpI to yeast proteins with membrane assembly functions has also been 2.1. Structural overview noted [80,81]. Recently a chaperone function was clearly shown for an AtpI protein. The AtpI protein from Propionigenium modestum was Bacterial ATP synthases contain eight proteins that are present in shown to be required for an assembly in Escherichia coli of a hybrid ATP the synthase at different stoichiometric ratios per complex. Like their synthase whose F1 domain was from thermophilic Bacillus PS3 and eukaryotic counterparts, bacterial ATP synthases have two domains, whose F0 domain was from P. modestum (Pm) [82]. His-tagged Pm-AtpI the F1 and F0 domains, as indicated in the schematic diagram in Fig. 1. was found to co-purify with both the c-subunit monomer and the The cytoplasmically located, soluble F1 domain contains three catalytic assembled c-ring, and a role of Pm-AtpI in the assembly of the ring was α and β subunit pairs (shades of purple in Fig. 1) and single γ, δ, and ε suggested. Further evidence for such a role was obtained from subunits (see reviews [6,8,24]). The membrane-embedded F0 experiments in which the P. modestum c-subunit was synthesized in domain is composed of a single a-subunit, two b-subunits and multiple vitro using purified components in the presence of liposomes [83]. c-subunits. The a, b and δ subunits (highlighted in blue in Fig. 1) Formation of a c-ring again depended upon the presence of Pm-AtpI. The comprise the stator for the rotary machine. The two b-subunits and the extent of the contribution of AtpI to the assembly of the c-rotor in native δ-subunit form a peripheral stalk. This stalk connects the two domains bacterial settings is not yet clear. Deletions or disruptions atpI in E. coli of the complex, spanning the ion-translocating a- and c-subunits of resulted in a reduced growth yield but the mutants retained significant 1364 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377

Fig. 2. Operons of a neutralophilic Bacillus, B. subtilis, and of an alkaliphilic Bacillus, B. pseudofirmus OF4. The genetic organization of these operons is described in the text.

2.3. Protonmotive force- or sodium motive force-dependent ATP synthesis by a rotary mechanism

The catalytic reaction carried out by ATP synthases/ATPases is shown below (Eq. (i)). The participation of a proton (H+)asa substrate in the ATP synthesis direction confers pH-dependence upon the reaction thermodynamics. When a very high external pH causes

the cytoplasmic pH to rise (e.g. in B. pseudofirmus OF4 at pHout N9.5), Fig. 1. A diagrammatic representation of a bacterial ATP synthase. The diagram depicts a the lower availability of substrate H+ will therefore affect ATP + H -translocating synthase such as found in aerobic alkaliphilic bacteria. The synthesis. ATP synthesis at high pH also faces additional challenges membrane-embedded F0 domain, that contains the proton-conducting stator and described in Section 3.2. The catalytic reaction that includes the rotor elements, and the cytoplasmic F1 domain that contains the catalytic sites, are + + shown. The stator elements of the synthase are shaded in blue and contain the coupling ions, multiple H or Na that are translocated during peripheral stalk described in the text. The central stalk is comprised of the γ (pink) and synthesis, is also shown below (Eq. (ii)); the P-side and N-side ε (tan) subunits and interacts with the loops of the oligomeric ring of c-subunits that is designate, respectively, the electrically positive and electrically the rotor for the rotary coupling mechanism for ATP synthesis (shown in green). As negative side of the membrane. The majority of bacterial synthases, energetically downhill H+ movement through the F energizes synthesis, rotation of 0 and the ATP synthases of animals and plants, are coupled to an the c-ring in the direction shown, leads to concomitant rotation of the interactive F1 subunits γ and ε that result in a conformational change in the three catalytic sites (at electrochemical gradient of protons, the protonmotive force (pmf). the α/β interfaces). The pmf is generated by proton-pumping complexes of the respiratory chain or by light-driven proteins and protein complexes that pump H+ across the membranes in which the synthases are found [89,90].The capacity for non-fermentative growth that requires ATP synthase F1F0-ATP synthases of a smaller group of bacteria are energized by an activity [84,85]. Deletion of atpI in alkaliphilic Bacillus pseudofirmus electrochemical gradient of Na+, sodium motive force (smf), that is OF4 similarly did not affect non-fermentative growth dramatically generated by Na+ pumping protein complexes in the membrane [6,8]. except under specific ionic conditions (see below) [86]. AtpI may have a There are motifs in the membrane-embedded c-subunits that identify less dominant role than the YidC chaperone family members that play a Na+-coupled ATP synthase and at least two types of H+-coupled ATP role in both the c-anda-subunit assembly in E. coli [87,88]. Possibly the synthases [43]. relative importance of the AtpI chaperone role varies among bacteria 3− 2− þ 4− depending upon the complement of chaperones that is present and/or ADP þ HPO4 þ H ↔ATP þ H2O ðiÞ upon specificconditions. The atp operon of alkaliphilic B. pseudofirmus OF4 has a small ORF þ þ 3− 2− þ 4− nH or NaPside þ ADP þ HPO4 þ H ↔ATP þ H2O upstream of the atpI gene, which was designated atpZ (Fig. 2) [86].The þ þ þ nH or NaNside ðiiÞ atpZ gene of B. pseudofirmus OF4 was shown to produce a hydrophobic protein product that is predicted to possess 2-TMH. Non-polar deletion The pmf or smf respectively power downhill translocation of H+ of atpI, atpZ or a double atpIZ deletion resulted in a defect in non- or Na+ that energizes ATP synthesis. The translocation starts with fermentative growth at pH 7.5 that was especially pronounced at sub- a-subunit-mediated uptake of the ions from the P-side of the optimal [Mg2+]; the relative deleterious effects of deletions was membrane and binding of the ion to successive c-subunits of the ΔatpIbΔatpZbΔatpZI. In view of the demonstration of a chaperone rotor at the interface of subunits a and c. After rotation of the rotor, role for P. modestum AtpI, the possibility arises that the Mg2+-related successive c-subunits resume interaction with the a-subunit and effects of AtpI status in B. pseudofirmus OF4 are an indirect effect of AtpI successive release of the ions from those c-subunits occurs. The on the assembly of the ATP synthase subunits or on the assembly of AtpZ ions then complete the path across the membrane into the bacterial in this organism. The direct role of AtpZ also needs to be more fully cytoplasm (see Fig. 1) [8,24,91]. No high resolution structural data are explored. AtpZ has numerous homologues that are thus far restricted to yet available for the a-subunit, but extensive biochemical and genetic the bacterial Firmicutes phylum, i.e. in low %GC Gram-positive bacteria evidence indicates that this ATP synthase subunit plays roles in such as those shown in Table 1. Not all members of the genera in which providing the proton path from outside the membrane surface to the atpZ is found have this gene and atpZ has not yet been observed in carboxylates of interacting c-subunits of the rotor. The ions are passed to operon sequences from several fermentative families of Firmicutes, e.g. the c-subunit carboxylates within the membrane. The c-subunit lactobacilli, staphylococci, streptococci and mycoplasma. This suggests carboxylates interact with a region of the a-subunit TMH4 where that the function of AtpZ is related to the physiological setting of a there is an essential arginine residue [92,93]. The positive charge of the specific sub-set of low %GC Gram-positive bacteria. In the genomes of conserved a-subunit arginine is proposed to facilitate the release of H+ 5 high %GC Gram-positive bacteria, a different gene is found between from protonated carboxylates of c-subunits completing a rotation of the atpI and atpB and is predicted to encode a hydrophobic peptide that ring and to create an environment in which re-protonation occurs exhibits sequence similarity to bacterial permeases [78]. [91,94–100]. The proton exit pathway to the bacterial cytoplasm has D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 1365

Table 1 Occurrence of AtpZ in selected bacteria from the Firmicute phylum (low % G+C Gram-positive bacteria).

Aerobes Accession Extremophile? F-ATP synthase Spacing between # amino % identity with % similarity with number coupling iona atpZ and atpIb acids B. pseudofirmus OF4 AtpZ B. pseudofirmus OF4 AtpZ

Bacillus pseudofirmus OF4 AAG48356 Alkaliphile H+ −7 74 Not applicable Not applicable Bacillus halodurans C-125 NP_244629 Alkaliphile H+ −3 75 42/74 (56%) 54/74 (72%) Bacillus clausii KSM-K16 YP_177351 Alkaliphile H+ −1 74 44/74 (59%) 59/74 (79%) Bacillus anthracis str. Ames NP_847713 No H+ 0 84 25/74 (33%) 47/74 (63%) Bacillus weihenstephanensis KBAB4 YP_001647885 No H+ −3 74 26/74 (35%) 48/74 (64%) Geobacillus kaustophilus HTA426 YP_149219 Thermophile H+ 5 73 29/73 (39%) 51/73 (69%) Bacillus sp. TA2.A1 Not available Thermophile; H+ 15 70 21/66 (31%) 37/66 (56%) alkaliphile

Anaerobes or facultative aerobes

Alkaliphilus metalliredigens QYMF YP_001318233 Alkaliphile Na+c 5 75 17/62 (27%) 37/62 (59%) Alkaliphilus oremlandii OhILAs YP_001514095 Alkaliphile Na+ 5 75 20/68 (29%) 38/68 (55%) Anoxybacillus flavithermus WK1 YP_002317048 Thermophile H+ −3 75 31/74 (41%) 47/74 (63%) Candidatus Desulforudis audaxviator YP_001718264 No H+ 13 99 22/66 (33%) 36/66 (54%) MP104C Carboxydothermus hydrogenoformans YP_361346 Thermophile H+ 2 72 17/56 (30%) 31/56 (55%) Z-2901 Clostridium difficile 630 YP_001089996 No Na+ 5 75 23/67 (34%) 35/67 (52%) Desulfotomaculum reducens MI-1 YP_001114485 No H+ 23 76 27/70 (38%) 40/70 (57%) Exiguobacterium sibiricum 255-15 YP_001815149 Psychrophile H+ −3 72 6/13 (46%)d 9/13 (69%)d Geobacillus thermodenitrificans YP_001127396 Thermophile H+ 7 60 20/55 (36%) 34/55 (61%) NG80-2 Pelotomaculum thermopropionicum SI YP_001213370 Thermophile H+ 15 81 21/61 (34%) 33/61 (54%) Examples of Bacillus species that lack an atpZ gene:

Oceanobacillus iheyensis HTE83, Bacillus amyloliquefaciens FZB42, Bacillus licheniformis ATCC 14580 DSM 13, Bacillus pumilus SAFR-032 SAFR032, Bacillus subtilis subsp. subtilis str. 168, Bacillus thuringiensis str. Al Hakam.

Examples of anaerobes that lack an atpZ gene:

x Clostridium botulinum A str. ATCC 19397, Enterococcus faecalis V583, Lactobacillus acidophilus NCFM, Natranaerobius thermophilus JW/NM-WN-LF.

a The coupling ion hypothesis is based on the absence (for H+) or presence (for Na+) of the Na+-binding motif in c-subunits known to bind Na+ (Pogoryelov, D. et al. 2009 Nature Struct. Molec. Biol. 16:1068–1074). b Nucleotide spacing is arbitrarily defined as +1 when the last nucleotide of the atpZ stop codon is adjacent to the first nucleotide of the start codon of the a-subunit; 0 (zero) nucleotides when those nucleotides exactly overlap and −1 if the last nucleotide of the atpZ stop codon overlaps with the second nucleotide of the start codon of the a-subunit. c The c-subunit of Alkaliphilus metalliredigens QYMF is 184 amino acids, more than twice the normal length of a c-subunit. Alignment with the Ilyobacter tartaricus c-subunit is indicative of Na+-binding. d Although the sequence identity and similarity is low, the size of the ORF and its overlap with the gene for the a-subunit is suggestive that this putative gene encodes an AtpZ. been proposed to be a discrete half channel within the a-subunit (as is (i.e., confer fragility) under another condition, e.g. near neutral pH. shown in Fig. 1) [6,101], but an alternative suggestion is that the exit Obligate alkaliphiles exhibit absolute “fragility” at near neutral pH and pathway is along the a-andc-subunit interface [8,99].Thea-andc- are often the best adapted at the extreme alkaline end of the range. subunit-mediated rotation of the c-rotor causes rotation of the γ and ε Facultative alkaliphiles strike a more temperate balance. Alkaliphilic subunits. These central stalk subunits interact with well-ordered bacteria can be isolated from natural environments such as highly cytoplasmic loops of the rotating c-ring so that torque is generated alkaline soda lakes or alkaline hyper-saline lakes like Lake Mono,

[102,103]. The rotating γ subunit tags successive β subunits of the F1, California. They can also be isolated from highly alkaline enrichments inducing conformational changes that enable ATP synthesis to proceed created by industrial activities, e.g. indigo dye plants, from highly [22,104,105]. alkaline segments of certain insect guts and from estuaries or soils that have periods or micro-environments of high alkalinity (e.g. 3. Alkaliphilic bacteria and the bioenergetic challenges to periods of evaporation in estuaries or clay particles with alkaline proton-coupled ATP synthesis, solute-transport and motility crevices in soil) [107–111]. Alkaliphilic bacteria are from diverse taxonomic groups and have diverse physiologies but share challenges 3.1. Alkaliphiles — diverse types of cytoplasmic pH homeostasis and functional adaptations of proteins that are secreted or highly exposed on the outer surface. They have Alkaliphilic bacteria grow optimally at pH values above 9, with received attention because of their bioenergetic profiles, for the useful extreme alkaliphiles growing optimally above pH 10. Obligate applications of their proteins [112] and their potential for bioreme- alkaliphiles are incapable of growing at near neutral pH values. diation [113]. A sampling of alkaliphiles whose genome sequences are Facultative alkaliphiles exhibit significant growth at near neutral pH complete or in progress is presented in Table 2. but also exhibit deficits in growth yield or growth rates relative to neutralophilic bacteria at the same pH [45,48,49]. This is likely to 3.2. Cytoplasmic pH homeostasis and the problem it creates for pmf- reflect the significant number of adaptations of alkaliphile proteins, driven ATP synthesis membranes and physiology that enable alkaliphiles to grow well at extremely high pH values and to tolerate sudden alkaline shifts. In 1977, Garland predicted that alkaliphilic bacteria would have to According to a general engineering principle referred to as either the maintain a cytoplasmic pH significantly below the external pH values “no free lunch” principle or the principle of “conservation of fragility” [114]. Experiments in many alkaliphilic strains confirm this expecta- [106], mechanisms that increase robustness to a particular condition, tion. Alkaliphiles characteristically possess an impressive capacity for e.g. growth capacity at high pH, inevitably compromise robustness alkaline pH homeostasis, maintaining cytoplasmic pH values up to 2.3 1366 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377

Table 2 Alkaliphilic eubacteria whose genomes have been or are in the process of being sequenced.

GenBank Genome sequencing status Gram + or − F-type ATP synthase coupling iona

Aerobes Arthrospira platensis str. Paraca ACSK00000000 Draft assembly − H+ Bacillus alcalophilus None In progress + H+ Bacillus clausii KSM-K16 AP006627.1 Complete + H+ Bacillus halodurans C-125 BA000004.3 Complete + H+ Bacillus pseudofirmus OF4 CP001878.1 Complete + H+ Oceanobacillus iheyensis HTE831 BA000028.3 Complete + H+

Anaerobes Alkalilimnicola ehrlichii MLHE-1 CP000453.1 Complete − H+ Alkaliphilus metalliredigens QYMF CP000724.1 Complete + Na+b Alkaliphilus oremlandii OhILAs CP000853.1 Complete + Na+ Bacillus selenitireducens MLS10 ABHZ00000000 Draft assembly + H+ Desulfonatronospira thiodismutans ASO3-1 ACJN00000000 Draft assembly − H+ Desulfurivibrio alkaliphilus AHT2 ACYL00000000 Draft assembly − H+ Dethiobacter alkaliphilus AHT 1 ACJM00000000 Draft assembly + (V-type) Natranaerobius thermophilus JW/NM-WN-LF c CP001034.1 Complete + Na+b Thioalkalivibrio sp. K90mix CP001905 Draft assembly − H+

a The coupling ion hypothesis is based on the absence (for H+) or presence (for Na+) of the Na+-binding motif in c-subunits known to bind Na+ (Pogoryelov, D. et al. 2009 Nature Struct. Molec. Biol. 16:1068–1074). b The c-subunits of Alkaliphilus metalliredigens QYMF and Natranaerobius thermophilus JW/NM-WN-LF are 184 and 185 amino acids , respectively, more than twice the normal length of an F-type c-subunit. Alignment of these c-subunits with the Ilyobacter tartaricus c-subunit is indicative of Na+-binding. c Natranaerobius thermophilus JW/NM-WN-LF is also a halophile and thermophile. pH units lower than the pH of the bulk medium under carefully pH- phobic proteins [48,49,127,129–131]. The uniquely complex structure controlled continuous culture conditions as well as in highly buffered of Mrp antiporters is likely to be especially suitable for this role. Mrp batch cultures [47,115–119].InFig. 3, the cytoplasmic pH of two antiporters are hypothesized to present a large surface on the outside neutralophilic bacteria, E. coli and B. subtilis, and four different of the inner membrane that facilitates H+ capture and funneling into alkaliphilic bacteria is shown at an alkaline external pH at which the antiporter [48,49,131]. Mechanisms for Na+ re-entry to supply growth is still optimal or near-optimal for that particular organism cytoplasmic Na+, the substrate for the high antiport activity, are [115,117,118,120–122]. Two of the alkaliphiles, aerobic Bacillus sp. essential to sustain antiport-dependent pH homeostasis, especially TA2.A1 (recently renamed Caldalkalibacillus thermarum TA2.A1) [123] when the external [Na+] is low [48,49,132–135]. The major Na+ re- and anaerobic Clostridium paradoxum [124], are thermophiles. Their entry pathways are shown in Fig. 3 (and see Section 3.3 below). A alkaliphily is less extreme than found in the other two examples that variety of additional mechanisms for cytoplasmic H+ retention are are non-thermophilic alkaliphiles, the cyanobacterium Spirulina (also also present in alkaliphiles although none can thus far replace the called Arthrospira) platensis [125] and the extreme facultative antiport-based component [48,49]. alkaliphile B. pseudofirmus OF4 [47]. The lesser alkaliphily of the For two of the four alkaliphile types depicted in Fig. 3, successful thermoalkaliphiles probably reflects trade-offs among adaptive pH homeostasis results in a thermodynamic problem vis a vis ATP strategies [126]. For all the depicted bacteria, and others where it synthesis by H+-coupled ATP synthases. Both Bacillus sp. TA2.A1 and has been tested, the capacity of neutralophilic as well as alkaliphilic B. pseudofirmus OF4 exhibit a reduced pmf relative to neutralophilic bacteria for maintaining a cytoplasmic pH below the external pH, bacteria because the “reversed ΔpH”, i.e. acid inside relative to the depends heavily upon electrogenic Na+(Li+)(K+)/H+ antiporters. outside, has an adverse effect on the total pmf. The other pmf These secondary active transporters utilize the energy of the component, the transmembrane electrical potential (Δψ,inside transmembrane electrical potential (Δψ) component of the pmf or negative relative to outside), rises at elevated pH. However, it does smf that is generated by primary ion pumps. The antiporters take up not increase enough to offset the chemiosmotically counterproductive external H+ in exchange for monovalent cations (Cat+) from the pH gradient [11,45,117,118]. By contrast to the aerobic, non- cytoplasm, with a H+/Cat+ N1. This makes it possible to acidify the fermentative alkaliphiles, C. paradoxum is not subject to this problem cytoplasm relative to the bulk medium in a Δψ-dependent fashion because in this fermentative anaerobe, the Na+-coupled ATP [48,49,120,127–129]. In alkaliphiles, antiporter-based pH homeosta- synthase/ATPase only functions in the hydrolytic direction, generat- sis is thus far specific for Na+ as the efflux substrate (and the ing a Δψ and controlling cytoplasmic Na+ levels as it pumps Na+ antiporters usually have an additional capacity for Li+/H+ antiport). outward [115,136]. The ATP synthase of the fourth alkaliphile in Fig. 3, This is not the case in neutralophiles, which use both Na+(Li+)/H+ cyanobacterial Spirulina platensis, is largely and perhaps entirely and K+/H+ antiporters (Fig. 3) [48,49]. The antiporter that has a sequestered in thylakoids that are not continuous with the cytoplas- dominant role in pH homeostasis of two extreme alkaliphiles is the mic membrane [137–139]. To the extent that this sequestration is hetero-oligomeric Mrp antiporter that contains 6–7 different hydro- extant, the low bulk pmf across the cytoplasmic membrane does not

Fig. 3. Diagrammatic illustration of the capacity for alkaline pH homeostasis and patterns of H+ and/or Na+ use for bioenergetic work in representative neutralophilic and alkaliphilic bacteria. The cytoplasmic pH at external pH values that support growth at optimal or near-optimal rates are shown in the top two panels for two neutralophilic bacteria, E. coli (left) [121] and B. subtilis (right) [234], in the middle two panels for the alkaliphilic cyanobacterium S. platensis (left) [122,140] and the thermoalkaliphilic aerobe Bacillus sp. TA2. A1 (right) [117,173], and in the bottom two panels for the thermoalkaliphilic anaerobe C. paradoxum (left) [115,151] and facultatively alkaliphilic B. pseudofirmus OF4 (right). Cation/ proton antiporters (CPAs) are indicated with their ion-coupling specificity [48]. Flagellar motility is entirely H+-coupled in E. coli, coupled to H+ and Na+ in B. subtilis and, where known, entirely coupled to Na+ in the alkaliphiles [48,49,147]. The two neutralophiles use a mix of Na+ and H+-coupled systems for ion-coupled solute-transport whereas the + + + + alkaliphiles use Na -coupling exclusively [48,49].Na -coupled solute uptake, motility and the NavBP, voltage-gated Na channel all have roles in Na circulation that supports + alkaline pH homeostasis in B. pseudofirmus OF4 [120,234,235];NavBP also has a role in chemotaxis [120]. For C. paradoxum, flagellar motility has been shown [124] and while Na - coupling of motility or of solute uptake have not yet been directly demonstrated, both are proposed [151]. The arcs outside the membrane are shown to indicate Secondary Cell Wall Polymers (SCWP) [236] or Outer Membrane (O.M.) [237] or O.M.-Trichome layers [125], respectively, in Bacillus species and in Gram-negative E. coli and S. platensis. D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 1367 pose a problem for ATP synthase function. The pmf across the challenge of carrying out OXPHOS. It was incorrectly expected that thylakoid membrane, largely in the form of a ΔpH, is much larger than alkaliphiles would need to maintain a cytoplasmic pH close to the that across the cytoplasmic membrane [122,140]. range maintained by non-alkaliphiles [114].Itturnsoutthat There is a further feature of alkaliphilic bacteria that was alkaliphiles grow well at cytoplasmic pH values that are not tolerated unanticipated by Garland [114] and is relevant to the bioenergetic at all by non-alkaliphiles. Extreme alkaliphiles maintain significant 1368 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 growth capacities at surprisingly high cytoplasmic pH values. The 3.4. Other bioenergetic work in alkaliphiles is Na+-coupled, anaerobic optimal cytoplasmic pH for neutralophilic bacteria such as E. coli and alkaliphiles pump out Na+ rather than H+ but ATP synthase-dependent Bacillus subtilis is 7.5–7.6. Neutralophiles that lack Na+/H+ anti- ATP synthesis in alkaliphiles is H+-coupled porters or lack antiport function because of protonophoric uncoupling are restricted to growth in a pH range from 6.3–7.7 [141,142]. Ion-coupled “bioenergetic work” such as ion-coupled solute Neutralophiles that possess active mechanisms of alkaline pH uptake and flagellar motility in alkaliphiles is coupled exclusively to homeostasis maintain a cytoplasmic pH of 7.5–7.6 up to external pH Na+ whereas it is coupled exclusively to H+ or to a mix of H+ and/or values of 8.5 (Fig. 3). At pH values above 8.5 the growth rate of Na+-coupled transport or “Mot” systems in neutralophilic bacteria neutralophiles usually slows and there is greatly reduced capacity for (Fig. 3) [45,147]. The use of Na+-coupled systems for solute uptake growth at pH≥9.0 [48,49]. Moreover, growth arrest occurs when the and motility is consistent with the notion that the smf is larger than cytoplasmic pH of E. coli rises to ∼pH 8 after sudden alkalinization of the available pmf under highly alkaline conditions. Na+ is used for the medium to 8.3 and occurs in B. subtilis when this organism is motility and transport even in many of the Bacillus alkaliphiles such as shifted suddenly to medium at pH 9.0 [143,144].Bycontrast, B. pseudofirmus OF4 that were isolated from environments in which alkaliphiles maintain a cytoplasmic pH of 7.5 and an optimal growth the Na+ concentration is not particularly high and special mechan- rate when the external pH is as high as 9.5. When the external pH is isms to capture Na+ for antiport are important [45,120]. It was even higher, the alkaliphile's pH capacity to retain a cytoplasmic pH of therefore anticipated that ATP synthesis in alkaliphilic bacteria would 7.6 is exceeded. However, these bacteria continue to grow in the also turn out to be Na+-coupled and was further suggested that presence of cytoplasmic pH values way above those that are neutralophilic organisms possessed the capacity to switch to Na+- compatible with neutralophile survival and/or growth. For example, coupling of ATP synthesis upon a drop in pmf [148–150]. ATP B. pseudofirmus OF4 grows optimally even when the cytoplasmic pH synthases of anaerobic alkaliphiles, most of which function as ATPases has risen to 8.2–8.3 (at pHout =10.5) and when pH 8.5-equilibrated physiologically, are the only alkaliphile ATP synthases/ATPases thus cells are suddenly shifted to an external pH of 10.5, they maintain this far found to be Na+-coupled [151]. This coupling choice is clearly cytoplasmic pH. B. pseudofirmus OF4 continues to exhibit significant adaptive for an alkaliphile for an ion-pumping ATPase. Use of Na+ non-fermentative growth at external pH values over 11, at which the pumping would meet the needs of Δψ and smf generation to support cytoplasmic pH is 9.5 [45,47,118]. Over the range of external pH from of Na+-coupled solute uptake and motility and would help protect 10.5 up past 11, the growth rate decreases in parallel to the rise in against excessive Na+ accumulation under circumstances of elevated cytoplasmic pH, underscoring the centrality of pH homeostasis in salt. Importantly, use of Na+ as the pumped ion would avoid the likely spite of the extraordinary tolerance of a high cytoplasmic pH. Little catastrophic loss of cytoplasmic H+ that could occur if the ATPase is known yet about adaptations of cytoplasmic components that activity was coupled to H+ instead. There is one recently character- underpin this tolerance, but there are several “plumbing problems” ized anaerobic alkaliphile, Alkaliphilus metalliredigens [113], that this tolerance raises with respect to synthesis of ATP. First, if an that catalyzes respiratory metal reduction and apparently possesses alkaliphile is to take advantage of OXPHOS as part of its metabolic aNa+-coupled ATP synthase, based on sequence motifs (see Table 2). strategy, it must minimize outward H+ leaks through the membrane This synthase probably completes a Na+ cycle that contains putative via any of the membrane proteins involved in the process during membrane-associated, Na+-translocating organic acid decarboxylases transient reductions of pmf. Second, mechanisms of H+ capture need [152]. to address not only the thermodynamic problem of a low pmf but By contrast, ATP synthases of aerobic or facultatively aerobic also the kinetic problem of H+ capture from a highly alkaline alkaliphiles whose physiological role is ATP synthesis, have thus far all exterior. been found to couple synthesis to H+ [45,47,153,154]. Moreover, this synthesis is inhibited by treatments that abolish the Δψ [155]. Coupling to H+ has similarly been found for Vibrio species. They had 3.3. How low is the pmf during non-fermentative growth of extreme been expected to have Na+-coupled synthases since they have one alkaliphiles at high pH? primary respiratory pump coupled to Na+ and have a larger smf than pmf [156]. The alkaliphile synthases exhibit no ability to use Na+ as an Thus far, the problem of a low pmf has been documented primarily alternative coupling ion [47,154,157]. This is in contrast with the Na+- in Gram-positive alkaliphiles of the Bacillus genus or related genera. In coupled synthases that are found in fermentative bacteria such as P. these alkaliphiles, the “reversed ΔpH”, acid inside relative to outside, modestum, which can use H+ for coupling to ATP synthesis when Na+ results from successful pH homeostasis. The reversed ΔpH detracts concentrations are low [158]. from the total pmf and there is incomplete compensation by a rise in Δψ. Possibly alkaliphiles will yet be found that have different proton- 4. Why do alkaliphilic bacteria use H+-coupled rather than pumping capacities and membrane properties such that a compen- Na+-coupled ATP synthases for OXPHOS? satory Δψ eliminates the adverse effect of pH homeostasis on the total pmf. For the two aerobic alkaliphilic Bacillus strains shown in Fig. 3 As already noted (see Eq. (i) in Section 2.3), the energy cost of ATP that grow well at pH 10–10.5, the effect of pH homeostasis on the pmf synthesis rises as the pH rises and the energy requirement for pH is significant. In pH-controlled batch cultures, thermoalkaliphilic homeostasis at high pH further challenges alkaliphiles. Perhaps these Bacillus sp. TA2.A1 cells exhibited a pmf of −164 mV during growth at elevated energy needs are the basis for use of pmf-coupled ATP pH 7.5, where growth is fermentative [123] and of −78 mV at pH 10; synthases. The pmf in alkaliphiles is usually generated during electron at pH 10, the organism can grow non-fermentatively and exhibited transport events that can cover a large redox span and maximize robust ATP synthesis [117]. In alkaliphilic B. pseudofirmus OF4 grown energy conservation from growth substrates [152]. It is notable that under continuous culture conditions with rigorous control of the except for the final redox site of the terminal oxidases, the redox medium pH, the pmf during growth at pH 7.5 was −136 mV and at pH potentials of respiratory chain components from alkaliphilic Bacillus 10.5 was −50 mV. Typical pmf values for neutralophiles, e.g. E. coli species are significantly lower than those of neutralophilic homo- and B. subtilis,atpH7–7.5 are ∼−130 to −140 mV [145,146], which is logues [159–161]. Use of sodium-coupled energetics in organisms comparable to the pmf of B. pseudofirmus OF4 at pH 7.5 [116,118].InB. without proton-tight membranes has been proposed to have pseudofirmus OF4, however, ATP synthesis was more robust at pH 10.5 preceded proton energetic during evolution [162]. However, among than at pH 7.5 even though the pmf was much lower at the higher pH current bacterial life forms, sodium energetics may be best suited to [116]. “boutique” situations in which the niche is highly specialized. Proton- D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 1369 based energetics may be required in niches that require particularly detectable hydrolytic activities of enzymes from B. pseudofirmus OF4 high energy expenditures and/or are highly competitive niches in and B. alcalophilus [153,154]. It was suggested that due to the which fast growth rates and high growth yields are important. combined challenges of high temperature and high pH, the thermo- A second consideration that may preclude the use of the known alkaliphile would be especially at risk of losing protons [173]. types of Na+-coupled ATP synthases by alkaliphiles is that those It appears that most if not all F-type ATPases share one inhibitory synthases can also couple to H+ [158]. In cells that have large mechanism, that of tight binding of MgADP [175], while other “reversed” pH gradients, inside more acidic than the outside, the mechanisms of inhibition are more specific to the type of organelle outwardly directed pH gradient might therefore inhibit synthesis of or organism. It is thought that the MgADP is bound to a high-affinity the ATP synthase even when Na+ is used as coupling ion. catalytic site. Exposure of the enzyme to inorganic phosphate, or Finally, there are indications that respiratory alkaliphiles and nucleotides, or a pmf can overcome the inhibition, probably by neutralophiles derive added value from using a H+-coupled ATP displacing the ADP [175,176]. In addition, mitochondria and chlor- synthase. At acidic pH, ATP synthases/ATPases that function in the oplasts each have special mechanisms to inhibit the ATPase activity. hydrolytic, H+-pumping mode are induced and have been shown to Mitochondria are unique in using a naturally occurring protein, the play a role in preventing adverse acidification of the cytoplasm [163– inhibitor protein, to regulate their ATPase activity. This protein, IF1,is 166]. Conversely, increased expression and activity of ATP synthases a small basic protein that inhibits hydrolytic activity below pH 8 but that function in the synthetic direction are implicated in a role in has no effect on ATP synthesis [177]; recently, the crystal structure of alkaline pH homeostasis by transcriptome observations and studies of a monomeric form of the inhibitor protein bound to mitochondrial F1 ATP synthase mutants. Alkali challenge of E. coli, B. subtilis, was reported [178]. The ATP synthase from chloroplasts differs from Desulfovibrio vulgaris,andCorynebacterium glutamicum are all all other ATP synthases in its γ subunit, which contains a unique associated with increased expression of H+-coupled ATP synthases regulatory disulfide that is reduced physiologically by a thioredoxin [76,167–170]. A mutant of B. subtilis that has lost function of its Mrp system during exposure to light. This results in activation of both its antiporter also exhibits increased expression of its ATP synthase [171]. hydrolytic and its synthetic activity. Chloroplast F1 can be reduced by The H+ taken up during ATP synthesis contributes to pH homeostasis dithiothreitol in vitro, which also stimulates ATPase activity [179]. in alkaliphiles. This was shown in studies of a panel of mutants in Structural rearrangements between γ and ε upon disulfide reduction alkaliphile-specific motifs of the B. pseudofirmus OF4 that have defects probably account for the activation of activity [180]. Interestingly, the of differing magnitude in their capacity to make ATP [172]. The cyanobacterial ATP synthase from alkaliphilic S. platensis shares a mutants and wild-type control cells were depleted of ATP and number of properties with the chloroplast enzyme, including equilibrated at pH 8.5, after which they were shifted to a malate stimulation of its hydrolytic activity by methanol [181] and enhanced containing medium at pH of 10.5. Although antiporter-based pH ATP synthesis at sub-optimal pmf values when reduced by dithio- homeostasis and ATP synthesis are both pmf-consuming processes, threitol [182,183]. However, it lacks the regulatory disulfide found in the immediate post-shift cytoplasmic pH was lowest, below 8.2, in a the organelle enzyme, leading to the suggestion that other cysteines mutant that synthesized ATP more rapidly than wild-type. The post- in the S. platensis γ subunit may be involved in this activation shift cytoplasmic pH of the remaining mutants was higher than wild- [182,183]. type and correlated with the extent of their deficits in ATP synthesis. Chloroplasts and many bacterial enzymes share a common

Contributions of ATP synthases to alkaline pH homeostasis would regulatory feature, inhibition by the endogenous ε subunit of the F1 create a selective advantage for use of H+-coupled ATP synthase as moiety. Either removal of the ε subunit or truncation of the C-terminal long as the bioenergetic challenges for their use can be overcome or end of ε in the F1 results in activation of hydrolytic activity in E. coli, bypassed. Bacillus sp. PS3, Bacillus sp. TA2.A1, or spinach chloroplasts [184]. From structural information on the isolated ε subunit and the γε + 5. Directionality of the H -coupled alkaliphile ATP synthases: complex [185–187] of E. coli F1, and from cross-linking experiments latency of ATPase activity is critical but not unique to alkaliphiles with the E. coli [188] and Bacillus sp. PS3 enzymes [189],itwas concluded that the C-terminal end of the ε subunit exists in two Before reviewing alkaliphile strategies to overcome the challenges conformational states. When ε is in a putative “extended” state, of low H+ concentration in the bulk and sub-optimal pmf conditions, stretched alongside the γ subunit, the enzyme's hydrolytic activity is we note mechanisms that are in place in alkaliphiles that prevent the blocked, but not the synthetic activity. When ε is in a putative use of the ATP synthase as an ATPase. Such controls are important in “contracted” conformation, away from γ, hydrolysis is active [184]. all ATP synthases, not just in alkaliphiles and not just in bacteria. Inhibition by the extended ε form is proposed to be mediated by Control of ATPase activity prevents depletion of the ATP pool under binding of basic amino acids in the C-terminal region of ε to the acidic conditions in which there is a drop in pmf. For alkaliphiles, stringent groups in the β subunit DELSEED region that interacts with the γ controls are further needed because loss of the ATP pool is not the only subunit. Replacement of either the ε basic residues or the β acidic major risk. The other risk is that at high pH–low pmf, the central residues with alanines results in activation of hydrolytic activity in process of pH homeostasis would be undercut by ATPase-dependent Bacillus sp. PS3 [190]. In the thermoalkaliphile, replacement of the 6 alkalinization of the cytoplasm. As noted earlier, while ATP synthases arginines in the C-terminal region increased the activity 5-fold, are generally described as reversible machines, this characterization although the enzyme could still be further stimulated 8-fold by LDAO does not apply to the physiological reality of many of them. For [191]. example, membrane vesicles or purified ATP synthases from aerobic Another basis for latency of the thermoalkaliphile ATP synthase, alkaliphilic Bacillus species catalyze only very low rates of ATP probably a dominant basis, was revealed in a structural study of the F1 hydrolysis, but the hydrolytic activity can be dramatically increased domain (lacking the δ subunit) [192]. Salt bridges were identified by solvents such as methanol or detergents such as octyl glucoside or between two successive arginines in the N-terminus of the γ subunit LDAO [153,173,174]. The purified, reconstituted ATP synthase from and two aspartates in the helix–turn–helix C-terminal domain of the β thermoalkaliphile Bacillus sp. TA2.A1 was shown to catalyze ATP subunit. These salt bridges result in a bent orientation of the γ subunit synthesis in response to an imposed membrane potential but it was relative to that found in other F1 structures and appear to prevent not possible to detect proton-pumping ATPase activity even when the rotation in the hydrolytic direction. The tilted γ subunit was ATPase activity was increased to high levels by LDAO [173]. The ATP visualized in remarkable electron micrographs of the purified ATP synthases of non-thermophilic alkaliphilic Bacillus species are less synthase, which showed that the γ subunit was bound to an edge of extremely latent, exhibiting proton-pumping together with low, but the c-ring and then angled up into the head piece [192]. Biochemical 1370 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 support for the effect of the salt bridges on activity was found by conclusion that any given organism has a constant c-ring stoichiom- mutation of the γ subunit arginines to glutamines, which created an etry that is determined by the primary structure of the subunit active enzyme [192]. [61,195–198]. This means that a facultative alkaliphile such as B. pseudofirmus OF4 will use an ATP synthase with the same c-ring

6. Resolution of the low pmf-high phosphorylation potential stoichiometry at the vastly different ΔGp/pmf ratios across its pH range dilemma: contribution of a high c-subunit stoichiometry from pH 7.5 to ≥10.5. Unless the c-subunit stoichiometry of extreme alkaliphiles is much higher than the current reported range, the low Different bacteria possess different numbers of c-subunits/ATP pmf problem that B. pseudofirmus OF4 faces at high pH will only be synthase. A complete 360° turn of the c-ring yields 3 ATP and the modestly addressed by its c-subunit stoichiometry. Moreover, an number of H+ or Na+ that move across to the N-side of the membrane energetic price will be exacted for use of an enzyme with a high c- during a full turn is equal to the number of c-subunits in the ring. subunit stoichiometry at pH 7.5 even if the stoichiometry is high but Therefore the H+/ATP ratio varies among different bacterial strains. within the current range. The use of a relatively inefficient enzyme for This has bioenergetic consequences because: ATP synthesis at pH 7.5, under conditions in which the pmf is not low, would disadvantage the alkaliphile at pH 7.5 relative to neutralophilic þ = Δ = ð Þ H ATP = Gp pmf iii organisms using enzymes with lower stoichiometries. For the alkaliphile this is an example of a trade-off in which an adaptation to the extreme environment results in poorer adaptation to the non- ΔG = ΔG0 + RT ln ½ATP = ½ADP ½P ðivÞ p i extreme environment. Other such trade-offs have been demonstrated [45,135]. The affects of these cumulative trade-offs probably account Δψ−ðÞ: = ⋅Δ ð Þ pmf = 2 3RT F pH v for the surprising finding that the molar growth yields of B. pseudofirmus OF4 on malate are comparable at pH 7.5 and 10.5

ΔGp is the phosphorylation potential that reflects the energy even though the energy costs for growth at pH 10.5 are higher required to sustain the [ATP]/[ADP][Pi] ratio. The bulk pmf (consisting [116,118]. of a transmembrane difference in electrical potential, Δψ, and a transmembrane difference in pH, ΔpH)istheelectrochemical 7. Resolution of the low pmf-high phosphorylation potential gradient assessed between the aqueous phases on the two sides of dilemma: hypotheses for sequestered proton transfer to the ATP the coupling membrane. synthase from primary proton pumps If an ATP synthase has a c-ring composed of 10 c-subunits, H+/ATP + and ΔGp/pmf are both 3.3. The efficiency is greater than that of a Na -coupling was not found in aerobic alkaliphiles and the rotors synthase with 13 (as in thermoalkaliphile Bacillus sp. TA2.A1 [63,65]) of alkaliphile ATP synthases are not expected to have c-subunit + or 15 (as in S. platensis [20,61]), for which the H /ATP as well as ΔGp/ stoichiometries high enough to resolve the discrepancy of the high pmf are respectively 4.3 and 5.0. However, since the ΔGp/pmf is ΔGp achieved by alkaliphilic bacteria at low bulk pmf. Rather, this highest in the synthases that have the most c-subunits/rotor ring, a unresolved energetic conundrum became a major paradigm for a high c-subunit stoichiometry would be expected to help overcome the large body of recent work that revisits a tenet of the original problem of a low pmf in alkaliphiles (see discussion in [61]). Indeed formulation of Mitchell's chemiosmotic hypothesis. Mitchell posited the 13 c-subunits of the thermoalkaliphile Bacillus sp. TA2.A1 rotor is that the pmf that drives H+-coupled bioenergetic work is entirely in toward the high end of the 10–15 c-subunit stoichiometries reported the form of a delocalized bulk electrochemical gradient between two to date [63,65]. This can account for part of the observed capacity of aqueous phases, e.g. the bacterial cytoplasm and the external growth this organism to synthesize ATP at pmf values that are lower than medium [89,199]. Williams challenged this tenet in his own those found in other organisms. However, the c-subunit stoichiometry formulation that was contemporaneous with Mitchell's, positing of 13 does not fully account for the observed ΔGp/pmf at pH 10. At pH charge densities and proton pathways along the membrane that 10, the pmf of Bacillus sp. TA2.A1 was −78 mV and the ΔGp was required proximity and specific properties of participating catalysts ∼430 mV, so ∼17 c-subunits/rotor would be needed instead of 13 to [200,201]. Other bioenergeticists as well as bacterial physiologists also account for the ΔGp observed [65,117]. For B. pseudofirmus OF4, there advanced experimental data and models for alternatives to com- would have to be 29 c-subunits per ring to account for observed ΔGp/ pletely delocalized energy-coupling to bulk electrochemical ion pmf ratios at pH 10.5 (ΔGp =478 mV and pmf=−50 mV) and the gradients [47,202–209]. As reviewed recently by Mulkidjanian et al. stoichiometry would have to be closer to 33 c-subunits/ring at [210], proposals of localized and/or surface-associated H+ transloca- pHN10.5 [116,118,172]. It currently seems unlikely that such high tion gained traction after experimental evidence using new technol- numbers of c-subunits will be found. Moreover, the two highest c-ring ogies showed a measurable time delay between the emergence of stoichiometries found to date are 14 in spinach [67–69] and 15 in pumped H+ on the outer surface of a coupling membrane and their S. platensis [20,61]. In both of these organisms, the ATP synthase is equilibration with the bulk aqueous phase [211,212]. Evidence from sequestered in organelle membranes and energized by a substantial experiments and modeling also emerged for a capacity for both H+ pmf that is largely in the form of a ΔpH. This raises the possibility that storage and H+ translocation near the surface [213–215]. Together, the highest c-subunit stoichiometries will turn out to be more closely delayed H+ equilibration with the bulk and near surface H+ associated with factors other than the magnitude of the pmf, e.g. the translocation create the opportunity for H+ consumers such as the preponderance of ΔpH over Δψ [8,11]. antiporters and ATP synthase of alkaliphiles to couple transport and Another point about c-ring stoichiometry is relevant to the profiles ATP synthesis to H+ that reach them at the surface before they of alkaliphiles. Before stable c-rings were isolated and visualized, the equilibrate with the bulk. Properties of the membrane proteins and possibility had been raised of a variable c-subunit stoichiometry for their surfaces, e.g. the large Mrp antiporter surface, and of the individual bacteria. Variable c-subunit stoichiometry was proposed as membrane lipids, e.g. the high cardiolipin content of B. pseudofirmus a possible adaptive strategy of bacteria that faced environments in OF4 membranes [45,47,216], could affect the retention and/or which the pmf varies, a group that would include facultative translocation of H+ near the surface [210,214,217]. In this scenario, alkaliphiles [193,194]. The hypothesis of a variable c-subunit stoichi- the “inadequate” bulk pmf of alkaliphiles would not be the relevant ometry in a particular organism is no longer considered tenable. driving force because the near surface pH would replace the external Studies of intact and partially fragmented rings as well as biochemical bulk pH for purposes of assessment of the effective transmembrane assessments of rings isolated under different conditions support the chemical gradient of H+. D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 1371

A specific model of interfacial potential barriers that impede H+ equilibration of H+ with the external medium and that such a barrier escape from the membrane surface region predicts that such a barrier has a bioenergetic impact. Cyanobacterial alkaliphiles whose ATP is common to all membranes and that the near surface pH, referred to synthase is sequestered in thylakoids, also lack the c- and a-subunit as pHs, could be as low as pH 6 [218,219]. It will be important to try to features possessed by the extremely alkaliphilic Bacillus species. assess the near surface pH experimentally in a native system such as Among the alkaliphilic Bacillus species, in which the alkaliphile- B. pseudofirmus OF4. A surface pH in the low end of the range permitted specific sequence features are found, the “fullness” of the recognized by the computational model of pHs may clash with the observed panel of sequence deviations that are found in particular alkaliphile properties of the organism. If the functional pH of the membrane strains correlates with how extreme the alkaliphily is in the strain. surface was pH 6.0 when the bulk external pH is very high, the total However, if the organism is a “poly-extremophile”, e.g. a thermoalk- pmf at pH 10.5 would be high since there is a Δψ of −180 mV at that aliphile, there are a few shared alkaliphile-specific sequence features pH. This would eliminate both the need for the special adaptations to but there are even more poly-extremophile deviations from the the alkaliphile OXPHOS machinery that the alkaliphilic bacilli possess consensus sequence that are different from those in bacteria that are (see Section 8 below) and the need for alkaliphiles to use only Na+- only alkaliphiles. Thus far, studies testing the physiological role of coupling for solute uptake and motility. A functional surface water alkaliphile-specific adaptations in ATP synthase in a native alkaliphile layer at pH 6 might also be difficult to reconcile with the progressive setting have all been conducted in B. pseudofirmus OF4, which is alkalinization of the cytoplasm at external pH values above pH 9.5. genetically tractable so that mutations can be introduced into the Perhaps the delocalized surface pH is lower than the external pH at chromosomal atp operon [44,172]. Use of the native setting is external pH values ≥10.5 because of the general property of interfacial important since, the adaptations evolved in a context with specific barriers to H+ equilibration and this offsets the alkaliphile low pmf homeostasis capacities, membrane lipids and respiratory chain problem but does not completely resolve it. Perhaps localized H+ components all in place [45,160,216,226]. microcircuits are also an important part of the energy-coupling solution in alkaliphiles and elsewhere. These circuits might involve 8.1. Adaptations at the heart of the rotor: c-subunit motifs mosaics or small coupling units that involve proximity of the H+ pumps and consumers, and rely upon the special features of the As shown in the alignment shown in Fig. 4, the c-subunits of protein participants and on properties of the membrane [207,208]. The extremely alkaliphilic Bacillus species such as B. pseudofirmus OF4, B. efficacy of localized H+ microcircuits has recently gained support from halodurans C-125 and B. alcalophilus have two major motifs that differ spectroscopic studies of model liposomal systems in which lipid from the consensus motif for non-alkaliphilic Bacillus species. The first headgroups behaved as H+ collecting antenna and interplay with of these is the AxAxAxA (or at least three Ala residues) replacing the membrane lipids increased the rate of H+ uptake by membrane- neutralophile consensus GxGxGxG motif in the middle of the N- associated proteins that catalyze such uptake [220,221]. terminal helix of the hairpin-like subunit. The more moderate The possibility of H+ transfers during OXPHOS during very close alkaliphile Bacillus clausii has GxAxGxA, only two Ala substitutions, encounters or dynamic physical interactions between one or more and the largely fermentative moderately alkaliphilic marine bacteri- respiratory chain components and the ATP synthase has also been um Oceanobacillus iheyensis has the non-alkaliphilic Bacillus consen- raised. Such interactions could support effective sequestered H+ sus sequence of GxGxGxG. By contrast, thermoalkaliphile Bacillus sp. transfers in alkaliphiles and even non-alkaliphiles [159,222,223]. TA2.A1 has a GxSxGxS, which is a different variant than that of the Some of the special adaptations that have been identified in alkaliphile-alone deviation from the consensus. The larger serine alkaliphile ATP synthase (see Section 8 below) may provide a gateway residues of the Bacillus sp. TA2.A1 subunit have been shown to for genetic-biochemical tests of various sequestration and microcir- increase the diameter of the thermoalkaliphile c-ring [63]. This may cuit models of H+ transfer. facilitate a stoichiometry of 13 subunits in this Bacillus or in alkaliphilic Bacillus strains in general, e.g. as opposed to 10 in yeast 8. Special adaptations of alkaliphile ATP synthase are required for or E. coli. However, use of an altered GxGxGxG motif that enlarges the function at high pH: an example of adaptive variations among ATP ring diameter cannot be a general requirement for a high c-subunit synthases stoichiometry since the largest stoichiometry to date, at 15 c- subunits/ring, is found in alkaliphilic cyanobacterium S. platensis, There is direct experimental evidence that special adaptations of which has the neutralophilic consensus GxGxGxG [20,63]. the ATP synthase play roles in OXPHOS in the native B. pseudofirmus A panel of single, double, triple and quadruple Ala→Gly mutations OF4 setting. The adaptations were recognized when this alkaliphile's was introduced into the AxAxAxA motif encoded by the atpE gene in atp operon was first sequenced and compared with sequence data the native chromosomal atp operon of alkaliphilic B. pseudofirmus from two other extreme alkaliphiles, Bacillus halodurans C-125 and OF4. Although there was no significant effect on the levels of Bacillus alcalophilus in comparison with sequences from neutralophi- membrane-associated β-subunit, ATPase (hydrolytic activity activat- lic bacteria. Several striking sequence deviations from the neutralo- ed by octylglucoside) or growth on glucose, the mutations clearly phile consensus sequence were found in regions of the a- and c- affected non-fermentative growth and ATP synthesis capacity. With subunits that had been shown to have roles in H+ translocation increasing numbers of Gly substitutions, there was progressively through the ATP synthase [45,224,225]. Further sequence data from a poorer non-fermentative growth both at pH 7.5 and 10.5, with the more diverse group of alkaliphilic bacteria, including alkaliphilic effect being somewhat greater at high pH. The quadruple mutant with Bacillus species as well as some cyanobacteria, thermoalkaliphiles and a complete GxGxGxG motif replacing the native AxAxAxA grew Gram-negative alkaliphiles have already been instructive. The normally on glucose but exhibited almost no non-fermentative alkaliphile-specific sequence deviations from the neutralophile growth. Almost no ATP synthesis was observed by the mutant consensus are found among Bacillus species that possess secondary synthase, as assayed in ADP+Pi-loaded right-side-out membrane cell wall polymers such as glycoprotein S-layers or teichoic/ vesicles from the quadruple mutant alkaliphile cells in comparison teichuronic acids that attach to the peptidoglycan (see Fig. 3) but with wild-type cells [44]. Perhaps the AxAxAxA motif is necessary for lack an outer membrane that is found in Gram-negative bacteria the assembly of an optimal, somewhat high c-subunit complement in [132–135]. So far, these sequence features are not apparent in the the particular rotor scaffold of alkaliphilic Bacillus species (Fig. 4). limited number of genome atp operon sequences available for Gram- Interestingly the “x” residues of the motif have some specificity. negative alkaliphiles. This raises the possibility that the outer The earliest attempt to probe the importance of the AxAxAxA motif in membrane of Gram-positive bacteria acts as a barrier to the B. pseudofirmus OF4 was carried out by the substitution of the entire 1372 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377

GAGIGNG found in Bacillus megaterium for the AGAIAVA found in B. 10.5, but not pH 7.5, was significantly impaired. However, ATP pseudofirmus OF4, and there was almost no ATP synthase found in the synthesis upon re-energization of starved cells was more robust than membranes [172]. Later mutagenesis showed that a single mutation that of wild-type at pH 10.5 and was accompanied by very strong of the first “x”, the Gly17 that follows the first Ala16 of the B. initial pH homeostasis. In vitro, a quick peak of ATP synthesis was 17 pseudofirmus OF4 c-subunit, to the Ala found in B. megaterium observed upon energization of ADP+Pi-loaded vesicles of this mutant caused a significant loss of membrane-associated enzyme [44]. The that was followed by loss of the ATP [172]. The presence of Thr33 near importance of the “x” residues in this motif is also clear in the Na+- the loop edge may affect how torque is generated in some subtle way translocating ATP synthases/ATPases in which a Gln residue between that results in strong initial torque that is quickly dissipated in the the final Gly residues of the GxGxGQG motif in those strains is part of context of the alkaliphilic Bacillus scaffold. the Na+-binding motif (see Fig. 4). The second major sequence deviation that was called the 8.2. Apparent adaptations in the ATP synthase a-subunit, the ion- “alkaliphile PxxExxP motif” by Arechaga and Jones [227] is the translocating stator subunit presence of two Pro residues that flank the key c-subunit carboxylate of the C-terminal helix (Glu54 in B. pseudofirmus OF4). Usually only a The alignment in Fig. 5 highlights (in blue) the Lys180 that is found single Pro is found three residues away on the periplasmic (C- in the a-subunits of alkaliphilic Bacillus species that grow non- terminal) side of the Glu. That Pro is replaced by Thr in S. platensis and fermentatively at high pH, including thermoalkaliphilic Bacillus TA2. by Gly in Na+-translocating enzymes (Fig. 4). It is the first Pro, Pro51, A1. In general the residue at this position (Gly218 in E. coli and other which is novel in the alkaliphile PxxExxP motif. This motif is again not non-alkaliphiles) is considered to be part of a trio of residues that has found in either thermoalkaliphilic Bacillus sp. TA2.A1 or O. iheyensis. critical roles in successful capture and translocation of H+ [8]. The Replacement of Pro51 by Ala resulted in almost complete loss of ATP second one is the neighboring Glu (Glu181 in alkaliphiles and Glu219 in synthetic capacity at pH 10.5 and a smaller deficit at pH 7.5 in vesicle E. coli) that is conserved among H+-, but not Na+-translocating ATP assays [172]; its replacement with Gly was compatible with synthases (Fig. 5). The third is highlighted (in blue) in several significant synthetic capacity but there was proton leakiness that alkaliphiles in which it is Gly, Gly212 in B. pseudofirmus OF4. The presumably accounts for significant growth deficits observed at high residue at this position is different in other bacterial types, Ser in non- pH even on glucose [44]. These results highlight the special alkaliphilic Bacillus species, His in E. coli and Asp in Na+-translocating importance of strategies that increase proton-tightness in the ATP synthases. Several lines of evidence indicate that the residues at alkaliphile setting. positions of the Lys180 and Gly212 of B. pseudofirmus OF4 interact A third sequence deviation of extreme alkaliphiles is a threonine within the ion uptake pathway from the external surface. This residue, Thr33, that directly precedes the conserved RQPE loop of the pathway leads to the a- and c-subunit interface at which H+ are c-subunit on its N-terminal side next to the loop Arg34. It is found only transferred to the rotor during ATP synthesis [172,228–230]. The in extremely alkaliphilic Bacillus species such as the three top species positive charge of the conserved Arg is critical in creating an shown in Fig. 4. Ala (or Gly) is otherwise usually found at that environment that facilitates the exchange of H+ at this interface position. Mutagenesis of the B. pseudofirmus OF4 Thr33 to Ala led to an and is also highlighted in Fig. 5 (red). Consequences of mutagenesis of interesting phenotype in which overall growth yield on malate at pH the Lys180 and/or Gly212 of B. pseudofirmus OF4 to the Bacillus

Fig. 4. Alignment of c-subunit of the ATP synthases from several alkaliphiles and neutralophiles. The organisms in blue are the alkaliphiles: the first three are extreme alkaliphiles, the B. clausii is a more moderate alkaliphile, Bacillus sp. TA2.A1 is a moderate thermoalkaliphile, and Oceanobacillus iheyensis is a primarily fermentative marine alkaliphile. The organisms in black are neutralophiles. The organism in green is the alkaliphilic cyanobacterium S. platensis. The bottom three organisms in brown contain Na+-coupled ATP synthases. The organisms with an asterisk are also shown in Fig. 3. Residue numbers are given for the B. pseudofirmus OF4 (top) and the E. coli c-subunits (bottom). Two helices marked in yellow are connected by a loop (RQPE, black shaded). The glycines of the conserved GXGXGXG motif in the N-terminal c-subunit helix of neutralophiles are partially or completely replaced by alanines in alkaliphilic Bacillus species but not thermoalkaliphilic Bacillus TA2.A1, which has distinct substitutions, or in alkaliphilic cyanobacterium S. platensis. The Pro51 of P51XXEXXP motif [227] in the C-terminal helix of extreme alkaliphiles substitutes for the glycine or alanine in neutralophiles. The residue in red, Glu or Asp, is the key carboxylate involved in the ion-binding site. The residues in green are part of the Na+ binding motif [43] although one of the residues is also present in the H+-coupled ATP synthase of S. platensis. The red arrow indicates the essential carboxylate, Glu54, that is involved in H+ binding. The National Center for Biotechnology Information (NCBI) gene accession numbers for the data shown are: B. pseudofirmus OF4, AAC08039; B. halodurans C-125, NP_244626; B. alcalophilus,AAA22255; B. clausii KSM-K16, YP_177348; Bacillus sp. TA2.A1, AAQ10085; O. iheyensis HTE831, BAC14936; B. licheniformis ATCC 14580, AAU42745; B. megaterium, AAA82521; B. subtilis subsp. subtilis str. 168, NP_391567; G. kaustophilus HTA426, YP_149216; E. coli K12 substr. DH10B, YP_001732558; S. platensis, 2WIE_A; P. modestum, CAA37840; I. tartaricus, AAM94908; and C. paradoxum, ABB13421. The cartoon model to the right of the alignment shows the two helices of the c-subunit monomer from B. pseudofirmus OF4 with the top of the cartoon (the loop) corresponding to the N-side of the membrane. D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 1373 consensus (Gly for Lys180 and Ser for Gly212) show that the presence of enrich our understanding of the plasticity of ATP synthase function. Lys180 in the putative H+ uptake path is critical for H+ capture and They will also continue to shed new light on basic mechanistic principles successful ATP synthesis at high pH but not as critical at near neutral that are common to many types of synthases and on particular pH. A further role of both Lys180 and Gly212, perhaps in concert, is mechanistic adaptations that are in place in specific groups of synthases. indicated by the finding that they help prevent H+ loss to the outside For example, the aerobic alkaliphilic Bacillus species have challenged bulk phase, e.g. during transient drops in pmf [172]. The effect of and enlarged models of H+-coupling in general and will be an important several replacements of Lys180 in the a-subunit of thermoalkaliphilic paradigm in which to test these models in a native setting. The Bacillus sp. TA2.A1 ATP synthase was also studied using an experi- alkaliphile also appears also to have specific adaptations in connection mental system in which the enzyme was expressed in E. coli. The with its unique and central challenge of alkaline pH homeostasis. thermoalkaliphile synthase does not support non-fermentative Among the many future goals of interest are the following. growth in E. coli but can be studied in ADP+Pi-loaded vesicles with Experimental approaches that provide defined parameters for intra- and extravesicular pH values that support synthetic activity. further models of surface-associated pmf or microcircuits need to be These experiments showed that this enzyme retains significant syn- developed in alkaliphile settings. These parameters will facilitate thetic ability with Arg and His as well as Gly replacing Lys180, with the evaluation of contributions of delocalized near surface H+ vs H+ pH profile for activity depending upon the pKa of the replacing residue whose localized transfer depends upon particular respiratory chain [231]. Preliminary evidence indicates that the B. pseudofirmus OF4 complexes, particular membrane lipids and/or a close proximity of enzyme does not tolerate Arg replacement at this position [232], the respiratory chain complex(es) to ATP synthases. One of the perhaps further reflecting the extensive differences between the parameters would be experimental estimation of the pH near surface adaptations of the H+-translocating subunits extremely alkaliphilic of the cytoplasmic membrane of B. pseudofirmus OF4 or a similar Bacillus and thermoalkaliphilic Bacillus strains (see Fig. 5). organism. Since extreme Gram-negative alkaliphiles appear not to

There is another region of particular interest in the a-subunit of require the F0 adaptations required for OXPHOS by Gram-positive extremely alkaliphilic Bacillus species that merits noting. The external alkaliphiles, it would be interesting to ascertain the extent to which loop between helices II and III of the a-subunit, from a circled Glu, Glu98 their bioenergetic challenges differ from those of the well studied to the putative border of helix III shown in Fig. 5 (and a bit further, to alkaliphilic Bacillus species and the role of the outer membrane. the circled Ser114) is generally more polar than the comparable region Further investigations of the specific adaptations of the ATP synthase of non-alkaliphilic Bacillus species or E. coli [172]. Replacement of the will continue to provide important information about how the alkaliphile loop part of this region with the sequence found in B. megaterium overcomes specific aspects of its bioenergetic challenges, which will also resulted in a significant loss of capacity for non-fermentative growth lead to a more refined understanding of what those challenges are. As and ATP synthesis at pH 10.5 but not at pH 7.5 [172]. This loop may play new structural data complement the work on these adaptations the a role in H+ capture on the external surface of the membrane, a insights will be much greater. Work has not yet been reported on equally possibility that will require more experimental investigation. interesting motifs that have been observed in respiratory chain complex features [159,226,233]. Such features and motifs may facilitate specific 9. Conclusions and perspectives partnering between respiratory chain complexes and alkaliphile ATP synthases either directly or in close proximity. The extensive structure–function data already gathered for ATP It remains possible that an extremely alkaliphilic bacterium or synthases reveal variations in the synthases from diverse organisms that archaean will emerge from some as-yet untapped environmental have adapted to a wide range of environmental challenges. Studies of niche that does compensate for the energetically adverse effect of these variations and the basis for their adaptive values will continue to alkaline pH homeostasis with a higher transmembrane electrical

Fig. 5. Alignment of a-subunits from several alkaliphiles and neutralophiles. The organisms are the same as in Fig. 4 except for omission of S. platensis. The names of the alkaliphiles are in blue, neutralophiles in black and bacteria with Na+-translocating ATP synthases in green. The numbering at the top is that of the B. pseudofirmus OF4 subunit and the numbering at the bottom is for the E. coli subunit. The conserved Arg in TMH-4 is highlighted in red, the alkaliphile-specific Lys180 of TMH-4 and Gly212 of TMH-5 are highlighted in blue. Three other residues that deviate from the Bacillus consensus sequence in extreme alkaliphiles are boxed and the distinct residue found in that position in the subunit from Bacillus sp. TA2.A1 is underlined. The NCBI gene accession numbers for the data shown are: B. pseudofirmus OF4, AF330160; B. halodurans C-125, NC_002570; B. alcalophilus, M84712; B. clausii KSM-K16, AP006627; B. selenitireducens MLS10, ZP_02169425; Bacillus sp. TA2.A1, AF533147; O. iheyensis HTE831, NC_004193; B. licheniformis ATCC 14580, NC_006322; B. megaterium, M23924; B. subtilis subsp. subtilis str. 168, NC_000964; G. kaustophilus HTA426, YP_149217; E. coli K12, NC_000913; I. tartaricus, AF522463; P. modestum, P21903; and C. paradoxum, ABB13420. The cartoon model to the right of the alignment shows a putative topological arrangement of 5 TMH of the B. pseudofirmus OF4 a-subunit with the C-terminus emerging on the N-side of the membrane. The circled Lys180 and Gly212 found in alkaliphilic Bacillus species are highlighted in blue, the circled conserved Arg172 is highlighted in red, and four other residues that are found in alkaliphilic Bacillus species, but not generally in non-alkaliphiles, are circled. 1374 D.B. Hicks et al. / Biochimica et Biophysica Acta 1797 (2010) 1362–1377 potential than heretofore observed. Or, an alkaliphilic organism may [28] K. Andries, P. Verhasselt, J. Guillemont, H.W. Gohlmann, J.M. Neefs, H. Winkler, J. Van Gestel, P. Timmerman, M. Zhu, E. Lee, P. Williams, D. de Chaffoy, E. Huitric, S. be found that resolves the energetic conundrum by using an ATP Hoffner, E. Cambau, C. Truffot-Pernot, N. Lounis, V. Jarlier, A diarylquinoline drug synthase rotor with an extraordinarily large number of subunits. It is active on the ATP synthase of Mycobacterium tuberculosis, Science 307 (2005) also important to probe the basis for the tolerance of a high 223–227. [29] A.H. Diacon, A. Pym, M. Grobusch, R. Patientia, R. Rustomjee, L. Page-Shipp, C. cytoplasmic pH in alkaliphiles and to probe the limits of such tolerance Pistorius, R. Krause, M. Bogoshi, G. Churchyard, A. Venter, J. Allen, J.C. Palomino, in diverse alkaliphiles. Perhaps it will be a major part of the resolution T. De Marez, R.P. van Heeswijk, N. Lounis, P. Meyvisch, J. Verbeeck, W. Parys, K. de of the bioenergetic problem in some organisms. Nature often surprises. Beule, K. Andries, D.F. Mc Neeley, The diarylquinoline TMC207 for multidrug- resistant tuberculosis, N. Engl. J. Med. 360 (2009) 2397–2405. [30] A. Koul, N. Dendouga, K. Vergauwen, B. Molenberghs, L. Vranckx, R. Willebrords, Z. Acknowledgements Ristic, H. Lill, I. Dorange, J. Guillemont, D. Bald, K. Andries, Diarylquinolines target subunit c of mycobacterial ATP synthase, Nature Chem. Biol. 3 (2007) 323–324. [31] M. Maeda, ATP synthases: bioinformatic based insights into how their The work that was conducted in the authors' laboratory was electrochemically driven motor comprised of subunits a and c might serve as supported by research grant GM28454 from the National Institute of a drug target, J. Bioenerg. Biomembr. 40 (2008) 117–121. General Medical Sciences of the National Institute of Health (to TAK). [32] S.P.S. Rao, S. Alonso, L. Rand, T. Dick, K. Pethe, The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic non-replicating Mycobacte- rium tuberculosis, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 11945–11950.

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