Arthur A. Guffanti

An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cad‐mium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.

The Na^+-dependence of alkaliphily in Bacillus

A Na(+) cycle plays a central role in the remarkable capacity of aerobic, extremely alkaliphilic Bacillus species for pH homeostasis. The capacity for pH homeostasis, in turn, appears to set the upper pH limit for growth. One limb of the alkaliphile Na(+) cycle consists of Na(+)/H(+) antiporters that achieve net H(+) accumulation that is coupled to Na(+) efflux. The major antiporter on which pH homeostasis depends is thought to be the Mrp(Sha)-encoded antiporter, first identified from a partial clone in Bacillus halodurans C-125. Mrp(Sha) may function as a complex. While this antiporter is capable of secondary antiport energized by an imposed or respiration-generated protonmotive force, the possibility of a primary mode has not been excluded. In Bacillus pseudofirmus OF4, at least two additional antiporters, including NhaC, have supporting roles in pH homeostasis. Some of these additional antiporters may be especially important for antiport at low [Na(+)] or at near-neutral pH. The second limb of the Na(+) cycle facilitates Na(+) re-entry via Na(+)/solute symporters and, perhaps, the ion channel associated with the Na(+)-dependent flagellar motor. The process of pH homeostasis is also enhanced, perhaps especially during transitions to high pH, by different arrays of secondary cell wall polymers in the two alkaliphilic Bacillus species studied most intensively. The mechanisms whereby alkaliphiles handle the challenge of Na(+) stress at very elevated [Na(+)] are just beginning to be identified, and a hypothesis has been advanced to explain the finding that B. pseudofirmus OF4 requires a higher [Na(+)] for growth at near-neutral pH than at very alkaline pH values.

MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional mot in Bacillus subtilis

The stator‐force generator that drives Na+‐dependent motility in alkaliphilic Bacillus pseudofirmus OF4 is identified here as MotPS, MotAB‐like proteins with genes that are downstream of the ccpA gene, which encodes a major regulator of carbon metabolism. B. pseudofirmus OF4 was only motile at pH values above 8. Disruption of motPS resulted in a non‐motile phenotype, and motility was restored by transformation with a multicopy plasmid containing the motPS genes. Purified and reconstituted MotPS from B. pseudofirmus OF4 catalysed amiloride analogue‐sensitive Na+ translocation. In contrast to B. pseudofirmus, Bacillus subtilis contains both MotAB and MotPS systems. The role of the motPS genes from B. subtilis in several motility‐based behaviours was tested in isogenic strains with intact motAB and motPS loci, only one of the two mot systems or neither mot system. B. subtilis MotPS (BsMotPS) supported Na+‐stimulated motility, chemotaxis on soft agar surfaces and biofilm formation, especially after selection of an up‐motile variant. BsMotPS also supported motility in agar soft plugs immersed in liquid; motility was completely inhibited by an amiloride analogue. BsMotPS did not support surfactin‐dependent swarming on higher concentration agar surfaces. These results indicate that BsMotPS contributes to biofilm formation and motility on soft agar, but not to swarming, in laboratory strains of B. subtilis in which MotAB is the dominant stator‐force generator. BsMotPS could potentially be dominant for motility in B. subtilis variants that arise in particular niches.

Effects of Nonpolar Mutations in Each of the Seven Bacillus subtilis mrp Genes Suggest Complex Interactions among the Gene Products in Support of Na+ and Alkali but Not Cholate Resistance

The Bacillus subtilis mrp (multiple resistance and pH) operon supports Na(+) and alkali resistance via an Na(+)/H(+) antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpF mutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans. Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na(+) efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na(+) resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na(+) sensitivity at both pH 7.0 and 8.3. The mrpA and mrpD mutants were particularly sensitive to alkaline pH even without added Na(+). While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships among mrp genes as well. All the mrp mutants, especially the mrpA, -B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type. Expression of an upstream gene, maeN, that encodes an Na(+)/malate symporter, was coordinately regulated with mrp, although it is not part of the operon.

Physiology of acidophilic and alkalophilic bacteria.

Publisher Summary This chapter reviews the physiology of acidophilic and alkalophilic bacteria. The greatest problem with respect to life at low pH values is the maintenance of a cytoplasmic environment far less acidic than the external milieu. Acidophiles could accomplish this in two ways, firstly, by pumping protons outward effectively and/or secondly, by possession of a cell-surface barrier extremely impermeable to protons. If the cytoplasmic pH value of acidophiles is maintained near neutrality, then no problems would be expected in connection with cytoplasmic enzymes or synthetic processes. However, extracellular enzymes, flagella, and all processes associated with the external membrane surface would have to function at extremely acid pH values. Unusual properties of the cell wall and membrane layer would be anticipated. Many bacteria are able to grow at pH values as low as 4.0, but the vast majority of such organisms can also grow at neutral pH values. More unusual are those bacteria that thrive at pH values below 3.0 and cannot grow at neutral pH. For extreme, obligately alkalophilic bacteria, the optimal external pH value for growth on most substrates is between pH 10.0 and 11.0. Growth at even higher pH values is also observed. The most obvious and pressing physiological problem is regulation of cytoplasmic pH value. Maintenance of a relatively acidic cytoplasm— that is, nearer to neutral pH value than the highly alkaline pH value of the medium, might bypass any need for unusual pH optima or stabilities for cytoplasmic enzymes or macromolecules.

Bacillus subtilis YqkI is a novel malic/Na+-lactate antiporter that enhances growth on malate at low protonmotive force.

Bacillus subtilis yheL encodes a Na+/H+ antiporter, whereas its paralogue,yqkI, encodes a novel antiporter that achieves a simultaneous Na+/H+ and malolactate antiport. B. subtilis yufR, a control in some experiments, encodes a Na+/malate symporter. YqkI complemented a malate transport mutant of Escherichia coliif Na+ and lactate were present. YheL conferred Na+ uptake capacity on everted membrane vesicles from an antiporter-deficient E. coli mutant that was consistent with a secondary Na+/H+ antiport, but YqkI-dependent Na+ uptake depended on intravesicular malate and extravesicular lactate. YqkI-dependent lactate uptake depended on intravesicular malate and extravesicular Na+. YqkI mediated an electroneutral exchange, which is proposed to be a malic−2-2H+ (or fully protonated malate)/Na+-lactate−1 antiport. Because the composite YqkI-mediated exchanges could be driven by gradients of the malate-lactate pair, this transporter could play a role in growth ofB. subtilis on malate at low protonmotive force. A mutant with a disruption of yqkI exhibited an abrupt arrest in the mid-logarithmic phase of growth on malate when low concentrations of protonophore were present. Thus growth of B. subtilis to high density on a putatively nonfermentative dicarboxylic acid substrate depends on a malolactate exchange at suboptimal protonmotive force.

A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore.

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na at elevated pH, was deficient in energy‐dependent Na extrusion. The capacity of the mutant JC901 for Na ‐dependent pH homeostasis was unaffected relative to the wild‐type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3 ‐terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane‐spanning regions at its C‐terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP‐binding proteins that are components of transport systems of the ATP‐binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p‐chlorophenylhydrazone (CCCP), and, more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na+‐sensitive phe‐notype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol‐resistance does not result from NatAB‐dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+

Energetics of Alkaliphilic Bacillus Species: Physiology and Molecules

The challenge of maintaining a cytoplasmic pH that is much lower than the external pH is central to the adaptation of extremely alkaliphilic Bacillus species to growth at pH values above 10. The success with which this challenge is met may set the upper limit of pH for growth in these bacteria, all of which also exhibit a low content of basic amino acids in proteins or protein segments that are exposed to the outside bulk phase liquid. The requirement for an active Na(+)-dependent cycle and possible roles of acidic cell wall components in alkaliphile pH homeostasis are reviewed. The gene loci that encode Na+/H+ antiporters that function in the active cycle are described and compared with the less Na(+)-specific homologues thus far found in non-alkaliphilic Gram-positive prokaryotes. Alkaliphilic Bacillus species carry out oxidative phosphorylation using an exclusively H(+)-coupled ATPase (synthase). Nonetheless, ATP synthesis is more rapid and reaches a higher phosphorylation potential at highly alkaline pH than at near-neutral pH even though the bulk electrochemical proton gradient across the coupling membrane is lower at highly alkaline pH. It is possible that some of the protons extruded by the respiratory chain are conveyed to the ATP synthase without first equilibrating with the external bulk phase. Mechanisms that might apply to oxidative phosphorylation in this type of extensively studied alkaliphile are reviewed, and note is made of the possibility of different kinds of solutions to the problem that may be found in new alkaliphilic bacteria that are yet to be isolated or characterized.

Energetic problems of extremely alkaliphilic aerobes

Over a decade of work on extremely alkaliphilic Bacillus species has clarified the extraordinary capacity that these bacteria have for regulating their cytoplasmic pH during growth at pH values well over 10. However, a variety of interesting energetic problems related to their Na(+)-dependent pH homeostatic mechanism are yet to be solved. They include: (1) the clarification of how cell surface layers play a role in a category of alkaliphiles for which this is the case; (2) identification of the putative, electrogenic Na+/H+ antiporter(s) that, in at least some alkaliphiles, may completely account for a cytoplasmic pH that is over 2 pH units lower than the external pH; (3) the determination of whether specific modules or accessory proteins are essential for the efficacy of such antiporters; (4) the mechanistic basis for the increase in the transmembrane electrical potential at the high external pH values at which the potential-consuming antiporter(s) must be most active; and (5) an explanation for the Na(+)-specificity of pH homeostasis in the extremely alkaliphilic bacilli as opposed to the almost equivalent efficacy of K+ for pH homeostasis in at least some non-alkaliphilic aerobes. The current status of such studies and future strategies will be outlined for this central area of alkaliphile energetics. Also considered, will be strategies to elucidate the basis for robust H(+)-coupled oxidative phosphorylation by alkaliphiles at pH values over 10. The maintenance of a cytoplasmic pH over 2 units below the high external pH results in a low bulk electrochemical proton gradient (delta p). To bypass this low delta p, Na(+)-coupling is used for solute uptake even by alkaliphiles that are mesophiles from environments that are not especially Na(+)-rich. This indicates that these bacteria indeed experience a low delta p, to which such coupling is an adaptation. Possible reasons and mechanisms for using a H(+)-coupled rather than a Na(+)-coupled ATP synthase under such circumstances will be discussed.

Mechanisms of cytoplasmic pH regulation in alkaliphilic strains of Bacillus

Abstract The central challenge for extremely alkaliphilic Bacillus species is the need to establish and sustain a cytoplasmic pH that is over two units lower than the highly alkaline medium. Its centrality is suggested by the strong correlation between the growth rate in the upper range of pH for growth, i.e., at values above pH 10.5, and the cytoplasmic pH. The diminishing growth rate at extremely high pH values correlates better with the rise in cytoplasmic pH than with other energetic parameters. There are also general adaptations of alkaliphiles that are crucial prerequisites for pH homeostasis as well as other cell functions, i.e., the reduced basic amino acid content of proteins or segments thereof that are exposed to the medium, and there are other challenges of alkaliphily that emerge from solution of the cytoplasmic pH problem, i.e., reduction of the chemiosmotic driving force. For cells growing on glucose, strong evidence exists for the importance of acidic cell wall components, teichuronic acid and teichuronopeptides, in alkaliphily. These wall macromolecules may provide a passive barrier to ion flux. For cells growing on fermentable carbon sources, this and other passive mechanisms may have a particularly substantial role, but for cells growing on both fermentable and nonfermentable substrates, an active Na1-dependent cycle is apparently required for alkaliphily and the alkaliphiles remarkable capacity for pH homeostasis. The active cycle involves primary establishment of an electrochemical gradient via proton extrusion, a secondary electrogenic Na+/H+ antiport to achieve net acidification of the cytoplasm relative to the outside pH, and mechanisms for Na+ re-entry. Recent work in several laboratories on the critical antiporters involved in this cycle has begun to clarify the number and characteristics of the porters that support active mechanisms of pH homeostasis.