Failure of an alkalophilic bacterium to synthesize ATP in response to a valinomycin-induced potassium diffusion potential at high pH

Guffanti, Arthur A.; Chiu, E; Krulwich, Terry A.

doi:10.1016/0003-9861(85)90695-2

Cited by 19 publications

(7 citation statements)

References 26 publications

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“…Perhaps in some marine aerobes there is a Na +-translocating ATP synthase, but in the extreme alkalophiles, such a Na+-dependent solution to the bioenergetic problem does not appear to obtain. In both whole cell experiments (Guffanti, Chiu & Krulwich, 1985) and experiments with ADP + Piloaded, right-side-out vesicles of B. alcalophilus (Guffanti et al, 1981b) and B. firmus RAB , no requirement for or stimulation by added Na + can be demonstrated for ATP synthesis. By contrast, ATP synthesis in these experimental systems is DCCD-sensitive and--in weakly buffered vesicles of B. alcatophitus--appears to involve influx of protons concomitant with ATP synthesis.…”

Section: Atp Synthesis Is Catalyzed By An Fifo-atpasementioning

confidence: 93%

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Bioenergetics of alkalophilic bacteria

Krulwich

1986

J. Membrain Biol.

101

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The central problem for organisms which grow optimally, and in some cases obligately, at pH values of 10 to 11, is the maintenance of a relatively acidified cytoplasm. A key component of the pH homeostatic mechanism is an electrogenic Na+/H+ antiporter which--by virtue of kinetic properties and/or its concentration in the membrane--catalyzes net proton uptake while the organisms extrude protons during respiration. The antiporter is also capable of maintaining a constant pHin during profound elevations in pHout as long as Na+ entry is facilitated by the presence of solutes which are taken up with Na+. Secondary to the problem of acidifying the interior is the adverse effect of the large pH gradient, acid in, on the total pmf of alkalophile cells. For the purposes of solute uptake and motility, the organisms appear to largely bypass the problem of a low pmf by utilizing a sodium motive force for energization. However, ATP synthesis appears not to resolve the energetics problem by using Na+ or by incorporating the proton-translocating ATPase into intracellular organelles. The current data suggest that effective proton pumping carried out by the alkalophile respiratory chain at high pH may deliver at least some portion of the protons to the proton-utilizing catalysts, i.e., the F1F0-ATPase and the Na+/H+ antiporter, by some localized pathway.

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Section: Atp Synthesis Is Catalyzed By An Fifo-atpasementioning

confidence: 93%

“…First, the phenomenon can be reproduced in membrane vesicles and is thus not dependent upon some cytoplasmic activity in the respiring cells (Guffanti et al, 1985)'. Second, an examination and evaluation has been made of the proton influx that accompanies the generation of large diffusion potentials.…”

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confidence: 99%

Bioenergetics of alkalophilic bacteria

Krulwich

1986

J. Membrain Biol.

101

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“…Lipids were prepared from right-side-out membrane vesicles that were made from whole cells by the lysozyme method of Kaback (11) as described previously (23). The vesicles thus prepared had undetectable levels of contaminating muramic acid (8). The concentration of membrane protein in each vesicle preparation was determined by the method of Lowry et al (22) with bovine serum albumin as the standard.…”

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confidence: 99%

Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp

Clejan¹,

Krulwich²,

Mondrus³

et al. 1986

J Bacteriol

183

101

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The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp. were characterized in a comparative study that included B. subtilis. Preparations of membrane lipids were made from pH 10.5-grown cells of all of the alkalophiles and from pH 7.5-or 7.0-grown cells of the two facultative strains and B. subtilis. The two obligate alkalophiles contained high ratios of membrane lipid to membrane protein, and the lipid fraction contained a high proportion of neutral lipid. These characteristics are probably not prerequisites for growth at very high pH since one or another of the facultative strains failed to show these properties at high pH. All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids.Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, including phosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the other major phospholipid. Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of the alkalophile preparations. Glycolipids and phosphoglycolipids were absent. The fatty acid composition of the total phospholipid and individual fractions revealed two features that distinguished between the obligate and facultative strains. Membranes from the obligately alkalophilic species contained a high concentration of branched-chain fatty acids, comparable to that in membranes from B. subtilis, as well as a relatively high content of unsaturated fatty acids. By contrast, the facultatively alkalophilic strains contained almost no unsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligate alkalophiles or B. subtilis.Bacteria and other microorganisms have been attractive experimental vehicles for studies of the nature and possible roles of individual membrane lipids. Among the bacteria that grow at extremes of pH, the membrane lipids of extreme acidophiles have been studied far more extensively (21) than those of alkalophiles. In a study of the total cellular lipids of alkalophilic Bacillus sp. A-007, Koga et al. (16) identified the major neutral lipids as diacylglycerols, squalene and dehydrosqualene, and the major polar lipids as phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin. These investigators also identified bis(monoacylglycero)phosphate in Bacillus sp. A-007 (26) and two other alkalophilic bacilli but failed to find this compound in a fourth alkalophilic Bacillus species (16).Currently, the basis for obligate alkalophily is not understood; some data suggest that the membranes of obligate alkalophiles lose integrity at near-neutral pH values (17). If, as this indicates, the membrane of these strains retains full barrier function only at alkaline pH values, the membrane lipids might reflect relevant properties. A comparative study of obligate and facultative strains should clarify this possibility. In addition, a comparative study of several obligate and facultative alkalophiles...

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“…For studies of ATP synthesis, the strains were starved at pH 7.5 as described previously (8), except that the cells used for the experiments (having been grown in the presence or absence of fatty acid supplements) were first washed with phosphate buffer (pH 7.5) containing 1% (wt/vol) bovine serum albumin. Synthesis of ATP was initiated by the addition of malate as described previously (7,9) in the presence or absence of any added inhibitors.…”

mentioning

confidence: 99%

“…Synthesis of ATP was initiated by the addition of malate as described previously (7,9) in the presence or absence of any added inhibitors. Cellular ATP levels were determined by a luciferin-luciferase assay (8).…”

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confidence: 99%

Incorporation of specific exogenous fatty acids into membrane lipids modulates protonophore resistance in Bacillus subtilis

et al. 1987

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Attempts to manipulate the level of C16:1 fatty acids in membrane phospholipids were made by using Bacillus subtilis and its protonophore-resistant mutants to test the hypothesis that C16:1 fatty acid levels relate to the bioenergetic properties of the mutant strains. Growth of the three mutants in the presence of palmitoleic acid restored the level of C16:1 fatty acids in the membrane lipids to somewhat above those found in the wild type.The palmitoleic acid was preferentially incorporated into diphosphatidylglycerol (cardiolipin) and phosphatidylethanolamine and was associated with increased levels of these phospholipids. These membrane preparations showed no increase in the levels of free fatty acids. The increase in C16:1 fatty acids achieved by growth in the presence of palmitoleic acid was accompanied by secondary changes in membrane lipids as well as a pronounced diminution in the protonophore resistance of growth and ATP synthesis. Other membraneassociated properties that had been observed in these mutants, e.g., elevated ATPase levels, were not altered coordinately with protonophore resistance and C16:1 fatty acid levels. Growth of the wild type in the presence of palmitic acid caused a modest elevation of the C16:0 of the membrane lipids and a modest increase in the protonophore resistance of growth and ATP synthesis. Growth of the wild type at elevated temperatures, in the absence of fatty acid supplementation, also enhanced its resistance to protonophores. The results support the hypothesis that specific changes in membrane lipid composition underlie the bioenergetic changes associated with protonophore resistance.In our previous work and that of others, protonophoreresistant mutant strains of Bacillus subtilis (9), Bacillus megaterium (2, 3, 7), and Escherichia coli (11,12,17) (14), ATP synthesis by proton-translocating ATPases is completely coupled to a A-IH+ composed of a transmembrane electrical potential (A+i) and a transmembrane pH gradient (4pH) that are generated across the coupling membrane between the cytosol and the bulk external phase. Thus a mutational change that alters the dependence of ATP synthesis on the bulk A-iH+ would be a potential challcnge to a strict chemiosmotic formulation.In the accompanying paper (9) we reported that three protonophore-resistant mutants of B. subtilis synthesized more ATP than the wild type did at suboptimal levels of the AJ.LH+. They also exhibited a number of other phenotypic changes involving the membrane (e.g., enhanced sensitivity to valinomycin and an increase in certain membraneassociated activities such as the ATPase). Since the mutants each appeared to have a single mutation, it was possible that the primary mutation caused a change in the properties of the coupling membrane, thus producing a pleiotropic phenotype with respect to membrane-associated functions. An analysis of membrane lipids was therefore undertaken. Two major changes in the fatty acid composition of membrane * Corresponding author.phospholipids were found in all three protonophor...

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Failure of an alkalophilic bacterium to synthesize ATP in response to a valinomycin-induced potassium diffusion potential at high pH

Cited by 19 publications

References 26 publications

Bioenergetics of alkalophilic bacteria

Bioenergetics of alkalophilic bacteria

Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp

Incorporation of specific exogenous fatty acids into membrane lipids modulates protonophore resistance in Bacillus subtilis

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