Effect of uncouplers on the bioenergetic properties of a carbonyl cyanide m-chlorophenylhydrazone-resistant mutant Escherichia Coli UV6

Sedgwick, Edward G.; Hou, Cynthia; Bragg, Philip D.

doi:10.1016/0005-2728(84)90046-x

Cited by 25 publications

(15 citation statements)

References 33 publications

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“…However, whatever these effects might be, they are rapidly and completely reversible. On the other hand, the concentration of CCCP required to prevent secretion is similar to those reported by others to be required to prevent both pmf-dependent solute uptake and the pmf-dependent export of preproteins across the cytoplasmic membrane (Enequist et al, 1981;Sedgwick et al, 1984). In control experiments, we found that the pmf-dependent uptake of lactose was not completely abolished unless cells were treated with 12.5 M CCCP.…”

Section: Discussionsupporting

confidence: 89%

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Possot

Letellìer

Pugsley

1997

Molecular Microbiology

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SummaryThe energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli. In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to Յ10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.

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Section: Discussionsupporting

confidence: 89%

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Possot

Letellìer

Pugsley

1997

Molecular Microbiology

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“…The characteristics of the mutants might support the view that protonophore action actually relates, at least in part, to an uncoupler-binding protein that may be associated with the ATPase (10,11,20). Alternatively, they might support one of the models of energy transduction in which the bulk A5LH+ is less directly coupled to bioenergetic work than some more localized, and perhaps less protonophoresensitive, pathway of proton flow (6,8).A small number of uncoupler-resistant mutant strains of Escherichia coli (16,17,37) and Bacillus megaterium (3, 4) have been isolated and described. These strains do not yet present a cohesive picture, the precise map positions of the mutations have not been determined, a possible change in passive proton permeability has not been excluded, and the primary biochemical change is unclear.…”

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confidence: 99%

“…A small number of uncoupler-resistant mutant strains of Escherichia coli (16,17,37) and Bacillus megaterium (3, 4) have been isolated and described. These strains do not yet present a cohesive picture, the precise map positions of the mutations have not been determined, a possible change in passive proton permeability has not been excluded, and the primary biochemical change is unclear.…”

mentioning

confidence: 99%

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis

et al. 1987

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Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 ,IM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps causally, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.During the past 15 years, the chemiosmotic model of energy coupling that had been advanced by Mitchell (28) almost 10 years earlier has achieved widespread confirmation and acceptance. According to this model, the energy made available by photosynthetic light capture, oxidation of substrates by the respiratory chain, or ATP hydrolysis by the membrane-associated F,FO-ATPase is transduced via an intermediary electrochemical proton gradient (4UH+). That is, these energy-producing processes are all associated with proton translocation across a relatively proton-impermeant coupling membrane such that a transmembrane pH gradient (ApH) and transmembrane electrical potential (AJi) are produced. In a bacterial cell or mitochondrion, the orientation of the 4iiiH+ is outside positive and acid relative to the interior of the cell or organelle. Completing the energetic circuit are the energy-consuming processes that are catalyzed by integral membrane porters or enzymes. These processes, e.g., ATP synthesis and ion-coupled solute uptake, utilize a...

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“…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.…”

mentioning

confidence: 99%

“…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) have been isolated on the basis of their capacity to grow aerobically on nonfermentable carbon sources in the presence of protonophore concentrations that inhibit the growth of wildtype parental strains. The major bioenergetic problem posed by such mutants is their apparent ability to synthesize ATP under conditions when the electrochemical proton gradient (AjiiiH+) is reduced to levels that will not support comparable synthesis in the wild type.…”

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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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Effect of uncouplers on the bioenergetic properties of a carbonyl cyanide m-chlorophenylhydrazone-resistant mutant Escherichia Coli UV6

Cited by 25 publications

References 33 publications

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis

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

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