Oxidative phosphorylation in Escherichia coli K12. An uncoupled mutant with altered membrane structure

1. A new mutant strain (AN228) of Escherichia coli K12, unable to couple phosphorylation to electron transport, has been isolated. The mutant allele (unc-405), in strain AN228, was found to map near the uncA and uncB genes at about minute 74 on the E. coli genome. 2. A transductant strain (AN285) carrying the unc-405 allele is similar to the uncA and uncB mutants described previously in that it is unable to grow on succinate, gives a low aerobic yield on limiting concentrations of glucose, has a normal rate of electron transport, is unable to couple phosphorylation to electron transport, and lacks ATP-dependent transhydrogenase activity. 3. Strain AN285 (unc-405) is similar to an uncA mutant, but different from an uncB mutant, in that it is unable to grow anaerobically in a glucose-mineral-salts medium, and membrane preparations do not have Mg(2+)-stimulated adenosine triphosphatase activity. 4. Strain AN285 (unc-405) does not form an aggregate analogous to the membrane-bound Mg(2+)-stimulated adenosine triphosphatase aggregate found in normal cells. In this respect it differs from strain AN249 (uncA(-)), which forms an inactive membrane-bound Mg(2+)-stimulated adenosine triphosphatase aggregate.     

Reconstitution of oxidative phosphorylation and the adenosine triphosphate-dependent transhydrogenase activity by a combination of membrane fractions from uncA? and uncB? mutant strains of Escherichia coli K12

1. Membrane preparations from both uncA(-) and uncB(-) mutant strains of Escherichia coli K12, in which electron transport is uncoupled from phosphorylation, were fractionated by washing with a low-ionic-strength buffer. The fractionation gave a ;5mm-Tris wash' and a ;membrane residue' from each strain. This technique, applied to membranes from normal cells, separates the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from the membrane-bound electron-transport chain and the non-energy-linked transhydrogenase activity. 2. Reconstitution of both oxidative phosphorylation and the ATP-dependent transhydrogenase activity was obtained by a combination of the ;membrane residue' from strain AN249 (uncA(-)) with the ;5mm-Tris wash' from strain AN283 (uncB(-)). 3. Valinomycin plus NH(4) (+) inhibited oxidative phosphorylation both in membranes from a normal strain of E. coli and in the reconstituted membrane system derived from the mutant strains. 4. The electron-transport-dependent transhydrogenase activity was located in the membrane residue and was de-repressed in both the mutant strains. 5. The spatial and functional relationships between the proteins specified by the uncA and uncB genes and the transhydrogenase protein are discussed.     

A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.     

Energy transduction in Escherichia coli. The effect of chaotropic agents on energy coupling in everted membrane vesicles from aerobic and anaerobic cultures.

1. The transduction of energy from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-ATPase was measured in everted membrane vesicles of Escherichia coli using the energy-dependent quenching of quinacrine fluorescence and the active transport of calcium. 2. Treatment of everted membranes derived from a wild-type strain with the chaotropic agents guanidine-HC1 and urea caused a loss of energy-linked functions and an increase in the permeability of the membrane to protons, as measured by the loss of respiratory-linked proton uptake. 3. The coupling of energy to the quenching of quinacrine fluorescence and calcium transport could be restored by treatment of the membranes with N,N'-dicyclohyexylcarbodiimide. 4. Chaotrope-treated membranes were found to lack Mg2+-ATPase activity. Binding of crude soluble Mg2+-ATPase to treated membranes restored energy-linked functions. 5. Membranes prepared from a wild-type strain grown under anaerobic conditions in the presence of nitrate retained respiration-linked quenching of quinacrine fluorescence and active transport of calcium after treatment with chaotropic agents. 6. Everted membrane vesicles prepared from an Mg2+-ATPase deficient strain lacked respiratory-driven functions when the cells were grown aerobically but were not distinguishable from membranes of the wild-type when both were grown under anaerobic conditions in the presence of nitrate. 7. It is concluded (a) that chaotropic agents solubilize a portion of the Mg2+-ATPase, causing an increase in the permeability of the membrane to protons and (b) that growth under anaerobic conditions in the presence of nitrate prevents the increase in proton permeability caused by genetic or chemical removal of the catalytic portion of the Mg2+-ATPase.     

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]     

The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability

Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis. These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala. Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain. Properties of membrane preparations from these strains were also similar to those from the coupled control strain. The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain. Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound. The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.     

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).     

Energy transduction in Escherichia coli: new mutation affecting the Fo portion of the ATP synthetase complex.

A mutation affecting the intrinsic membrane portion (BFo) of the ATP synthetase complex is described. The phenotype is different from previously reported BFo mutants. This mutation results in the ability of membranes lacking the extrinsic membrane portion (BF1) of the ATP synthetase complex to maintain a transmembrane pH gradient. Unlike other BFo mutants, this strain, NR71, is capable of utilizing ATP hydrolysis for the formation of a transmembrane pH gradient.     

Energy transduction in Escherichia coli: physiological and biochemical effects of mutation in the uncB locus.

The transduction of energy through biological membranes was investigated in Escherichia coli strains defective in the ATP synthetase complex. Everted vesicles prepared from strains containing an uncA or uncB mutation were compared with those of the parental strain for their ability to couple energy derived from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-adenosine triphosphatase, as measured by the energy-dependent quenching of quinacrine fluorescence or the active transport of 45Ca2+. Removal of the Mg2+-adenosine triphosphatase from membranes derived from the parental or an uncA strain caused a loss of energy-linked functions and a concomitant increase in the permeability of the membrane for protons. Proton impermeability was restored by treatment with N,N'-dicyclohexylcarbodiimide. When membranes of the uncB strain were treated in a similar manner, there was no loss of respiratory-driven functions, nor was there a change in proton permeability. These observations suggest that the uncB mutation specifically results in alteration of an intrinsic membrane protein channel necessary for the generation of utilzation of the electrochemical gradient of protons by that complex. Loss of the function of the proton channel is believed to prevent the transduction of energy through the ATP synthetase complex.     

Glutamate transport in membrane vesicles of the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system.     

The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K 12

1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg(2+)+Ca(2+)-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.     

Mutants of Escherichia coli K12 unable to grow on non-fermentable carbon substrates.

Among a number of mutants unable to utilize non-fermentable carbon substrates, scoring for membrane ATPase and for ATP-driven transhydrogenase activity permitted to distinguish two phenotypes: (A) mutants lacking ATPase and ATP-driven transhydrogenase; (B) one mutant with an ATPase which behaved according to several criteria as released into solution instead of being membrane bound, a.o it exhibited no ATP-driven transhydrogenase activity. All A and B mutants exhibited a common nutritional pattern. The ATPase-deficient group, when scored for ATPase-binding sites on its membrane particles revealed three different subgroups: (1) mutants having free ATPase-binding sites, (2) mutants with ATPase-binding sites made available by the procedure which releases ATPase from wild-type membrane, and (3) mutants with no detectable ATPase-binding sites. Membranes of the mutant B with unbound ATPase also exhibited a deficiency in ATPase-binding sites, but its soluble ATPase was also found unable to bind to ATPase-binding sites of wild type membranes. The double alteration, namely abnormal or inactive ATPase and absence of ATPase-binding sites on the membrane is compatible with a single mutational defect.     

The F1F0-ATPase of Escherichia coli. The substitution of alanine by threonine at position 25 in the c-subunit affects function but not assembly

A mutant strain of Escherichia coli carrying a mutation in the uncE gene which codes for the c-subunit of the F1F0-ATPase has been isolated and examined. The mutant allele, designated uncE513, results in alanine at position 25 of the c-subunit being replaced by threonine. The mutant F1F0-ATPase appears to be fully assembled and is partially functional with respect to oxidative phosphorylation. The ATPase activity of membranes from the mutant strain is resistant to the inhibitor dicyclohexylcarbodiimide, but this is due to the F1-ATPase being lost from the membranes in the presence of the inhibitor. Mutant membranes from which the F1-ATPase has been removed have a greatly reduced proton permeability compared with similarly treated normal membranes. The results are discussed in relation to a previously proposed mechanism of oxidative phosphorylation.     

Colicins and bacterial membranes: structures and functions. I. Effects of colicins on the protein composition of the membranes of sensitive and tolerant Escherichia coli.

Treatment of Escherichia coli K12 C600 with colicin K or E1, but not E3, caused changes in the protein composition of the bacterial cytoplasmic membrane and an impairment of the membrane-associated ATP-linked transhydrogenase activity. The major compositional changes were loss and/or reduction in the levels of protein bands 4, 8, 9, 10, 13, and 18 with approximate molecular weights of 122,000, 81,000, 75,000, 73,000, 62,000, and 44,000, respectively. Colicin K or E1 treatment had no significant effect on the protein composition or the ATP-linked transhydrogenase activity of the cytoplasmic membranes of the isogenic tolerant strain E. coli K12 C600 TolII (A592). The cytoplasmic membranes of the untreated tolerant mutant were characteristically devoid of protein bands 4 and 13. It is proposed that protein bands 4 and/or 13 participate in colicin action by acting as receptors for colicins at the cytoplasmic membrane level. Some observations on the structural and functional heterogeneity of the cytoplasmic membrane preparations were made.     

Beta-subunit of Escherichia coli F1-ATPase. An amino acid replacement within a conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins

A mutant strain KF87 of E. coli with a defective beta-subunit (Ala-151----Val) of F1-ATPase was isolated. The mutation is within the conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins. The mutant F1-ATPase had a much higher rate of uni-site hydrolysis of ATP than the wild type, and about 6% of the wild-type multi-site activity. The mutant enzyme showed defective transmission of conformational change(s) between the ligand- and aurovertin-binding sites.     

The maintenance of the energized membrane state and its relation to active transport in Escherichia coli.

1. An ATPase mutant of Escherichia coli and two partial revertants of that mutant were examined for the ability to generate a high energy membrane state with D-lactate or ATP, as measured by the quenching of the fluorescent dye quinacrine. 2. All three strains showed reductions in the aerobically-driven quenching of fluorescence compared to the wild type, but the reduction could be reversed by the addition of eitherN,N'-dicyclohexylcarbodiimide or the crude soluble ATPase of the wild type. 3. The mutant exhibited a decreased ability to accumulate sugars and amino acids and showed an increased permeability to protons. 4. One partial revertant showed a slight increase in active transport and a slight decrease in proton permeability. 5. The other partial revertant showed a large increase in transport ability and a large decrease in proton permeability. 6. A model is proposed in which the conformation of the Mg-2+-ATPase is important in the utilization of energy derived from the electron transport chain and this function is independent of the catalytic activity of the Mg-2+-ATPase.     

Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli. Implications regarding the nature of energy coupling by F1-ATPases

Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+. The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E. coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1. Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships. This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement. The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release. It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation. The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity. Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate. Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis. From all the data, a model is derived to describe the mechanism of ATP synthesis. ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.     

The F1F0-ATPase of Escherichia coli. The substitution of alanine by tyrosine at position 25 in the c-subunit affects function but not assembly

A site-directed mutation in the gene which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of Ala-25 by Tyr, has been constructed and characterized. A plasmid carrying the mutation was used to transform strain AN943 (uncE429). The resulting strain is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield. Membranes prepared from the mutant possess low levels of ATPase activity and are proton-impermeable. The F1-ATPase activity was found to be inhibited by 80% when bound to the membrane. When carried on a plasmid, the mutation is dominant in complementation tests with all mutant unc alleles tested and when transformed into wild-type strain AN346, the mutation results in an uncoupled phenotype. A mutant which overcomes this dominance was isolated and found to possess an 11-amino-acid deletion extending from Ile-55 to Met-65 within the c-subunit. These results are discussed in relation to the previously isolated Ala-25 to Thr mutant (Fimmel, A.L., Jans, D.A., Hatch, L., James, L.B., Gibson, F. and Cox, G.B. (1985) Biochim. Biophys. Acta 808, 252-258) and in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).     

Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation.

1. The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied. 2. Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport. 3. One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine. 4. The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport. 5. The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains. 6. The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium. The unc-405 mutant, however, required the addition of fumarate for growth and for uptake. 7. The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon. Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium. However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.