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.     

Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.     

Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation.

A plasmid was isolated which included the region of the Escherichia coli chromosome carrying the known genes concerned with oxidative phosphorylation (unc genes). This plasmid was used to prepare partial diploids carrying normal unc alleles on the episome and one of the three mutant alleles (unc A401, uncB402 or unc-405) on the chromosome. These strains were compared with segregants from which the plasmid had been lost. Dominance of either normal ormutant unc alleles was determined by growth on succinate, growth yields on glucose, Mg-ATPase (Mg2+-stimulated adenosine triphosphatase) activity, atebrin-fluorescence quenching, ATP-dependent transhydrogenase activity and oxidative phosphorylation. In all the above tests, dominance of the normal allele was observed. However, in membranes from the diploid strains which carried a normal allele and either of the mutant alleles affecting Mg-ATPase activity (uncA401 or unc-405), the energy-linked functions were only partially restored.     

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.     

The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed 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).     

Tributyltin-driven enhancement of the DCCD insensitive Mg-ATPase activity in mussel digestive gland mitochondria

The lipophilic pollutant tributyltin (TBT), other than inhibiting the DCCD (N,N′-dicyclohexylcarbodiimide) and oligomycin-sensitive Mg-ATPase activities in digestive gland mitochondria from the Mediterranean mussel Mytilus galloprovincialis, at higher than 1.0 μM concentrations in vitro promotes an increase in the ATPase activity fraction refractory to inhibitors of F_O moiety, namely oligomycin and DCCD. By exploring the mechanisms involved in the TBT promoted enzyme desensitization to DCCD, we pointed out intriguing differences in the enzyme desensitization to the two inhibitors. Differently from oligomycin, the TBT promoted enzyme desensitization to DCCD is independent of the redox state of thiol groups of the enzyme complex and strongly temperature dependent, being significantly elicited only at temperatures above the break of the Arrhenius plots (around 18 °C). Such differences may cast light on multiple TBT interaction modes with the enzyme complex. The TBT-driven increase in the activation energy of the Mg-ATPase activities insensitive to inhibitors of F_O sector suggests that the temperature-dependent incorporation of the lipophilic toxicant within the lipid bilayer may deeply affect the membrane-bound complex functionality. Accordingly, incorporated TBT may cause structural changes in the intramembrane F_O subunits, thus weakening or even preventing DCCD binding to the enzyme complex.     

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.     

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.     

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F1F0-ATPase disrupt ATP-driven proton translocation

Three missense mutants in subunit a of the Escherichia coli F1F0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp119----His, Ser152----Phe, or Gly197----Arg substitutions in subunit a. Function was not completely abolished by any of the mutations. The F0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F1F0-ATPase. The H+ translocation capacity of F0 was reduced in all three mutants. ATP-driven H+-translocation was also reduced, with the response in the Gly197----Arg mutant being almost nil and that in the Asp119----His and Ser152----Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly197----Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp119----His and Ser152----Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp231----stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB+ plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp231----stop) background strain was shown to bind F1-ATPase nearly normally despite lacking subunit a in its membrane.     

Oxidative phosphorylation in Escherichia coli K-12: The genetic and biochemical characterisation of a strain carrying a mutation in the uncB gene

1. A mutant strain of Escherichia coli unable to grow with succinate as sole carbon source was isolated. This mutant was found to carry a mutation in a gene (designated uncB) mapping at about minute 73.5 on the E. coli chromosome and close to the uncA gene which is probably the structural gene for (Mg²⁺,Ca²⁺)-stimulated ATPase.2. The uncB401 allele was transduced into two other strains of E. coli and the transductants compared with the parent strains.3. Strains carrying the uncB401 allele have low aerobic growth yields when grown on limiting concentrations of glucose, but unlike mutations in the uncA gene, mutations in the uncB gene do not impair anaerobic growth on a glucose-mineral salts medium.4. Oxidase activities in membranes from the normal strains and strains carrying the uncB401 allele were similar.5. Measurement of P/O ratios indicated that a mutation in the uncB gene causes uncoupling of phosphorylation associated with electron transport with d-lactate as substrate.6. (Mg²⁺,Ca²⁺)-stimulated ATPase activities in the normal strains and in strains carrying the uncB401 allele are similar.7. Estimation of the energy-linked and non-energy-linked transhydrogenase activities in membrane preparations from both the normal and mutant strains indicated that the protein affected by a mutation in the uncB gene is essential for the functioning of the ATP-dependent energy-linked transhydrogenase.8. It is concluded that two proteins, specified by the uncA and uncB genes, are essential for phosphorylation coupled to d-lactate oxidation and also for the energy-linked transhydrogenase activity using ATP as the energy source.     

A Fifth Gene (uncE) in the Operon Concerned with Oxidative Phosphorylation in Escherichia coli

Three mutant unc alleles (unc-408, unc-410, and unc-429) affecting the coupling of electron transport to oxidative phosphorylation in Escherichia coli K-12 have been characterized. Genetic complementation analyses using previously defined mutant unc alleles indicated that the new mutant unc alleles affect a previously undescribed gene designated uncE. The phenotype of strains carrying the uncE408 or uncE429 allele is similar in that Mg(2+)-adenosine triphosphatase activity is only found in the cytoplasmic fraction, and membranes do not bind the F(1) portion of adenosine triphosphatase purified from a normal strain. In contrast, adenosine triphosphatase activity is present both in the cytoplasm and on the membranes from a strain carrying the unc-410 allele, and normal F(1) binds to F(1)-depleted membranes from this strain. The adenosine triphosphatase solubilized from membranes of a strain carrying the unc-410 allele reconstituted ATP-dependent membrane energization in F(1)-depleted membranes from a normal strain. Genetic complementation tests using various Mu-induced unc alleles in partial diploid strains show that the uncE gene is in the unc operon and that the order of genes is uncB E A D C. The unc-410 allele differs from the uncE408 and uncE429 alleles in that complementation tests with the Mu-induced unc alleles indicate that more than one gene is affected. It is concluded that this is due to a deletion which includes part of the uncE gene and another gene, or genes, between the uncE and uncA genes.     

Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli.

The effects of two protease inhibitors on the solubilization of the membrane-bound Mg2+-adenosine triphosphatase (Mg-ATPase) of Escherichia coli were investigated. p-Aminobenzamidine prevented the solubilization of the Mg-ATPase during treatment of membranes with low-ionic-strength buffers containing ethylenediaminetetraacetic acid. p-Aminobenzamidine did not prevent subsequent solubilization of the Mg-ATPase by treatment of the membranes with chloroform. This method of solubilization yielded a preparation of similar apparent molecular weight but with a 10-fold-increased specific activity as compared with the Mg-ATPase solubilized by washing with low-ionic-strength buffer. However, in contrast to the latter preparation, the chloroform-solubilized Mg-ATPase did not reconstitute ATP-dependent energization of stripped membranes, which were prepared by low-ionic-strength washing in the absence of p-aminobenzamidine. Another protease inhibitor, epsilon-amino-n-caproic acid, did not effect the solubilization of the Mg-ATPase, but did inhibit the loss of activity occurring during concentration, by ultrafiltration, of the Mg-ATPase solublized by the low-ionic-strength treatment.     

Reconstitution of energy-dependent transhydrogenase in ATPase-negative mutants of Escherichia coli

The aerobic-driven and ATP-driven energy-dependent transhydrogenase activities of membrane particles from two different Ca²⁺, Mg²⁺-activated ATPase-negative mutants of $\text{E.}$$\text{coli}$ were examined. The activities were low or absent in one of the mutants (DL-54). Reconstitution of the aerobic-driven reaction could be obtained by addition to particles from this mutant of DCCD or of a coupling factor prepared from the parent strain. The coupling factor also restored the ATP-driven reaction. In the other mutant (N144) the aerobic-driven activity was unimpaired, and was not affected by DCCD or by the coupling factor. The difference between the two mutants could be rationalized if the coupling factor ATPase had both a stabilizing and an enzymic function.     

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.     

Biochemical and mutational analysis of a gingipain-like peptidase activity from Prevotella ruminicola B(1)4 and its role in ammonia production by ruminal bacteria.

A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.     

O-acetylserine and O-acetylhomoserine sulfhydrylase of yeast; studies with methionine auxotrophs.

The nutritional requirements of three yeast mutants, previously shown to possess low O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) sulfhydrylase activities, were reinvestigated. It was thus found that one mutant (strain No. 16), previously identified as a homocysteine auxotroph, is in fact a double mutant requiring both cysteine and OAH. In agreement with the previous assignment, the other two strains (strains No. 13 and 17) were shown to be true cysteine auxotrophs. These results can best be explained by assuming the cystathionine pathway to be the main route of homocysteine synthesis in this organism. It was further found that extracts of the three mutants contain genetically modified OAS-OAH sulfhydrylases with much reduced catalytic activities. Modified sulfhydrylase was partially purified from strain No. 16 by the same procedure as for the wild-type enzyme. Both OAS and OAH sulfhydrylase activities of the mutant enzyme were copurified and behaved identically on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the purified mutant enzyme were shown to be very similar to those of the wild-type enzyme, except that the catalytic activities of the former were only 3-5% of those of the latter, and that the ratio of OAH sulfhydrylase to OAS sulfhydrylase activity was somewhat lower in the former than in the latter.     

Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

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.