The role of arginine in the conserved polar loop of the c-subunit of the Escherichia coli H(+)-ATPase.

The Arg-41 of the c-subunit of the F0F1-ATPase of Escherichia coli has been changed by site-directed mutagenesis to Glu, Leu or Lys. None of the mutants can carry out oxidative phosphorylation. No detectable F1-ATPase activity is found on the membranes and only small amounts in the cytoplasm. Two-dimensional gel electrophoresis shows that in all three mutant strains the assembly of the F0F1-ATPase has been affected. When plasmids carrying the mutant genes, together with other normal unc genes, were inserted into strains each carrying a mutation in one of the unc genes other than uncE their capacity for oxidative phosphorylation was reduced or eliminated, the effect being most pronounced with the uncG and uncC mutants and least pronounced with the plasmid giving the Arg-->Lys substitution. The c-subunit is a multimer in the ATP synthase complex and it appears that a mixture of normal and mutant gene products allows assembly of a functional complex.

Mutational analysis of the glycine-rich region of the c subunit of the Escherichia coli F0F1 ATPase.

Eight strains carrying amino acid substitutions within the c subunit of the F0F1 ATPase of Escherichia coli have been constructed by using site-directed mutagenesis. Three strains carrying the substitutions Gly-23----Leu, Ala-24----Leu, and Gly-38----Leu, which reside in or near the highly conserved glycine-rich region of the c subunit, are unable to carry out oxidative phosphorylation. Membranes prepared from these strains possess basal levels of ATPase activity. In contrast, strains carrying the substitutions Ile-30----Phe, Gly-33----Leu, Gly-58----Leu, and Lys-34----Val and the Lys-34----Val, Glu-37----Gln double substitution were found to possess a coupled phenotype similar to that of the wild type.

Mutations within the uncE gene affecting assembly of the F1F0-ATPase of Escherichia coli.

Site-directed mutagenesis has been used to construct two mutations within the uncE gene, which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of glycine-27 by leucine and of glycine-32 by leucine. Strains carrying each mutation are unable to grow on minimal medium containing succinate as the sole carbon source and possess an uncoupled growth yield. Membranes prepared from strains carrying each mutation possess low levels of ATPase activity and are proton-impermeable. The c-subunit in each mutant strain appears to assemble into the F0-ATPase and disrupt the normal assembly of the F1-ATPase. The results are discussed in relation to a previously proposed model for the F0 sector [Cox, Fimmel, Gibson & Hatch (1986) Biochim. Biophys. Acta 849, 62-69].

The F1F0-ATPase of Escherichia coli. The substitution of glycine by valine at position 29 in the c-subunit affects function but not assembly.

Site-directed mutagenesis has been used to construct two mutations within the uncE gene, coding for the c-subunit of the F1F0-ATPase, resulting in the substitution of Gly-29 by Val and Gly-18 by Leu. The strain carrying the Gly-29----Val substitution is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield, while the strain carrying the Gly-18----Leu substitution possesses a wild-type phenotype. Membranes prepared from the strain carrying the Gly-29----Val substitution possess low levels of ATPase activity and are proton-impermeable. The F1-ATPase activity of this strain was found to be inhibited by approx. 75% when bound to the membrane. These 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).

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).

Amino acid substitutions in the epsilon-subunit of the F1F0-ATPase of Escherichia coli.

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature epsilon-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F0. Purified F1-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F1-ATPase. Partial revertant strains were isolated in which Pro-47 of the epsilon-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II beta-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III beta-turn. Space-filling models of the beta-turn (residues 46-49) in the normal, mutant and partial revertant epsilon-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the epsilon-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F1-ATPase.

The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits.

A model for the mechanism of ATP synthase was proposed previously (Cox, G.B., Jans, D.A., Fimmel, A.L., Gibson, F. and Hatch, L. (1984) Biochim. Biophys. Acta 768, 201-208) in which the b subunit of the Fo of Escherichia coli rotated. The driving force was proposed to be an interaction between two charged residues in the membrane, namely, Lys-23 of the b subunit and Asp-61 of the c subunit. To test this proposal the Lys-23 of the b subunit was replaced by threonine using site-directed mutagenesis. The resulting mutant, although it had an impairment in the assembly of the F1F0-ATPase, was normal with respect to oxidative phosphorylation. The role of the a subunit, which had been previously proposed to be a structural one, was reassessed by examination of the possible secondary and tertiary structure of the analogous proteins from several sources. Not only did these subunits appear to have very similar structures, but in each there was a highly conserved helical arm on one of the transmembrane helices which could form a proton channel if it interacted with the Asp-61 of the c subunit. A revised model is therefore presented in which five transmembrane helices from the a subunit and two from the b subunit are surrounded by a ring of c subunits. The highly conserved nature of the structures of the a, b and c subunits from various organisms suggests that the model may have relevance for ATP synthases from bacterial plasma membranes, mitochondria and chloroplasts.

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.

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.

An acidic or basic amino acid at position 26 of the b subunit of Escherichia coli F1F0-ATPase impairs membrane proton permeability: suppression of the uncF469 nonsense mutation.

The uncF469 allele differed from normal in that a G----A base change occurred at nucleotide 77 of the uncF gene, resulting in a TAG stop codon rather than the tryptophan codon TGG. Two partial revertant strains were isolated which retained the uncF469 allele but formed a partially functional b-subunit, due to suppression of the uncF469 nonsense mutation. From the altered isoelectric points of the b-subunits from these strains, it was concluded that the suppressor gene of partial revertant strain AN1956 inserts an acidic amino acid for the TAG codon, and that the suppressor gene of partial revertant strain AN1958 inserts a basic amino acid. The membranes of both partial revertant strains showed impaired permeability to protons on removal of F1-ATPase. The membranes of both strains, however, were able to carry out oxidative phosphorylation, and the ATPase activities of both were resistant to the inhibitor dicyclohexylcarbodiimide.

An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function.

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.

Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene.

The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined. The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence. The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427. The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E. coli genetic map. A promoter region has been identified on the basis of homology with the consensus sequence of other E. coli promoter regions. However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively.

Oxidative phosphorylation by mutant Escherichia coli membranes with impaired proton permeability.

The effect on the function of the Escherichia coli F1F0-ATPase of the substitution of leucine-31 by phenylalanine in the c-subunit of the enzyme was examined. The assembly of the mutant c-subunit requires an increased gene dosage [Jans, Fimmel, Langman, James, Downie, Senior, Ash, Gibson & Cox (1983) Biochem. J. 211, 717-726], and this was achieved by incorporation of the uncE408 or uncE463 alleles on to F-plasmids or multicopy plasmids. Membranes from strains carrying either the uncE463 or uncE408 alleles on F-plasmids or multicopy plasmids were capable of carrying out oxidative phosphorylation. In particular, membranes from strain AN1928 (pAN162, uncE463) gave phosphorylation rates and P/O ratios equal to or greater than those obtained for the control strain AN1460 (pAN45, unc+). However, the mutant membranes, on removal of the F1-ATPase, appeared to be proton-impermeable. The ATPase activity of the mutant membranes was also resistant to the inhibitor dicyclohexylcarbodi-imide.

The F1F0-ATPase of Escherichia coli. Substitution of proline by leucine at position 64 in the c-subunit causes loss of oxidative phosphorylation.

The uncE410 allele differs from the normal uncE gene in that C leads to T base changes occur at nucleotides 190 and 191, resulting in proline at position 64 in the c-subunit of the F1F0-ATPase being replaced by leucine. Two partial-revertant strains were isolated in which alanine-20 of the c-subunit was replaced by proline, owing to a G leads to C base change at nucleotide 58. These c-subunits, coded for by the uncE501 and uncE502 alleles, therefore contained two amino acid changes, namely proline-64 leads to leucine, and alanine-20 leads to proline. Membranes prepared from the partial-revertant strains lacked ATP-dependent atebrin-fluorescence-quenching activity but were able to carry out oxidative phosphorylation. The ATPase activity of the F1-ATPase was inhibited when bound to membranes from strains carrying the uncE410, uncE501 and uncE502 alleles. It is concluded that a bend in the helix axis in one of the arms of the c-subunit hairpin structure is required for integration of the c-subunit into a functional F1F0-ATPase.

Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli.

The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the 'Helical Hairpin Hypothesis' of Engelman & Steitz [(1981) Cell 23,411-422].

Use of chlC-lac fusions to determine regulation of gene chlC in Escherichia coli K-12.

Gene fusions between the lac structural genes and the chlC locus were isolated, and the regulation of lac gene expression was studied. The fused lac genes were induced by nitrate anaerobically and repressed by the presence of oxygen.

Isolation of a lambdadcys transducing bacteriophage and its use in determining the regulation of cysteine messenger ribonucleic acid synthesis in Escherichia coli K-12.

A defective specialized lambda transducing phage carrying the cysJ, cysI, cysH, and cysD genes has been isolated from a secondary-site lysogen. Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization studies utilizing this phage have been carried out to detect cysteine-specific messenger RNA (cys mRNA) synthesized in vivo. A vivo. A 3.5- to 9-fold increase in the rate of synthesis of cys mRNA has been detected in the derepressed wild-type (Cys+) strain grown on glutathione compared with a repressed control grown on cystine. Pleiotropic cysE and cysB mutants grown on glutathione were found to possess rates of synthesis of cys mRNA that were significantly lower than their derepressed isogenic parent. The addition of O-acetyl-L-serine to the cysE strain produced a 5.5-fold increase in the rate of synthesis of cys mRNA. These results indicate that cysteine biosynthesis is controlled at the level of transcription by the inducer O-acetylserine, the cysB protein and cyst(e)ine.

Isolation and characterization of cysK mutants of Escherichia coli K12.

cysK mutants, deficient in O-acetylserine sulphydrylase A [O-acetyl-L-serine acetate-lyase (adding hydrogen-sulphide); EC 4.2.99.8], were isolated as strains resistant to selenite or giving a black colour reaction on bismuth citrate indicator medium. All were resistant to the inhibitor I,2,4-triazole. Four independent mutants were found which possessed lowered levels of O-acetylserine sulphydrylase activity and also partially constitutive levels of NADPH-sulphite reductase [hydrogen-sulphide: NADP+ oxidoreductase; EC I.8.I.2]. Strains containing both a cysE mutation and a cysK mutation lacked the constitutive levels of NADPH-sulphite reductase showing that these levels were due to the in vivo concentration of the inducer, O-acetylserine. The cysK locus was found to be 81% cotransducible with the ptsI gene.