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J R Guest

Publications and source records attributed to J R Guest.

At least 163 records · Page 9Linked to original sources
PubMed1984

Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase.

The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene. The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14-base-pair intercistronic region containing a good ribosomal binding site. The absence of a typical terminator sequence and the presence of an IS-like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon. The IS-like sequence is homologous with other intercistronic sequences including that between the sdhB and sucA genes, the overall gene organisation being: sdhB-IS-sucAsucB-IS-. The patterns of codon usage indicate that sucB may be more strongly expressed than sucA, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex. The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein. Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceF gene product) indicates that each contains two analogous domains, an amino-terminal lipoyl domain linked to a carboxy-terminal catalytic and subunit binding domain. The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated approximately 100-residue lipoyl binding regions containing two short (approximately 19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one approximately 100-residue lipoyl binding region, with approximately 27% homology to each of the three comparable regions in E2p, and no detectable internal repeats. The catalytic and subunit binding domains, each approximately 300 residues, have an overall homology of 34% and, consistent with their combination of analogous and specific functions, some regions are more homologous than others. Both sequences feature segments rich in proline and alanine. In E2p these occur at the carboxy-terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline-alanine segment is found approximately 50 residues into the subunit binding domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed1984

Cyclic AMP and anaerobic gene expression in E. coli.

The expression of the fumarate reductase system of Escherichia coli is completely dependent on the presence of adenylate cyclase or cyclic AMP, but the cyclic AMP receptor protein (CRP) is not required. This suggests that cyclic AMP may function as an effector for a second gene activator protein, possibly Fnr, and thus form part of a redox-sensitive regulatory mechanism controlling the expression of anaerobic respiratory functions.

Adenylyl Cyclases↗
PubMed1984

Complete nucleotide sequence of the fumarase gene fumA, of Escherichia coli.

The nucleotide sequence of a 2.41 kb fragment of E. coli DNA containing the fumA gene encoding fumarase was determined using the dideoxy chain termination method. The initiation and termination sites of fumA transcription were located using RNA:DNA hybridisation with single-stranded M13 probes. The length of the fumA transcript was estimated as 1760 +/- 15 nucleotides and the fumA coding region of 1647 base pairs (549 codons) was identified. The deduced molecular weight of the fumarase protein (Mr = 60163) is in good agreement with that of the protein identified by the maxicell procedure. The DNA fragment also contains the 5' end of the manA gene encoding mannose 6-phosphate isomerase, which is transcribed with the opposite polarity to fumA .

Amino Acid Sequence↗
PubMed1984

Structural relationship between glutathione reductase and lipoamide dehydrogenase.

The elucidation of the primary structure of the Escherichia coli lipoamide dehydrogenase (EC 1.8.1.4) by sequencing the corresponding structural gene (lpd) has enabled a detailed structural comparison between lipoamide dehydrogenase and the related disulphide oxido-reductase, human erythrocyte glutathione reductase (EC 1.6.4.2). Some 28% of the amino acid residues were found to be identical and a striking degree of homology was apparent throughout the polypeptide chains. It was concluded that the two enzymes possess very similar three-dimensional structures with particularly strong conservation of residues around the FAD and NAD(P) binding sites and at the redox centres of the molecules. Significant amino acid substitutions occur in the substrate binding pocket and these include an extra 18 amino acid residues at the C terminus of lipoamide dehydrogenase. Under physiological conditions, lipoamide dehydrogenase and glutathione reductase act in opposite directions, passing reducing equivalents to NAD+ or from NADPH (respectively), and two key substitutions near the redox centre could be associated with this difference in function. This study represents the first direct structural comparison between two related enzymes that are NADP+-linked (glutathione reductase) and NAD+-linked (lipoamide dehydrogenase). The differential recognition of these two cofactors could be explained in terms of amino acid substitutions. A divergent evolutionary relationship between the two enzymes including their NAD and NADP binding domains is fully supported by this analysis.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli.

A 1.6-kb MspI-HindIII chromosomal DNA segment, carrying the complete coding region of the 6-phosphomannose isomerase (PMI) structural gene, manA, and the 5' end of the gene encoding the major fumarase activity, fumA, of Escherichia coli K-12, has been sequenced using the chain termination method. The start points of manA and fumA transcripts were located by S1 mapping using 32P-labelled M13 ssDNA probes, and the two genes were shown to be transcribed divergently. The sequence of the 390 amino acid residues comprising the PMI monomer has been deduced, and the predicted Mr of 42 716 agrees with that for the protein of Mr 42 000 identified previously by the maxicell procedure.

Amino Acid Sequence↗
PubMed1984

Characterization of the fumarase gene of Bacillus subtilis 168 cloned and expressed in Escherichia coli K12.

The fumarase (citG) gene of Bacillus subtilis 168 has been identified in a collection of lambda phages carrying EcoRI-generated fragments of B. subtilis DNA. Regions of the cloned DNA have been subcloned into plasmid vectors, and the ability of prophages and multicopy plasmids to complement Escherichia coli and B. subtilis fumarate mutations has been examined. Two EcoRI fragments of 1.5 and 5.1 kb are both required for fumarase expression in E. coli and B. subtilis. The level of fumarase activity from a single copy of the B. subtilis citG gene expressed in E. coli is approximately one-tenth of that from the normal E. coli gene; the level is increased by expression from a pBR322-derived multicopy plasmid. The citG gene has been located within the cloned DNA by transposon mutagenesis and by expression studies, which have also identified a polypeptide of Mr 49000 as the product of the citG gene. The properties of a truncated derivative of this polypeptide have indicated the direction of transcription of the citG gene.

Autoradiography↗
PubMed1984

Cloning of the aspartase gene (aspA) of Escherichia coli.

The aspartase gene (aspA) of Escherichia coli has been isolated in two plasmids, pGS73 and pGS94, which contain segments of bacterial DNA (12.5 and 2.8 kb, respectively) inserted into the tet gene of the vector pBR322. The plasmids were constructed by sequential sub-cloning from a larger ColE1-frd+ hybrid plasmid. The location of the aspA gene confirmed predictions based on a correlation between the genetic and restriction maps of the corresponding region. The aspartase activities of plasmid-containing aspA mutants were amplified four- to sixfold relative to aspA+ parental strains. The aspA gene product was tentatively identified as a polypeptide of Mr 55 000, which is somewhat larger than previous estimates (Mr 45000 to 48000) for aspartase.

Ammonia-Lyases↗
PubMed1983

Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12.

The nucleotide sequence of a 1980-base-pair segment of DNA, containing the lpd gene encoding the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The lpd structural gene comprises 1419 base pairs (473 codons, excluding the initiating AUG codon). It is preceded by a good promoter and an excellent ribosome binding site and it ends with a typical rho-independent terminator sequence. The results confirm that the lpd gene is an independent gene linked to, but not part of, the ace operon that encodes the E1 and E2 components of the pyruvate dehydrogenase complex. The location and transcriptional polarity of the lpd gene relative to the restriction map of the corresponding region of DNA, are completely consistent with previous genetic and post-infection labelling studies. The composition, Mr (50554 or 51274 if the FAD cofactor is included), amino-terminal sequence and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with previous studies on the purified enzyme. The enzyme also exhibits a remarkable degree of sequence homology with peptides of the pig heart enzyme and with other pyridine nucleotide disulphide oxidoreductases whose sequences have been defined: human erythrocyte glutathione reductase and plasmid-encoded mercuric reductase.

Amino Acid Sequence↗
PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component.

The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain-termination method. The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho-independent terminator. The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon. The amino terminus, carboxy-terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein. The predicted molecular weight (Mr = 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78000-89000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration. The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain. The lipoyl domain corresponds to the amino-terminal segment of the protein. It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues. The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy-terminal segment.

Acetyltransferases↗
PubMed1983

Nucleotide sequence of the promoter region of the citrate synthase gene (gltA) of Escherichia coli.

The gltA gene, specifying the citrate synthase (EC 4.1.3.7) of Escherichia coli, has been isolated and the nucleotide sequence of a 752 basepair segment containing the gltA promoter and encoding 96 aminoterminal residues of the protein has been defined using the dideoxy/M13 method. The results confirm the location and transcriptional polarity of the gltA gene and indicate that the gltA transcript may contain a long leader sequence of 302-306 nucleotides upstream from the coding region.

Base Sequence↗
PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component.

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Amino Acids↗
PubMed1983

Homology between CAP and Fnr, a regulator of anaerobic respiration in Escherichia coli.

The fnr gene is essential for the expression of anaerobic respiratory metabolism in Escherichia coli. Genetic and biochemical studies support the view that its product. Fnr, is a transcriptional regulatory protein specific for genes encoding anaerobic respiratory functions (fumarate, nitrate and nitrite reductases, hydrogenase, etc.). In this respect Fnr may be considered analogous to the well-characterized catabolite gene activator protein (CAP), which mediates the control of catabolite-sensitive gene transcription. With a view to identifying its function, the fnr gene has recently been cloned and the primary structure of the Fnr protein deduced from the nucleotide sequence. This has revealed the presence of three regions of sequence homology with CAP. One corresponds to the DNA-binding site, a region of about 20 highly conserved amino acids that is believed to form a characteristic three-dimensional structure in several transcriptional regulators. The other regions of homology are in the nucleotide binding domain of CAP but the residues that interact with cAMP are not identical in Fnr. These homologies suggest that Fnr and CAP may have similar three-dimensional structures and that the regulation of anaerobic energy metabolism may involve interaction between Fnr and an unidentified effector molecule.

Amino Acids↗
PubMed1983

Hybrid plasmids containing the pyruvate dehydrogenase complex genes and gene-DNA relationships in the 2 to 3 minute region of the Escherichia coli chromosome.

A sample of colonies from the Clarke-Carbon ColE1-Escherichia coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, delta (aroP aceE aceF lpd). Two ColE1-lpd+ hybrid plasmids were identified: pGS2 (ColE1-ace lpd+; 24 kb) and pGS5 (ColE1-lpd+; 14 kb). Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex, whereas pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter). These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24.2 kb segment of bacterial DNA in the nadC-lpd region. A further 13 sites (six enzymes) were defined in a 5.4 kb sub-segment containing the lpd gene. lambda phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7.78 kb sub-segment flanked by AccI and NruI sites.

Bacteriophage lambda↗
PubMed1983

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.

Adenosine Triphosphatases↗
PubMed1983

Cloning, mapping, and expression of the fumarase gene of Escherichia coli K-12.

Two classes of fumarase-transducing phages, lambda fumA and lambda fumB, were isolated from populations of recombinant phages containing HindIII fragments of Escherichia coli DNA; they were isolated by virtue of their ability to complement the metabolic lesion of a fumarase-negative mutant. The strongly complementing lambda fumA phages contained a 6.2-kilobase HindIII fragment encoding: the fumA gene, located at 35.5 min in the E. coli linkage map and expressing the major fumarase activity; the mannosephosphate isomerase gene, manA; and an unidentified gene, g48. The three genes were located relative to the restriction map of the cloned fragment and the genetic linkage map (terC-g48-fumA-manA-uidAoR), their transcription polarities were defined as anticlockwise in the chromosome, and the molecular weights of the corresponding gene products were established: fumA, 61,500; manA, 42,000; g48, 48,000. Organisms containing the fumA gene sub-cloned in multicopy plasmids overproduced fumarase up to 50-fold. The weakly complementing class of transducing phages, lambda fumB, contained several genes in an 8.2-kilobase HindIII fragment, including one (fumB) that determines a minor fumarase activity. Complementation by fumB was only observed in high-copy situations such as transduction plaques and in strains containing a multicopy plasmid in which 40% of normal fumarase activity was detected. The basis for the complementation by fumB was not defined.

Bacteriophage lambda↗
PubMed1982

Nucleotide sequence of the fnr gene and primary structure of the Enr protein of Escherichia coli.

The nucleotide sequence of a 1.64 kb fragment of E. coli DNA containing the fnr gene (regulatory gene for fumarate and nitrate reduction) was determined using the dideoxy chain termination method. The fnr coding region (750 bp) was identified, and the initiation and termination points of fnr transcription were located by RNA:DNA hybridisation with single-stranded M13 probes. The DNA fragment also contained the 5' end of a separately transcribed gene of unknown function. The deduced molecular weight (27947) of the Fnr protein was in agreement with that of the protein identified by the maxicell procedure, and the primary structure contained regions of homology with several transcriptional regulator proteins.

Amino Acid Sequence↗