RESUMO
Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.
Assuntos
Vetores Genéticos , Bactérias Gram-Negativas/genética , Plasmídeos , Sequência de Bases , Clonagem Molecular , Conjugação Genética , Escherichia coli/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Especificidade da EspécieRESUMO
A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contained five different cob genes named cobN to cobQ and cobW. Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cob proteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibiting both cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified as cob(I)alamin adenosyltransferase. CobN is proposed to play a role in cobalt insertion reactions. Four other open reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not lead to a cobalamin-minus phenotype.
Assuntos
Alquil e Aril Transferases , DNA Bacteriano/química , Genes Bacterianos , Complexos Multienzimáticos/genética , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/genética , Pseudomonas/genética , Transaminases/genética , Transferases/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Teste de Complementação Genética , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/química , Mutagênese Insercional , Nucleotidiltransferases/biossíntese , Fosfotransferases/biossíntese , Transaminases/biossíntese , Vitamina B 12/biossíntese , Vitamina B 12/química , Vitamina B 12/genéticaRESUMO
A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertion-mediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Genes Bacterianos , Pseudomonas/genética , Vitamina B 12/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , DNA Bacteriano/química , Desoxirribonuclease HindIII , Teste de Complementação Genética , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Mutagênese Insercional , Vitamina B 12/biossínteseRESUMO
Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P. denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.
Assuntos
Proteínas de Bactérias , Cobamidas/genética , Genes Bacterianos , Complexos Multienzimáticos , Transferases de Grupos Nitrogenados , Nucleotidiltransferases , Pentosiltransferases/genética , Pseudomonas/genética , Transferases/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Cobamidas/biossíntese , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Desoxirribonuclease EcoRI , Desoxirribonuclease HindIII , Amplificação de Genes , Teste de Complementação Genética , Cinética , Dados de Sequência Molecular , Peso Molecular , Pseudomonas/enzimologiaRESUMO
An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified in Methanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield. The enzyme had an apparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, as estimated by gel electrophoresis under denaturing conditions. The Km value for uroporphyrinogen III was 52 nM. The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III into precorrin-2. Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet and B. Cameron, J. Bacteriol. 171:4222-4231, 1989), M. ivanovii SUMT did not show substrate inhibition at uroporphyrinogen III concentrations of up to 20 microM. Oligonucleotide probes from limited peptide sequence information were used to clone the corresponding gene. The encoded polypeptide showed more than 40% strict homology with P. denitrificans SUMT. The M. ivanovii SUMT structural gene is likely to be, as is P. denitrificans cobA, involved in corrinoid synthesis.
Assuntos
Euryarchaeota/genética , Metiltransferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Euryarchaeota/enzimologia , Genes Bacterianos , Cinética , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Pseudomonas/enzimologia , Pseudomonas/genética , Homologia de Sequência do Ácido Nucleico , EspectrofotometriaRESUMO
A Bacillus megaterium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P. denitrificans SUMT, substrate inhibition at uroporphyrinogen III concentrations above 0.5 microM, suggesting a common regulation for aerobic cobalamin-producing organisms.
Assuntos
Bacillus megaterium/genética , Escherichia coli/genética , Metiltransferases/genética , Plasmídeos , Sequência de Aminoácidos , Bacillus megaterium/enzimologia , Sequência de Bases , Escherichia coli/enzimologia , Genes Bacterianos , Metiltransferases/química , Dados de Sequência MolecularRESUMO
A 8.7-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and the genetic analysis revealed that this fragment carries eight different cob genes (cobF to cobM). Six of these genes have the characteristics of translationally coupled genes. cobI has been identified as S-adenosyl-L-methionine (SAM):precorrin-2 methyltransferase structural gene because the encoded protein has the same NH2 terminus and molecular weight as those of the purified enzyme. From protein homology with CobA and CobI, two SAM-dependent methyltransferases of the cobalamin pathway, it is proposed that cobF, cobJ, cobL, and cobM code for other methyltransferases involved in the cobalamin pathway. In addition, purified CobF protein has affinity for SAM, as expected for a SAM-dependent methyltransferase. Accumulation of cobalamin precursors in Agrobacterium tumefaciens mutants complemented by any of these eight genes suggest that, apart from cobI, whose function is identified, the products of all these genes are implicated in the conversion of precorrin-3 into cobyrinic acid.
Assuntos
DNA Bacteriano/genética , Genes Bacterianos , Pseudomonas/genética , Uroporfirinas/metabolismo , Vitamina B 12/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Códon/genética , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Plasmídeos , Pseudomonas/metabolismo , Replicon , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Vitamina B 12/biossínteseRESUMO
A 5.4-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment carries five different cob genes (cobA to cobE). Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT. For the same reasons the cobB gene was shown to be the structural gene for cobyrinic acid a,c-diamide synthase. Genetic and biochemical data concerning cobC and cobD mutants suggest that the products of these genes are involved in the conversion of cobyric acid to cobinamide.