Assuntos
Colífagos/genética , Corynebacterium/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Treonina/biossíntese , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Regulação Viral da Expressão Gênica , Genes Bacterianos , Genes Virais , Plasmídeos , TemperaturaRESUMO
Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.
Assuntos
Oxirredutases do Álcool/genética , Corynebacterium/genética , Regulação Bacteriana da Expressão Gênica , Homosserina Desidrogenase/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/genética , Brevibacterium/enzimologia , Brevibacterium/genética , Clonagem Molecular , Corynebacterium/enzimologia , Corynebacterium/metabolismo , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Homosserina/biossíntese , PlasmídeosRESUMO
A library of EcoRI DNA fragments from Brevibacterium flavum was constructed using plasmid vector. The genes complementing ThrA2 and ThrB mutations in Escherichia coli were identified in the library. The gene thrA2 of B. flavum codes for mutant enzyme homoserine dehydrogenase insensitive to inhibition by threonine. The genes thrA2 and thrB are localized wihtin the EcoRI fragment 4.1 kb long and are expressed under the control of their own promoters in E. coli cells. Structural and functional analysis of cloned C. glutamicum gene ilvA was performed. The gene of C. glutamicum complemented ilvA mutation in E. coli and appeared to be localized within the EcoRI--SacI DNA fragment 1.6 kb in size. Using E. coli minicells we have demonstrated that the gene ilvA of C. glutamicum controls the synthesis of polypeptide of relative molecular mass 50 kD.
Assuntos
Ácido Aspártico/biossíntese , Brevibacterium/genética , Escherichia coli/genética , Genes Bacterianos , Glutamatos/biossíntese , Ácido Aspártico/análogos & derivados , Southern Blotting , Brevibacterium/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/metabolismo , Plasmídeos , Mapeamento por RestriçãoRESUMO
Molecular cloning and expression of Corynebacterium glutamicum genes complementing Escherichia coli mutations thrA2 and ilvA was performed. It was demonstrated that the thrA2 gene of C. glutamicum is located close to thrB on EcoRI DNA fragment 4.1 kb long. The fragment was cloned in pUC18 vector. The thrA2 gene is expressed in the recombinant plasmid pOBT3 under control of the vector pUC18 Plac promoter. In E. coli minicells, the genes thrA2 and thrB determined synthesis of proteins of Mr 43kD and 25 kD, respectively. A gene complementing ilvA mutation of E. coli was identified in a library of EcoRI C. glutamicum DNA fragments. This library was constructed using plasmid vector. It was shown that the ilvA gene of C. glutamicum is located inside the 3.6 kb EcoRI fragment and is expressed using its own promoter.
Assuntos
Clonagem Molecular , Corynebacterium/genética , Escherichia coli/genética , Genes Bacterianos , Teste de Complementação Genética , Mutação , DNA/genética , Regulação da Expressão Gênica , Vetores Genéticos , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas , Mapeamento por Restrição , Transdução GenéticaRESUMO
Cloning of genes for threonine and lysine biosynthesis from Corynebacterium glutamicum was performed in Escherichia coli cells using the plasmid vector lambda pSL5. The cloned genes are identified via complementation of thrB and lysA mutations. The gene complementing thrB of E. coli is located within a 4.1 kb EcoRI fragment of C. glutamicum chromosomal DNA. All the recombinant phasmids complementing the lysA gene of E. coli contain common 2.2 kb and 3.3 kb EcoRI C. glutamicum DNA fragments. The cloned DNA fragments hybridize with identical EcoRI fragments of C. glutamicum chromosomal DNA.
Assuntos
Aminoácidos/biossíntese , Clonagem Molecular/métodos , Corynebacterium/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Aminoácidos/genética , Bacteriófagos/genética , Cromossomos Bacterianos , Corynebacterium/metabolismo , DNA Bacteriano/genética , Escherichia coli/metabolismo , Genes Bacterianos , Teste de Complementação Genética , Vetores Genéticos , Lisina/biossíntese , Lisina/genética , Hibridização de Ácido Nucleico , Plasmídeos , Treonina/biossíntese , Treonina/genéticaRESUMO
The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.
