RESUMO
Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.
Assuntos
Dioxigenases , Genes Bacterianos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Fenóis/metabolismo , Catecol 2,3-Dioxigenase , Mapeamento Cromossômico , Clonagem Molecular , Elementos de DNA Transponíveis , Escherichia coli/genética , Expressão Gênica , Oxigenases de Função Mista/genética , Mutagênese Insercional , Oxigenases/genética , FenolRESUMO
pFDX1 is a recombinant plasmid which carries a foreign gene xylE. By selecting for kanamycin-resistant mutants of Bacillus stearothermophilus CU21(pFDX1) at higher temperature, a variant strain CU21-163 was obtained. This strain harbors a mutant plasmid pFDX163, which was formed by insertion of a 2.0kb H-fragment from the CU21 genome onto the plasmid pFDX1. pFDX163 was supposed to be integrated into the CU21 chromosome via homologous recombination of H-fragments. The CU21-163 strain consists of two cell types, i.e. y-cell and w-cell. The expression level of xylE gene in the former is higher than that in the latter. The progeny of a y-cell always contains some w-cells, while that of a w-cell contains y-cells. This is supposed to be due to a phase variation of CU21-163. Analysis on the amount of free and integrated plasmid DNA in different DNA samples of CU21-163 cells allows us to draw the conclusion that there are both free and integrated plasmids in the y-cells, whereas only integrated ones in the w-cells.
Assuntos
Geobacillus stearothermophilus/genética , Plasmídeos , Southern Blotting , Catalase/análise , Geobacillus stearothermophilus/efeitos dos fármacos , Geobacillus stearothermophilus/enzimologia , Canamicina/farmacologia , Hibridização de Ácido NucleicoRESUMO
Using the gene expression vector pFDC11 for Bacillus stearothermophilus CU21, a recombinant plasmid pFDX1 was constructed, which carries the Pseudomonas-derived xylE gene coding for Catechol 2,3-dioxygenase (CatO2ase). CatO2ase activity can be detected from CU21 (pFDX1) cells grown at 48 degrees C. This result indicates that the promoterless xylE gene can be expressed in a thermophilic host under the direction of a promoter from a thermophilic bacterium. By selecting Kmr mutants at elevated growth temperature, a segregant CU21-161 was obtained, the xylE gene expression of which at 55 degrees C and 60 degrees C was much higher than that of the parental strain. A method for CatO2ase assay with suspension of intact cells was also reported in this paper.
Assuntos
Dioxigenases , Expressão Gênica , Genes Bacterianos , Geobacillus stearothermophilus/genética , Catecol 2,3-Dioxigenase , Geobacillus stearothermophilus/enzimologia , Oxigenases/genética , Oxigenases/metabolismoRESUMO
A low copy number mutant of ColE1-like plasmid of Escherichia coli was reported in this paper. The recombinant plasmid pPGVT3 derived from the vector pUC4 was unstable in the E. coli host strain DF2145. No transformant could be obtained at 40 degrees C when DF2145 was transformed with pPGVT3. By in vitro mutagenesis of pPGVT3 plasmid DNA with hydroxylamine which induces point mutations, a mutant plasmid pPGVT3HA was obtained, which was stable in DE2145. The mutation site was localized within the pUC moiety of pPGVT3HA. The copy number of the mutated pUC vector and its derivatives were found to be reduced. The effect of low copy number mutation on the stability of the recombinant plasmids was discussed.