[Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells].
Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.
Kozlova
E V
EV
Suvorova
E S
ES
Romanov
V P
VP
Boronin
A M
AM
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D007778:Lactobacillus / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D013379:Substrate Specificity
D010105:Oxygenases
Klonirovanie i ékspressiia v kletkakh Escherichia coli gena plazmida pBS195, determiniruiushchego oksigenaznuiu aktivnost'.