CLONING AND EXPRESSION IN ESCHERICHIA-COL I-CELLS OF A PLASMID PBS195GENE THAT DETERMINES THE ACTIVITY OF OXYGENASE

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated fro m long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of t he strain Escherichia coli HB1O1 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are pres ent in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in an y known metapyrocatechases, were found in cells of E. coli. Hybridizat ion with a P-32-labeled fragment containing the NahC gene revealed a r egion of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS 195. Deletion variants of this plasmid that lost oxygenase activity co nfirmed the location of the oxygenase gene in this region. The gene re sponsible for oxygenase activity in the plasmid was cloned on the pUC1 9 vector in E. coli cells. The expression of the cloned gene is contro lled by the lac promoter of this vector, Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synth esized in E. coli mini-cells, showed that this activity requires the p articipation of a polypeptide with molecular mass of 34 kDa.