Kozlova E V, Suvorova E S, Romanov V P, Boronin A M
Genetika. 1995 Feb;31(2):170-3.
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.
在从长寿者体内分离出的一株乳酸杆菌中检测到的质粒pBS195具有广泛的宿主范围，包括革兰氏阳性和革兰氏阴性微生物[1]。携带该质粒的大肠杆菌HB101菌落与儿茶酚发生颜色反应。这表明介导加氧酶活性的基因存在于该质粒中。在大肠杆菌细胞中发现，由pBS195介导的该酶具有高活性水平以及在任何已知间苯二酚酶中均未检测到的底物特异性。用含有NahC基因的32P标记片段进行杂交，结果显示与质粒pBS195的1.6kb EcoR I - BamH I片段存在同源区域。该质粒失去加氧酶活性的缺失变体证实了加氧酶基因位于该区域。将质粒中负责加氧酶活性的基因克隆到大肠杆菌细胞中的pUC19载体上。克隆基因的表达受该载体的lac启动子控制。物理分析、杂交分析、缺失分析以及对在大肠杆菌微小细胞中合成的多肽的分析表明，这种活性需要一种分子量为34 kDa的多肽参与。
