Neidle E L, Ornston L N
J Bacteriol. 1986 Nov;168(2):815-20. doi: 10.1128/jb.168.2.815-820.1986.
Catechol 1,2-dioxygenase (EC 1.13.1.1), the product of the catA gene, catalyzes the first step in catechol utilization via the beta-ketoadipate pathway. Enzymes mediating subsequent steps in the pathway are encoded by the catBCDE genes which are carried on a 5-kilobase-pair (kbp) EcoRI restriction fragment isolated from Acinetobacter calcoaceticus. This DNA was used as a probe to identify Escherichia coli colonies carrying recombinant pUC19 plasmids with overlapping sequences. Repetition of the procedure yielded an A. calcoaceticus 6.7-kbp EcoRI restriction fragment which contained the catA gene and bordered the original 5-kbp EcoRI restriction fragment. When the catA-containing fragment was placed under the control of the lac promoter on pUC19 and induced with isopropylthiogalactopyranoside, catechol dioxygenase was formed in E. coli at twice the level found in fully induced cultures of A. calcoaceticus. A. calcoaceticus strains with mutations in the catA gene were transformed to wild type by DNA from lysates of E. coli strains carrying the catA gene on recombinant plasmids. Thus, A. calcoaceticus strains with a mutated gene can be used in a transformation assay to identify E. coli clones in which at least part of the wild-type gene is present but not necessarily expressed.
邻苯二酚1,2 -双加氧酶（EC 1.13.1.1）是catA基因的产物，它催化通过β-酮己二酸途径利用邻苯二酚的第一步反应。介导该途径后续步骤的酶由catBCDE基因编码，这些基因位于从醋酸钙不动杆菌中分离出的一个5千碱基对（kbp）的EcoRI限制性片段上。该DNA被用作探针来鉴定携带具有重叠序列的重组pUC19质粒的大肠杆菌菌落。重复该过程得到了一个醋酸钙不动杆菌的6.7 kbp EcoRI限制性片段，它包含catA基因并与原始的5 kbp EcoRI限制性片段相邻。当将含catA的片段置于pUC19上的lac启动子控制下并用异丙基硫代半乳糖苷诱导时，大肠杆菌中形成的邻苯二酚双加氧酶水平是完全诱导的醋酸钙不动杆菌培养物中所发现水平的两倍。携带重组质粒上catA基因的大肠杆菌菌株裂解物中的DNA将catA基因突变的醋酸钙不动杆菌菌株转化为野生型。因此，具有突变基因的醋酸钙不动杆菌菌株可用于转化试验，以鉴定其中至少存在部分野生型基因但不一定表达的大肠杆菌克隆。
