MOLECULAR AND FUNCTIONAL-ANALYSIS OF THE TOL PLASMID PWWO FROM PSEUDOMONAS-PUTIDA AND CLONING OF GENES FOR THE ENTIRE REGULATED AROMATIC RING META-CLEAVAGE PATHWAY
MOLECULAR AND FUNCTIONAL-ANALYSIS OF THE TOL PLASMID PWWO FROM PSEUDOMONAS-PUTIDA AND CLONING OF GENES FOR THE ENTIRE REGULATED AROMATIC RING META-CLEAVAGE PATHWAY
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DOI：
10.1073/pnas.78.12.7458
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
TIMMIS, KN
中科院分区：
文献类型：
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作者：
FRANKLIN, FCH;BAGDASARIAN, M;TIMMIS, KN
通过转座诱变和基因克隆研究了恶臭假单胞菌质粒pWWO-WWO-161的遗传结构，该质粒编码降解甲苯和相关芳烃的酶。分解代谢基因定位于2个簇，1个用于上游途径（烃→烃）。羧酸）酶和另一种用于较低途径（羧酸→）。三羧酸循环）酶，其由14-脱氢酶DNA片段分开。因此，分解代谢基因的物理组织反映了它们的功能组织为2个调节块。将pWWO-161 DNA片段Sst I片段C和片段D克隆到宽宿主范围载体中以产生质粒pKT 530。该杂合体编码甲苯甲酸加氧酶和所有Meta裂解途径酶，并且它使恶臭假单胞菌mt-2和大肠杆菌K-12细胞能够在间甲苯甲酸作为唯一C源上生长。pKT 530质粒还携带xylS（其中产物被假定为调节较低途径基因的表达的基因）和与该产物相互作用的途径的控制序列，因为儿茶酚2，3-加氧酶合成在恶臭假单胞菌和大肠杆菌中均由间甲苯酸特异性诱导。杆菌有证据表明，启动子操纵基因的Meta途径的功能与RNA聚合酶或xylS产品的E。与恶臭假单胞菌的酶或产物相比，
The genetic organization of the P. putida plasmid pWWO-WWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, was investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to 2 clusters, 1 for upper pathway (hydrocarbon .fwdarw. carboxylic acid) enzymes and the other for lower pathway (carboxylic acid .fwdarw. tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into 2 regulatory blocks. The pWWO-161 DNA fragments SstI fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole C source. The pKT530 plasmid also carries xylS (a gene in which the product is postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida.