Keil H, Lebens M R, Williams P A
J Bacteriol. 1985 Jul;163(1):248-55. doi: 10.1128/jb.163.1.248-255.1985.
Pseudomonas putida MT15 contains a 250-kilobase-pair (kbp) TOL plasmid pWW15, encoding toluene and xylene catabolism, which undergoes large spontaneous deletions to give two classes of mutants with altered catabolic phenotypes (H. Keil and P. A. Williams, J. Gen. Microbiol, 131:1023-1033, 1985). Two structural genes for catechol 2,3-oxygenase (C23O) were cloned from pWW15. The gene for C23OI was located on the 2.1-kbp XhoI fragment Xh, whereas that for C23OII was found on the 11.5-kbp BamHI fragment BJ. The two restriction fragments and the subcloned regions of them showed no similarity in the pattern of restriction digestion, nor did they hybridize with each other. The substrate specificities of the two enzymes were also substantially different. The two structural genes were separated on pWW15 by about 100 kbp. In plasmid pWW15-510 of a B5 mutant, the 90-kbp deletion in the plasmid removed most of the intervening DNA, but it also deleted 80% of the gene for C23OI from its 3' end. Thus, only C23OII was expressed in the host MT15-510. Conversely, in RP4::pWW15 cointegrate plasmid pWW15-1003, only the C23OI gene was present. The expression of C23O activity from these two derivative plasmids and from the wild-type pWW15 showed that only C23OI was induced by growth in the presence of m-toluate, whereas both activities were induced in the only C23OI was induced by growth in the presence of m-toluate, whereas both activities were induced in the presence of m-xylene. These findings cast doubt on the earlier hypothesis that the deletions in B3 and B5 mutants remove a regulatory gene by which m-toluate induces the enzymes necessary for its own catabolism.
恶臭假单胞菌MT15含有一个250千碱基对（kbp）的TOL质粒pWW15，编码甲苯和二甲苯分解代谢，该质粒会发生大量自发缺失，产生两类分解代谢表型改变的突变体（H. 凯尔和P. A. 威廉姆斯，《普通微生物学杂志》，131:1023 - 1033，1985）。从pWW15中克隆了儿茶酚2,3 - 加氧酶（C23O）的两个结构基因。C23OI的基因位于2.1 kbp的XhoI片段Xh上，而C23OII的基因位于11.5 kbp的BamHI片段BJ上。这两个限制性片段及其亚克隆区域在限制性消化模式上没有相似性，它们彼此也不杂交。这两种酶的底物特异性也有很大差异。这两个结构基因在pWW15上相隔约100 kbp。在B5突变体的质粒pWW15 - 510中，质粒中的90 kbp缺失去除了大部分间隔DNA，但也从其3'端删除了80%的C23OI基因。因此，在宿主MT15 - 510中仅表达C23OII。相反，在RP4::pWW15共整合质粒pWW15 - 1003中，仅存在C23OI基因。来自这两个衍生质粒和野生型pWW15的C23O活性表达表明，只有C23OI在间甲苯酸盐存在下生长时被诱导，而在间二甲苯存在下两种活性均被诱导。这些发现对早期的假设提出了质疑，即B3和B5突变体中的缺失去除了一个调节基因，通过该基因间甲苯酸盐诱导其自身分解代谢所需的酶。
