Savard P, Charest H, Sylvestre M, Shareck F, Scholten J D, Mariano D D
Université du Québec, Institut national de la recherche scientifique--Santé, 245, Pointe-Claire, Canada.
Can J Microbiol. 1992 Oct;38(10):1074-83. doi: 10.1139/m92-176.
The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp. strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440. In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme. The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 degrees C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E. coli-pPSA843 cells and approximately 28 units per 100 g of P. putida-pPSA843 cells. Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 degrees C) prepared from the E. coli and P. putida clones was unstable and at least 20-fold lower than that observed with the whole cells. The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products. Analysis of dehalogenase activity in omega insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment. Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system. Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products. Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed.
在早期研究中，编码假单胞菌属CBS3菌株4 - 氯苯甲酸脱卤酶的基因，通过黏粒载体pPSA843克隆到大肠杆菌DH1中，随后转移至4 - 氯苯甲酸脱卤酶缺陷型菌株恶臭假单胞菌KT2440中。在本文中，我们报告了这些克隆体中4 - 氯苯甲酸脱卤酶的表达情况以及活性酶的多肽组成。悬浮于3.2 mM 4 - 氯苯甲酸（30℃）中的全细胞的脱卤酶活性，测定结果为每100 g大肠杆菌 - pPSA843细胞约27个单位（每分钟产生的4 - 羟基苯甲酸微摩尔数），每100 g恶臭假单胞菌 - pPSA843细胞约28个单位。从大肠杆菌和恶臭假单胞菌克隆体制备的新鲜细胞提取物（pH 7.4，30℃）中的脱卤酶活性不稳定，且至少比全细胞中观察到的活性低20倍。通过克隆的脱卤酶基因的选择性表达和基因翻译产物分析，确定了脱卤酶的多肽成分。对ω插入突变体和缺失突变体中的脱卤酶活性分析，将脱卤酶基因限定在9.5 kb DNA片段的4.8千碱基（4.8 kb）区域。在一个大细胞系统中，从克隆的4.8 kb DNA片段选择性表达脱卤酶基因，揭示出一种30 kDa的多肽是脱卤酶系统的成分之一。使用T7聚合酶启动子系统选择性表达脱卤酶基因，揭示出30 kDa的多肽以及57 kDa和16 kDa的多肽产物。确定在缺失突变体中翻译的三种多肽中的哪一种，提供了编码基因在单条DNA链上的相对位置以及它们的转录方向。
