Cuskey S M, Wolff J A, Phibbs P V, Olsen R H
J Bacteriol. 1985 Jun;162(3):865-71. doi: 10.1128/jb.162.3.865-871.1985.
A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.
将含有一组指定碳水化合物分解代谢基因的铜绿假单胞菌PAO染色体的6.0千碱基EcoRI片段克隆到多拷贝质粒pRO1769中。该载体在链霉素抗性决定簇内含有一个用于克隆的独特EcoRI位点和一个编码庆大霉素抗性的选择基因。用嵌合质粒pRO1816转化的铜绿假单胞菌PAO突变体恢复了在葡萄糖上生长的能力，并且与特定突变相对应的以下酶或转运活性缺陷得到了互补：glcT1，葡萄糖转运和周质葡萄糖结合蛋白；glcK1，葡萄糖激酶；以及edd-1，6-磷酸葡萄糖酸脱水酶。另外两个与glcT1和edd-1共转导的碳水化合物分解代谢标记未被质粒pRO1816互补：zwf-1，葡萄糖-6-磷酸脱氢酶；以及eda-9001，2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶。然而，当原养型菌株PAO1的转化细胞在没有碳水化合物诱导剂的情况下生长时，所有这五种通常可诱导的活性都在明显升高的基础水平上表达。载体质粒pRO1769对转化的突变体或野生型细胞中这些活性的表达没有影响。因此，pRO1816中的染色体插入片段包含edd和glcK结构基因、至少一个对葡萄糖主动转运系统表达必不可少的基因（glcT）以及其他调节五个成簇的碳水化合物分解代谢基因表达的位点。pRO1816中的插入片段也互补了葡萄糖阴性恶臭假单胞菌突变体中的edd-1突变，但没有互补另一个突变体中的eda-1缺陷。此外，pRO1816导致了葡萄糖激酶高比活性的表达，葡萄糖激酶是这些恶臭假单胞菌菌株中天然缺乏的一种酶。
