Schell M A, Roberts D P, Denny T P
Department of Microbiology, University of Georgia, Athens 30602.
J Bacteriol. 1988 Oct;170(10):4501-8. doi: 10.1128/jb.170.10.4501-4508.1988.
A major endopolygalacturonase excreted by Pseudomonas solanacearum was purified to greater than 95% homogeneity and shown to have an isoelectric point of 9.0 and a subunit molecular mass of 52 kilodaltons (kDa). The gene encoding this enzyme (pglA) was isolated from a genomic library of P. solanacearum DNA based on its expression in Escherichia coli and shown to be contained on a 1.8-kilobase DNA fragment. The identity of the pglA gene product and the 52-kDa polygalacturonase was demonstrated by immunoadsorption and isoelectric focusing experiments. The cloned pglA gene was apparently expressed from its own promoter in E. coli and its product was partially secreted into the periplasm. The pglA gene was insertionally inactivated in vitro and used to mutate the chromosomal pglA gene of P. solanacearum by marker exchange mutagenesis. The resulting mutant strain was deficient in production of the 52-kDa polygalacturonase and took twice as long to wilt and kill tomato plants as the wild-type parent in plant bioassay experiments. Complementation in trans with the wild-type cloned pglA gene restored virulence to near wild-type levels. The data indicate that the pglA gene is important, but not absolutely necessary, for pathogenesis.
由青枯假单胞菌分泌的一种主要胞内多聚半乳糖醛酸酶被纯化至均一性大于95%，其等电点为9.0，亚基分子量为52千道尔顿（kDa）。基于该酶在大肠杆菌中的表达，从青枯假单胞菌DNA的基因组文库中分离出编码此酶的基因（pglA），并证明其位于一个1.8千碱基的DNA片段上。通过免疫吸附和等电聚焦实验证实了pglA基因产物与52-kDa多聚半乳糖醛酸酶的一致性。克隆的pglA基因在大肠杆菌中显然是从其自身启动子表达的，其产物部分分泌到周质中。pglA基因在体外被插入失活，并用于通过标记交换诱变使青枯假单胞菌的染色体pglA基因发生突变。在植物生物测定实验中，所得突变株缺乏52-kDa多聚半乳糖醛酸酶的产生，使番茄植株枯萎和死亡的时间是野生型亲本的两倍。用野生型克隆的pglA基因进行反式互补可使毒力恢复到接近野生型水平。数据表明，pglA基因对致病作用很重要，但不是绝对必需的。
