Ostroff R M, Vasil M L
Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver 80262.
J Bacteriol. 1987 Oct;169(10):4597-601. doi: 10.1128/jb.169.10.4597-4601.1987.
The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.
铜绿假单胞菌的磷脂酶C（PLC）基因编码一种热不稳定的分泌型溶血素，它是Pi调节操纵子的一部分。通过插入四环素抗性基因盒，在体外对PLC的结构基因plcS进行了突变。采用基因置换技术，将突变的plcS基因导入铜绿假单胞菌染色体，取代野生型基因。通过Southern杂交证实了突变序列对野生型序列的精确置换。命名为PLC S的突变菌株不溶血，并且缺乏一种与野生型PLC大小相对应的78千道尔顿的蛋白质。然而，PLC S中存在一种额外的磷脂酶活性，它能够水解对硝基苯基磷酰胆碱（一种合成的PLC底物）和磷脂酰胆碱。这种酶活性不是由突变的plcS基因产生的截短产物导致的。PLC S的磷脂酶活性被鉴定为一种非溶血型PLC。
