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首页> 外文期刊>Infection and immunity >Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor.
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Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor.

机译：金黄色葡萄球菌磷脂酰肌醇特异性磷脂酶C的克隆，表达和诱变：潜在的葡萄球菌致病因子。

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摘要

Staphylococcus aureus secretes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) which is able to hydrolyze the membrane lipid PI and membrane protein anchors containing glycosyl-PI. The gene for PI-PLC (plc) was cloned from S. aureus into Escherichia coli. Oligonucleotide probes based on partial protein sequence and polyclonal antibodies raised against the purified protein were used to identify positive clones. E. coli transformed with a plasmid containing the plc gene expressed PI-PLC enzyme activity which was abolished by mutagenesis with a tetracycline resistance gene. The plc gene was present in all 15 S. aureus strains examined but not in any of 6 coagulase-negative staphylococcal species. The plc gene contained 984 bp and coded for a mature protein with a calculated molecular mass of 34,107 Da. Amino acid sequence comparisons indicated that the staphylococcal plc gene was similar (51 to 56%) to the PI-PLCs from Bacillus cereus, Bacillus thuringiensis, and Listeria monocytogenes. The recombinant PI-PLC expressed in E. coli was purified and exhibited biochemical properties identical to those of the native PI-PLC from S. aureus. PI-PLC production was decreased in agr mutant strains of S. aureus. However, PI-PLC production by both agr+ and agr mutant strains exhibited a similar dependence on the type of medium used. These data suggested that PI-PLC production was regulated by both agr-dependent and agr-independent mechanisms.
机译：金黄色葡萄球菌分泌磷脂酰肌醇（PI）特异性磷脂酶C（PI-PLC），该酶能够水解膜脂质PI和含有糖基PI的膜蛋白锚。将PI-PLC（plc）基因从金黄色葡萄球菌克隆到大肠杆菌中。基于部分蛋白质序列的寡核苷酸探针和针对纯化蛋白质的多克隆抗体被用于鉴定阳性克隆。用含有plc基因的质粒转化的大肠杆菌表达PI-PLC酶活性，该活性通过与四环素抗性基因的诱变而被消除。 plc基因存在于所检查的所有15株金黄色葡萄球菌菌株中，但不存在于6种凝固酶阴性葡萄球菌物种中的任何一种中。 plc基因包含984 bp，编码一个成熟蛋白，分子量为34107 Da。氨基酸序列比较表明，葡萄球菌的plc基因与蜡状芽孢杆菌，苏云金芽孢杆菌和单核细胞增生李斯特氏菌的PI-PLC相似（51％至56％）。纯化了在大肠杆菌中表达的重组PI-PLC，其生化特性与金黄色葡萄球菌的天然PI-PLC相同。在金黄色葡萄球菌的agr突变株中PI-PLC的产量降低。但是，由agr +和agr突变菌株产生的PI-PLC对所用培养基的类型显示出相似的依赖性。这些数据表明，PI-PLC的生产受农业相关和非农业相关机制的调节。

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