金黄色葡萄球菌金属磷酸酶的遗传特征分析及功能鉴定

目的对金黄色葡萄球菌(简称金葡菌)一种新型金属磷酸酶(metallophosphatase,MPP)SA1662进行遗传特征分析及生物学功能鉴定。方法从金葡菌基因组DNA中克隆SA1662基因,通过序列比对和构建进化树分析其遗传特征;构建重组表达质粒,转化大肠埃希菌BL21,IPTG诱导表达,表达的重组蛋白经Ni柱纯化后,测定酶活性。应用同源重组技术构建SA1662基因缺失株,分析SA1662基因缺失对金葡菌生长、万古霉素敏感性、细菌自溶性和细菌生物菌膜形成的影响,探讨SA1662的生物学功能。结果 SA1662蛋白具有MPP类蛋白结构域,并含有特征性金属离子结合位点,与金葡菌和施维茨葡萄球菌中相关蛋白有更近的亲缘关系。该蛋白酶活性依赖于Mg^2+和Mn^2+,特别是Mg^2+。与野生型菌株相比,SA1662基因缺失后,细菌生长速度减慢,万古霉素敏感性、TritonX-100诱导下的细菌自溶性和细菌生物菌膜形成能力均下降。结论 SA1662蛋白为一种新型的金葡菌MPP,在细菌抗逆性中起着重要作用。

Genetic characteristics and function of metallophosphatase from Staphylococcus aureus

Objective To analyze the genetic characteristics and identify the biological function of SA1662, a metallophosphatase(MPP) from Staphylococcus aureus. Methods SA1662 gene was cloned from the genomic DNA of S. aureus,of which the genetic characteristics was analyzed by sequence alignment and construction of phylogenetic tree.Recombinant expression vector was constructed and transformed to E. coli BL21 and induced with IPTG. The expressed recombinant protein was purified by nickel ion affinity chromatography and determined for enzyme activity. The bacterial strain with SA1662 gene deleted was constructed by homologous combination technique,based on which the effects of SA1662 gene deletion on the growth,sensitivity to vancomycin,autolysis and biofilm formation of S. aureus were evaluated,and the biological function of SA1662 was investigated. Results Genetic analysis showed that SA1662 protein had the structural domain of MPP protein and contained the characteristic metal ion binding site,which was more closely related to the proteins in S. aureus and S. schweiz. The enzyme activity of recombinant SA1662 protein was dependent on magnesium ion and manganese ion,especially magnesium ion. Compared with wild-type strain,the strain with SA1662 gene deleted grew slowly,of which the sensitivity to vancomycin as well as autolysis under induction of TritonX-100 and biofilm formation ability decreased. Conclusion SA1662 protein was a novel S. aureus MPP,which played an important role in bacterial stress resistance.