牙龈卟啉单胞菌FtsZ第322位的甘氨酸在细胞分裂中的作用

Gly322 in the FtsZ of Porphyromonas gingivalis is important for cell division

目的:探讨牙龈卟啉单胞菌FtsZ(PgFtsZ)第322位的甘氨酸在细菌细胞分裂过程中的作用。方法:通过site-d irected mutagenesis技术,采用m egaprim er方法,使用三个引物进行两轮的PCR,构建Pg-FtsZ第322位甘氨酸的点变异型质粒pYW 9(ZG322P,携带PgFtsZ第322位甘氨酸由脯氨酸取代的点变异型基因)和pYW 10(ZG322H,携带PgFtsZ第322位甘氨酸由组氨酸取代的点变异型基因),然后将质粒pYW 9、pYW 10和pEZ1(携带野生型PgFtsZ的基因)分别转化到Escherichia coliBL21(DE3)pLysS中进行表达、分离和纯化目的蛋白质。同时将载体pET3 a也转化到E.coliBL21(DE3)pLysS中作为阴性对照。进一步通过免疫印迹法鉴定构建的点变异型ZG322P和ZG322H。E.coli的形态通过显微镜观察。结果:野生型PgFtsZ的过表达导致E.coli细胞分裂的明显抑制,含质粒pEZ1的E.coli和仅含有载体作为对照的E.coli相比延长大约20倍。然而点变异型ZG322P和ZG322H的过表达并未引起E.coli细胞分裂的抑制,即含质粒pYW 9的E.coli和含质粒pYW 10的E.coli表现出正常的形态,与仅含有载体作为对照的E.coli细胞的形态相似。免疫印迹分析表明野生型PgFtsZ、点变异型ZG322P、ZG322H在相同位置出现阳性带,并且表达水平相似。结论:牙龈卟啉单胞菌FtsZ第322位的甘氨酸在细菌细胞分裂中起着重要的作用。

AIM ： To analyze the role of Gly322 in the FtsZ of Porphyromonas gingivalis for cell division. METHODS： Two point mutation plasmids of Gly322, pYW9 （ZG322P） and pYW10 （ZG322H） , were constructed by site-directed mutagenesis by using the “megaprimer” method, which utilized three primers and two rounds of PCR. The pYW9, pYW10, pEZ1 and pET3a vector alone were introduced into Escherichia coli BL21 （DE3）pLysS, respectively. The mutant proteins were expressed in E. coli BI21 （DE3）pLysS, purified by HiTrap SP column and identified by immunblot method. E. coli morphology was observed under light microscope. RESULTS： We found that overexpression of the wild-type PgFtsZ caused a significant inhibition of cell division in E. coli. E. coli cells carrying pEZI were elongated about 20-fold compared with the E. coli cells containing the control vector. However, overexpression of ZG322P and ZG322H did not cause inhibition of cell division. E. coli cells carrying pYW9 and pYW10 had normal appearances,and were similar to E. coil cells carrying the control vector, unlike the wild-type PgFtsZ. Immunoblot analysis showed that the wild-type and the two point mutant proteins were expressed at similar levels and same position. CONCLUSION： These results suggest that Gly322 in the PgFtsZ is important for cell division.