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Development of self-disruptive E. coli cells by using phage encoded lysis genes

使用噬菌体编码的裂解基因开发自我破坏性大肠杆菌细胞

基本信息

批准号：
09555250
负责人：
TANJI Yasunori
金额：
$ 6.46万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09555250/
关键词：
Bacleriophage Bacillus E. coli Lysis Endolysine Holin Disruption T4ファージ

项目摘要

Bacteriophages specific to Bacillus amyloliqueficiens, a gram-positive bacterium, were isolated from a local sewage treatment center. Using a lysis assay, a gene was isolated and its nucleotide sequence revealed one open reading frame of 375 bp. Over expression of the cloned gene yielded a 13 kDa lysis protein. Two additional proteins, endolysine and holin, responsible for the lysis of B. amyloliqueficiens were also identified from the same phage. Endolysine degrades peptidoglycan of the cell. Holin degrades the cytoplasmic membrane thus allowing endolysine to reach the periplasm and gain access to the peptidoglycan layer. Right after the induction of the holin expression, growth of E. coli cells was halted. On the other hand, expression of endolysine did not give any detectable change on E. coli cells. Lysis genes cloned in pET26b(+) vector were expressed in BL21(DE3) host cell. Expressed protern is expected to be translocated into the periplasmic space driven by signal peptide fused in frame at N-terminal end of lysis protein. Immediate cell disruption was observed when the holin was expressed in the logarithmic growth phase. The production of endolysine with the N-terninal fusion of signal peptide did not cause immediate cell lysis but caused morphological change of the BL21 cells. Resuspension of this endolysine producing E. coli cell pellet with pure water caused cell lysis followed by β-galactosidase release to the medium. Furthermore, exogenous effects of the purified endolysinon growth of B. amyloliqueficiens and B. subtiils were observed.

从当地的污水处理中心分离出一种革兰氏阳性菌--解淀粉芽孢杆菌的噬菌体。用裂解法分离出一个基因，其核苷酸序列显示一个375 bp的开放阅读框。过表达克隆的基因产生了一个13 kDa的裂解蛋白。另外两种蛋白质，内赖氨酸和孔蛋白，负责B的裂解。还从同一噬菌体中鉴定出了解淀粉菌。内赖氨酸降解细胞的肽聚糖。Holin降解细胞质膜，从而允许内赖氨酸到达周质并进入肽聚糖层。在诱导holin表达后，E.大肠杆菌细胞停止。另一方面，内赖氨酸的表达在E. coli细胞。将裂解酶基因克隆到pET 26 b（+）载体中，在BL 21（DE 3）中进行表达。预期表达的蛋白质在与裂解蛋白N-末端框内融合的信号肽的驱动下移位到周质空间中。当holin在对数生长期表达时，观察到细胞立即破裂。信号肽N端融合产生的内赖氨酸不会立即引起细胞溶解，但会引起细胞形态学改变。将产内赖氨酸的E.大肠杆菌细胞团与纯水混合后，细胞裂解，然后β-半乳糖苷酶释放到培养基中。此外，纯化的内溶素对B. amyloliqueficiens和B.观察到枯草芽孢杆菌。