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

从当地一家污水处理中心分离到革兰氏阳性菌淀粉液化芽孢杆菌的特异性噬菌体。用裂解试验分离到一个基因，其核苷酸序列显示一个375bp的开放阅读框。该克隆基因的过度表达产生了一个13 kDa的裂解蛋白。从同一噬菌体中还鉴定出了另外两种蛋白质，内切酶和Holin，它们负责裂解淀粉液化芽孢杆菌。内毒素能降解细胞中的肽聚糖。霍林降解细胞质膜，从而允许内毒素到达周质，并进入肽聚糖层。在诱导Holin表达后，大肠杆菌细胞的生长立即停止。另一方面，内切酶在大肠杆菌细胞上的表达没有发生任何可检测到的变化。克隆到pET26b(+)载体中的裂解基因在BL21(DE3)宿主细胞中表达。在N-末端融合的信号肽的驱动下，表达的蛋白有望进入周质间隙。当Holin在对数生长期表达时，细胞立即被破坏。信号肽的N-三端融合产生的内毒素不会立即引起细胞溶解，但会引起BL21细胞的形态变化。产生内毒素的大肠杆菌细胞颗粒用纯水重新悬浮，导致细胞裂解，随后β-半乳糖苷酶释放到培养基中。此外，还观察了纯化的内溶素对淀粉液化芽孢杆菌和枯草芽孢杆菌生长的影响。

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.