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

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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.