Controlled expression of lysis genes encoded in T4 phage for the gentle disruption of Escherichia coli cells

Abstract

Two lysis genes, gene-t and gene-e, encoded in bacteriophage T4 were cloned in vectors pUC118 and pET26b(+), respectively. Immediately after the induction of gene-t expression, growth of Escherichia coli JM 109 cells was halted. Since the expression of gene-t was toxic to the cells, the expressed gene-t product (gp-t) could not be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the other hand, expression of gene-e cloned in vector plasmid pUC118 did not give rise to any detectable change in E. coli JM 109 cells; normal cell growth was observed, and the gene-e product (gp-e) was identified by SDS-PAGE. Lysis genes cloned in vector pET26b(+) were expressed in BL21 (DE3) host cells carrying plasmid pLysS, which encodes T7 lysozyme. It is thought that the expressed protein is translocated into the periplasmic space driven by the signal peptide of the peIB outermembrane protein of E. coli fused in the frame at the N-terminal end of the lysis protein. Immediate cell disruption was observed when the gene-t cloned in pET26b(+) was expressed in the logarithmic growth phase. β-Galactosidase activity was observed in the centrifuged supernatant of the cell culture producing gp-t. Almost 100% of the activity of the β-galactosidase produced in the BL21(DE3)pLysS cells was identified in the supernatant 30 min after the start of the induction period of gene-t, indicating that complete cell disruption had occurred at that time. The production of gp-e with the N-terminal fusion of the signal peptide of pelB did not cause immediate cell lysis, but it did result in a morphological change in the BL21(DE3)pLysS cells, from rod-shaped to elliptical. Resuspension of the gp-e-producing BL21(DE3)pLysS cell nellet with pure water caused cell lysis followed by the release of β-galactosidase into the medium. Almost 100% of the β-galactosidase activity was identified in the resuspended supernatant.