CLONING, SEQUENCE-ANALYSIS, AND EXPRESSION OF THE GENES ENCODING LYTIC FUNCTIONS OF BACTERIOPHAGE-PHI-G1E

The lysis genes of a Lactobacillus phage phi g1e were cloned, sequence d, and expressed in Escherichia coli. Nucleotide sequencing of a 3813- bp phi g1e DNA revealed five successive open reading frames (ORF), Ror f50, Rorf118, hol, and lys and Rorf175, in the same DNA strand. By com parative analysis of the DNA sequence, the putative hol product (holin ) has an estimated molecular weight is 14.2 kDa, and contains two pote ntial transmembrane helices and highly charged N- and C-termini, resem bling predicted holins (which are thought to be a cytoplasmic membrane -disrupting protein) encoded by other phages such as mv1 from Lactobac illus bulgaricus, phi adh from Lactobacillus gasseri, as well as monoc ins from Listeria. On the other hand, the putative phi g1e lys product (lysin) of 48.4 kDa shows significant similarity with presumed murami dase, known as a cell wall peptidoglycan-degrading enzyme, encoded by the Lactobacillus phage mv1 and phi adh, the Lactococcus lactis phage phi LC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli, the phi g1e lysin and/or holin decreased th e cell turbidity significantly, suggesting that the phi g1e hol-lys sy stem is involved in cytolytic process.