Cloning, sequence analysis, and expression of the genes encoding lytic functions of Bacteriophage Fg1e

摘要

The lysis genes of a Lactobacillus phage Fgle were cloned, sequenced, and expressed in Escherichia coli. Nucleotide sequencing of a 3813-bp Fgle DNA revealed five successive open reading frames (ORF), Rorf50, Rorf118, hol, and lys and Rorf175, in the same DNA strand. By comparative analysis of the DNA sequence, the putative hol product (holin) has an estimated molecular weight is 14.2 kDa, and contains two potential transmembrane helices and highly charged N- and C-termini, resembling predicted holins (which are thought to be a cytoplasmic membrane-disrupting protein) encoded by other phages such as mvl from Lactobacillus bulgaricus, Fadh from Lactobacillus gasseri, as well as monocins from Listeria. On the other hand, the putative Fgle lys product (lysin) of 48.4 kDa shows significant similarity with presumed muramidase, known as a cell wall peptidoglycandegrading enzyme, encoded by the Lactobacillus phage mvl and Fadh, the Lactococcus lactis phage FLC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli, the Fgle lysin and/or holin decreased the cell turbidity significantly, suggesting that the Fgle hol-lys system is involved in cytolytic process.