CLONING, MOLECULAR CHARACTERIZATION, AND EXPRESSION OF THE GENES ENCODING THE LYTIC FUNCTIONS OF LACTOCOCCAL BACTERIOPHAGE-PHI-LC3 - A DUALLYSIS SYSTEM OF MODULAR DESIGN

The genes encoding the lysis proteins of Lactococcus lactis bacterioph age phi LC3 were cloned, sequenced, and expressed in Escherichia coli. The phi LC3 lysis genes, lysA and lysB, encode a membrane-disrupting protein (LysA) of 88 amino acids, and a cell wall degrading protein (L ysB) of 429 amino acids, which shares significant sequence similarity with lysins from the Streptococcus pneumoniae phages Cp-1, Cp-7, and C p-9, and lactobacillus delbrueckii phage mv1. Both LysA and LysB funct ion in E. coli, as judged by lysis of the E. coli host cells and by ly tic activity against lactococcal cells when the cloned lysA and lysB g enes are expressed. The LysA protein possesses two putative transmembr ane helices and highly charged N- and C-termini, and is structurally s imilar to phage holins that are known to induce lesions in the inner m embrane through which phage endolysin can be released to its cell wall substrate. The C-terminal end of LysB contains two highly homologous sequence repeats of 43 amino acids. The LysB repeats show strong seque nce similarity to repeats found in lytic enzymes from other Gram-posit ive bacteria and from Bacillus subtilis phage phi 29 and PZA, as well as in some functionally unrelated proteins, and they are possibly invo lved in binding of the enzyme to the cell wall substrate. The organiza tion of the dual phi LC3 lysis system supports earlier suggestions tha t exchange of modular units is an important principle in protein evolu tion.