FUNCTIONS INVOLVED IN BACTERIOPHAGE-P2-INDUCED HOST-CELL LYSIS AND IDENTIFICATION OF A NEW TAIL GENE

Successful completion of the bacteriophage P2 lytic cycle requires pha ge-induced lysis of its Escherichia coli host, a process that is poorl y understood. Genetic analysis of lysis-deficient mutants defined a si ngle locus, gene X, which lies within the largest late transcription u nit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that span s the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host c ell lysis. Sequence analysis revealed five open reading frames, initia lly designated orf19 through orf23. All of the existing lysis mutants- ts60, am12, am76, and am218-were located in oral, which must therefore correspond to gene K. The K gene product has extensive amino acid seq uence similarity to the product of gene R of bacteriophage lambda, and it exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four oth er newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 ( lysB) are nonessential. Gene Y encodes a polypeptide with striking sim ilarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holi n mutant, The gene products of lysA and lysB, although nonessential, a ppear to play a role in the correct timing of lysis, since a lysA ambe r mutant caused slightly accelerated lysis and a lysB amber mutant sli ghtly delayed lysis of nonpermissive strains. Gene X must encode a tai l protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells in fected with P2 head mutants but not with tail mutants.