Lysis and Lysis Inhibition in Bacteriophage T4: rV Mutations Reside in the Holin t Gene

The T-even bacteriophages employ a powerful strategy to maximize the yield of virus particles when a population of susceptible Escherichia coli cells is encountered (7, 14). Cells are initially infected by single phage particles and, at 37°C with aeration, lyse 25 to 35 min later, releasing on the order of 102 particles per cell. Eventually, the ratio of released particles to unlysed cells exceeds 1, whereupon cells are infected by one particle and then repeatedly “superinfected” by additional particles. When the interval between infection and superinfection is at least a few minutes, the superinfecting particles trigger lysis inhibition. Lysis is then delayed for up to several hours, and the burst size approaches 103 particles per cell. Lysis inhibition involves several genes (2, 40). e, a late gene whose protein-encoding DNA sequence resides at kb 66.493 to 66.985 on the standard map (25), encodes an endolysin (gpe in T4 gene product terminology) that accumulates but does not act until released into the periplasmic space; gpe is a lysozyme that degrades the murein layer of the cell envelope (30). Cells singly infected with e mutants cease metabolism after about 30 min at 37°C and synthesize no more phage but neither lyse nor form plaques. t, a late gene that resides at kb 160.219 to 160.873, encodes a 218-residue protein (gpt) (28) that acts as a holin, that is, a (regulated) pore allowing the lysozyme access to the murein layer (27, 32, 40). Cells singly infected with the canonical t amber mutants accumulate endolysin, but rarely lyse, and form tiny plaques or no plaques at all, depending on the plating medium (references 15 and 16 and this report). Unlike e mutants, such t mutants continue to synthesize phage particles long after the normal time of lysis, but “t” refers not to “timing” or “trigger” but to the unfortunate Tithonus, who was granted immortality but not protection against aging (15, 16). In rapid-lysis (r) mutants, superinfection fails to induce lysis inhibition, resulting in large, sharp-edged plaques instead of the smaller r+ plaques that are surrounded by a dense haze of superinfected, lysis-inhibited cells (14); however, the larger r plaques contain roughly 10-fold-fewer phages than do r+ plaques (10). In the present context, the key r genes are rI and rV. rI is an early gene at kb 59.192 to 59.483, and it has been suggested that gprI senses some primary aspect of superinfection (31). rV was first defined by a single allele mapping to the region between 38 and motA and producing an r phenotype only at higher temperatures (18, 23); additional rV alleles are described in this report. Among the other r genes, rIV and rVI are poorly defined genetically (25, 39) and null mutations in rIIA, rIIB, and rIII produce an r phenotype on some host cells but not on others (2, 31) and thus are not essential for lysis inhibition. These r genes are not further considered here. Here we show that t and rV mutations are alleles of the same gene. Although rV was discovered in 1965 (23) and t in 1970 (15), we call the gene t because it encodes the holin. We also characterize patterns of lysis and lysis inhibition produced by the two kinds of t alleles and suggest how rI and t alleles may interact.