Phage Biology

Patrick Paddison,# Katherine Gailbreath, Holly Dressman,* Steve Abedon,§ David Hudson, Eric Mosser, Burton Guttman, Jim Neitzel & Elizabeth Kutter. Evergreen State College, Olympia, WA ; *NIEH, Research Triangle Park, NC; § Dept. of Microbiology, Ohio State University, Mansfield, OH #Current Address: Seattle Project, Fred Hutchinson Cancer Center, Seattle, WA

As first described by Gus Doermann in 1948, T-even phages add a special touch to their control of lysis: binding of other T-even phages to previously-infected cells causes extensive delay in lysis, even when cells are starting to break open. Rapid lysis (r) mutants lack this response, giving large, sharp-edged plaques under appropriate conditions. The resultant rI, rII, and rIII loci played key roles in early T4 genetics, but the mechanisms of lysis inhibition and normal lysis timing remain shrouded in mystery. We here identify the rI gene as ORF tk.-2, which encodes an 11.1 kDa protein (pI 4.8). The hydrophobicity plot suggests the gpRI N-terminus may be membrane-spanning, in contrast to the hydrophilic rIII protein (which is 9.3 kDa, pI 8.5). RI lies 3.3 kb from an early promoter and 245 bp below a late promoter, showing transcriptional regulation similar to that of rIII.. (Gene e is transcribed late in the early direction, but an RNA stem-loop structure impairs translation of the e early transcript.) As with most phages, lysis in T4 requires a "holin" (encoded by gene t) in addition to lysozyme (gene e); e-mutant infected cells lose membrane potential and stop making phage at the normal lysis time unless superinfected, while t-mutant infections produce phage for hours, breaking open very inefficiently even when lysozyme is made. A likely hypothesis is that rI plays a key role in functional control of this holin, since rI mutants block lysis inhibition on all host strains tested, while rII and rIII mutations block lysis inhibition only on B; it is not clear what strain differences are responsible. We are now implementing a variety of approaches to try and understand the interactions of all of these factors in regulating the timing of lysis.

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