S gene expression and the timing of lysis by bacteriophage lambda

Abstract

The S gene of bacteriophage encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2. . . N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.

Micron-scale holes terminate the phage infection cycle

“…In addition, even at these enormous sizes, the perimeters of the holes are consistent with the numbers of S105 holins present at the time of lysis. Because each holin has three TMDs, which, as α-helices, are ∼1 nm in diameter, current estimates of ∼1,000 S105 molecules (35,36) could correspond to ∼3 μm of perimeter if all the proteins are in the perimeter and each TMD participates. To settle this issue, the ideal approach would be to tag S105 with a fluorescent moiety and use correlative microscopy to demonstrate the presence of the holin in the lesion.…”

A signal-arrest-release sequence mediates export and control of the phage P1 endolysin

“…Some experiments used RY8653 (MC4100 phoR dsbA::kan1 zih12::Tn10), kindly provided by T. J. Silhavy, Princeton University, Princeton; RY1531 (MC4100 secA ts ), kindly provided by J. Beckwith (Harvard Medical School, Boston) (10); or TG1 [FЈ traD36 proAB lacI q ⌬(lacZ) M15 ͞supF hsdD5 thi ⌬(lac Ϫ proAB)], a phenotypically PhoA Ϫ host (11). Standard conditions for the growth of cultures and the monitoring of lysis kinetics have been described (9,12). When appropriate, the presence of active, cytosolic endolysin in nonlysing cultures was tested for by the addition of CHCl 3 .…”

Active Bax and Bak are functional holins

“…The pS105 plasmid ( Fig. 2A) contains the wellcharacterized l lysis cassette, consisting of the lysis genes S105, R (endolysin), Rz, and Rz1, downstream from its native l late promoter, pR9 (Chang et al 1995;Smith et al 1998). The D(SR) cells harbor a thermally inducible l prophage, which is lysis-defective due to deletions of both S (S105) and R genes.…”