Functional Analysis of a Class I Holin, P2 Y

Abstract

Y is the putative holin gene of the paradigm coliphage P2 and encodes a 93-amino-acid protein. Y is predicted to be an integral membrane protein that adopts an N-out C-in membrane topology with 3 transmembrane domains (TMDs) and a highly charged C-terminal cytoplasmic tail. The same features are observed in the canonical class I lambda holin, the S105 protein of phage lambda, which controls lysis by forming holes in the plasma membrane at a programmed time. S105 has been the subject of intensive genetic, cellular, and biochemical analyses. Although Y is not related to S105 in its primary structure, its characterization might prove useful in discerning the essential traits for holin function. Here, we used physiological and genetic approaches to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. Taken together, these results suggest that class I holins share a set of unusual features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. However, Y holin function requires the integrity of its short cytoplasmic C-terminal domain, unlike for S105. Finally, instead of encoding a second translational product of Y as an antiholin, as shown for lambda S107, the P2 lysis cassette encodes another predicted membrane protein, LysA, which is shown here to have a Y-specific antiholin character. Holins are small phage-encoded membrane proteins that control the length of the bacteriophage infection cycle by determining the time of host lysis (1, 2). Almost all of the experimental work done on holin function at the physiological or molecular level has been focused on a few holin genes: lambda S, phage 21 S 21 , and T4 t. These genes encode the holins S105, S 21 68, and T, which are, respectively, the prototypes of three distinct holin classes, distinguished by their experimentally determined membrane topologies (Fig. 1A). S105 has class I topology, with three transmembrane domains (TMDs) arranged N-out C-in, whereas S 21 68 has class II topology, with two TMDs arranged N-in C-in. As of the last major review, ϳ90% of the Ͼ100 genes from phages and prophages of eubacteria that have been assigned as putative holins fit into either class I or class II, based on predicted topology using widely accepted algorithms (1). These predicted holins are very diverse, with 12 and 14 unrelated sequence families grouped into classes I and II, respectively. Class III topology, with a single TMD and a large periplasmic domain, is restricted to the sequence homologs of protein T, the holin of T4. These proteins are found only in phages of the large myophage class of T4-like phages and in the large siphophage class of T5-like phages.Despite this diversity, holins share some universal functional features, based mainly on extensive work that has been done with these three holin paradigms. Holin genes are turned on at the beginning of viral morphogenesis, so that the holins accumulat...

Bacteriophage P2

“…Gene Y encodes a holin, which allows the P2 lysozyme, the product of gene K, to access the peptidoglycan. 43,44 The lysA gene product functions as an antiholin, needed to delay the action of gpY until the optimal lysis time. 43,44 Amber mutants in lysB exhibit delayed lysis in non-permissive cells.…”

“…43,44 The lysA gene product functions as an antiholin, needed to delay the action of gpY until the optimal lysis time. 43,44 Amber mutants in lysB exhibit delayed lysis in non-permissive cells. 43 The lysC gene, which overlaps the lysB gene, was originally identified by a mutation that overcomes the P2 growth defect conferred by a temperature-sensitive mutation in the b' subunit of RNA polymerase.…”

The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

“…Because RI is a specific inhibitor of T, it is formally a member of a diverse class of proteins designated antiholins (23)(24)(25)(26). Moreover, since RI inhibits T only under certain physiological conditions, it is the only antiholin known that transduces environmental information to effect real-time control of holin function and thus of the length and fecundity of the phage infection cycle (21).…”

Probing the Structure of the S105 Hole

“…Similar nonspecificity and permissiveness for the large endolysin chimeras had been noted for other holins, the Y holin from coliphage P2 and the T holin from coliphage T4 (9,10). Recently, cryo-electron microscopy (cryo-EM) studies revealed that both of these holins formed similar micrometer-scale interruptions in the inner membrane (IM) (11).…”

“…Recently, another holin, P2 Y, although lacking detectable similarity to S105, has been shown to adapt the same topology and follow the same general pathway, including triggering to form micrometer-scale holes (9). It also has two TMDs that have faces with widely disparate hydrophobic characters, but in this case, they are TMD2 and TMD3 rather than TMD1 and TMD3, as in S105.…”