Endolysin Regulation in Phage Mu Lysis

Endolysin Regulation in Phage Mu Lysis

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ABSTRACT Bacteriophage Mu is a paradigm coliphage studied mainly because of its Figure Skating - Accessories - Skate Guards use of transposition for genome replication.However, in extensive nonsense mutant screens, only one lysis gene has been identified, the endolysin gp22.This is surprising because in Gram-negative hosts, lysis by Caudovirales phages has been shown to require proteins which disrupt all three layers of the cell envelope.

Usually this involves a holin, an endolysin, and a spanin targeting the cytoplasmic membrane, peptidoglycan (PG), and outer membrane (OM), respectively, with the holin determining the timing of lysis initiation.Here, we demonstrate that gp22 is a signal-anchor-release (SAR) endolysin and identify gp23 and gp23.1 as two-component spanin subunits.

However, we find that Mu lacks a holin and instead encodes a membrane-tethered cytoplasmic protein, gp25, which is required for the release of the SAR endolysin.Mutational analysis showed that this dependence on gp25 is conferred by lysine residues at positions 6 and 7 of the short cytoplasmic domain of gp22.gp25, which we designate as a releasin, also facilitates the release of SAR endolysins from other phages.

Moreover, the entire length of gp25, including its N-terminal transmembrane domain, belongs to a protein family, DUF2730, found in many Mu-like phages, including those with cytoplasmic endolysins.These results are discussed in terms of models for the evolution and mechanism of releasin function and a rationale for Mu lysis without holin control.IMPORTANCE Host cell lysis is the terminal event of the bacteriophage infection cycle.

In Gram-negative hosts, lysis requires proteins that disrupt each of the three cell envelope components, only one of which has been identified in Mu: the endolysin gp22.We show that gp22 can be characterized as a SAR endolysin, a muralytic enzyme that activates upon release from the membrane to degrade the cell wall.Furthermore, we identify genes 23 and 23.

1 as spanin subunits used for outer membrane disruption.Significantly, we demonstrate that Mu is the first known Caudovirales phage to lack a holin, a protein that disrupts the Vacuum Floor Mop Tool inner membrane and is traditionally known to release endolysins.In its stead, we report the discovery of a lysis protein, termed the releasin, which Mu uses for SAR endolysin release.

This is an example of a system where the dynamic membrane localization of one protein is controlled by a secondary protein.