Abstract
Holin/endolysin-mediated lysis of phage T4 of Escherichia coli is tightly regulated by the antiholins RI and RIII. While regulation by the cytoplasmic RIII plays a minor role, the periplasmic antiholin RI binds tightly to the holin T and is believed to directly sense periplasmic phage DNA from superinfections as a trigger for the inhibition of lysis. RI has been reported to contain a non-cleavable signal peptide that anchors the protein to the membrane. Lysis is believed to be induced at some stage by a membrane depolarization that causes a release of RI into the periplasm without cleavage of the signal anchor. For the current model of phage lysis induction, it is thus a fundamental assumption that the N-terminal trans-membrane domain (TMD) of RI is such a signal anchor release (SAR) domain. Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. The signal peptide of RI can also mediate the normal translocation of a well-characterized Sec substrate, PhoA, into the periplasm. This simplifies the current view of phage lysis regulation and suggests a fundamentally different interpretation of the recently published structure of the soluble domains of the RI–T complex.
Highlights
Lytic phages tightly regulate the lysis of their host cells primarily by controlling the access of phageencoded muralytic enzymes, the endolysins, to the bacterial cell wall (Cahill and Young, 2019)
In the course of studies on signal anchor release” (SAR) domains, we initially found that signal peptide prediction by SignalP 5.0 (Almagro Armenteros et al, 2019) strongly differed between RI and the P1 SAR endolysin Lyz (Figure 1A): the N-terminus of Lyz contained no likely signal peptide cleavage site, with only a 40% probability cleavage site after Gly24, whereas RI was predicted to have a very likely cleavage site after Ala24 (95% probability)
Other pinholin-associated endolysins such as the SAR endolysin R of phage P21 resembled the P1 endolysin in having no likely cleavage site, and these SAR domains clearly differed from the N-terminal domain of RI (Supplementary Figure 1)
Summary
Lytic phages tightly regulate the lysis of their host cells primarily by controlling the access of phageencoded muralytic enzymes, the endolysins, to the bacterial cell wall (Cahill and Young, 2019). Endolysins can be either released via holes formed by membrane proteins that are collectively termed “canonical holins”, or they can be secreted by the general secretory (Sec) system and remain inactive as membrane-anchored proteins that can be released and activated upon membrane depolarization by associations of so-called “pinholins” (Young, 2014) As the latter endolysins are anchored to the cytoplasmic membrane via signal peptides that can exit the membrane without cleavage, these special signal peptides are termed “signal anchor release” (SAR) domains (Xu et al, 2005). In addition to the intrinsic and dual-start timing systems, many holins are regulated by specific antiholins that somehow sense superinfections and delay lysis This is thought to be a strategy to increase the chance for lysis in an environment with a lower phage abundance and potential higher abundance of non-infected hosts (Ramanculov and Young, 2001a).
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