The two‐step lysis system of pneumococcal bacteriophage EJ‐1 is functional in Gram‐negative bacteria: triggering of the major pneumococcal autolysin in Escherichia coli

Abstract

The holin function Ejh of the pneumococcal bacteriophage EJ-1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli, and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.

Characterization of Streptococcus gordonii prophage PH15: complete genome sequence and functional analysis of phage-encoded integrase and endolysin

“…This appears to be also the case for PH15. ORF54 is predicted to contain two transmembrane helices and was most similar to the holins from S. mitis phage Sk137 (Llull et al, 2006) and S. pneumoniae phage EJ-1 (Díaz et al, 1996). ORF55 has an amidase-5 domain, suggesting that it cleaves the peptidoglycan at the amide bond between N-acetylmuramic acid and L-alanine of the stem peptide.…”

Reconstitution of Holin Activity with a Synthetic Peptide Containing the 1–32 Sequence Region of EJh, the EJ-1 Phage Holin

“…EJh, 1 the holin of the temperate pneumococcal EJ-1 phage, is an 85-amino acid polypeptide chain ascribed to the type II holin subgroup (14). As shown for S holin, EJh expression is lethal for the host cell (14).…”

“…As shown for S holin, EJh expression is lethal for the host cell (14). In contrast to S holin, the EJh polypeptide chain lacks a defined dual start, and its sequence analysis only allows the prediction of two transmembrane regions of higher hydrophobicity.…”

Insights into the regulation of bacteriophage endolysin: multiple means to the same end

“…A similar property of autolysin induction by phage was previously reported in the case of Micrococcus lysodeikticus infected with phage N1 (Goepfert & Naylor, 1967). When the holin of pneumococcal bacteriophage EJ-1 was expressed in E. coli, it also activated LytA autolysin and caused cell death (Díaz et al, 1996). These experiments suggest that bacterial cell death after phage infection, occurring due to interference in cell wall biosynthesis or cell wall disruption, requires depolarization of the cell membrane (Bayles, 2000(Bayles, , 2003Martínez-Cuesta et al, 2000;Clementi et al, 2012;Sadykov & Bayles, 2012).…”

“…For example, the endolysin of pneumococcal bacteriophage EJ-1 that infects Gram-positive bacteria could act on E. coli cell wall. Interestingly, a twofold increase in the activity was observed when a CBD substrate, choline, was added in the reaction mixture (Díaz et al, 1996).…”