Enzymatic property and stabilization mechanism of LysBT1, a novel polyextremotolerant endolysin with a C-terminal S-layer homology domain

Phage-encoded endolysins are getting increasing attention because of their potential to serve as alternative antimicrobials to combat antibiotic-resistant bacteria. Here, we report a novel endolysin LysBT1, which is encoded by a prophage of thermophilic <i>Brevibacillus thermoruber</i> WF146 and comprises an N-acetylmuramoyl-L-alanine amidase domain and an S-layer homology (SLH) domain not found in known endolysins. LysBT1 is not only extremely thermostable, retaining more than 60% activity after 1 h incubation at 95°C, but also highly stable over a wide pH range of 4.0-11.0. Moreover, the thermostability of LysBT1 could be enhanced by EDTA or reducing agents. Although none of the seven cysteine residues of LysBT1 participate in disulfide bond formation, six of them, including the catalytic Zn²⁺-coordinating Cys156, are involved in stabilizing the enzyme at elevated temperatures. The SLH domain contributes to the thermostability of LysBT1 and mediates cell surface binding of the enzyme to facilitate enzymatic lysis of strain WF146 cells via increasing local enzyme concentration around the substrate. LysBT1 is capable of trimerization, where the SLH domains are predicted to form a three-prong spindle-like trimer similar to that in S-layer proteins. The SLH domain of LysBT1 could bind to cell surfaces of both Gram-positive and Gram-negative bacteria. LysBT1 can lyse not only Gram-positive strain WF146, <i>Geobacillus stearothermophilus</i>, and <i>Bacillus subtilis</i> but also Gram-negative <i>Escherichia coli</i> and <i>Acinetobacter baumannii</i> with the aid of EDTA or citric acid. EDTA also facilitates LysBT1 to lyse <i>Bacillus cereus</i>, probably because EDTA-induced disorganization of the S-layer allows LysBT1 to access and hydrolyze the peptidoglycan.IMPORTANCEThe emergence of antibiotic-resistant bacteria has led to an urgent requirement to develop novel antimicrobials, and endolysins are regarded as ideal alternatives to antibiotics. The thermostability of endolysins plays an important role in the feasibility of enzymatic bacteriolysis. However, reports on thermostable endolysins are limited, and little is known about their stabilization mechanisms. Our results demonstrate that the thermophile-derived prophage endolysin LysBT1 is highly thermostable and functional under polyextreme (multiple forms of stress) conditions, enabling the enzyme to lyse both Gram-positive and Gram-negative bacteria in synergy with outer membrane permeabilizer. Moreover, we found that the unique S-layer homology domain of LysBT1 contributes to the stability, activity, oligomerization, and cell-wall binding ability of the enzyme. This study not only characterizes a novel endolysin but also provides new clues about the stabilization mechanisms of endolysins.