A Second Endolysin Gene Is Fully Embedded In-Frame with the lysA Gene of Mycobacteriophage Ms6

Abstract

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA 241) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin384) or the shorter (Lysin241) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various Gram-positive bacteria and mycobacteria.

Molecular Dissection of Phage Endolysin

“…We examined these proteins for their in vitro PG hydrolytic activity using M. ysodeikticus cells as substrate immobilized in a polyacrylamide gel. The procedure, known as zymography, is a well established method for assaying the cell wall lysis activity of the enzyme in question, and M. lysodeikticus cells are commonly used as substrate in these experiments (36,41). The cell lysis activity of the proteins appeared as a clear zone after staining the gel with methylene blue (Fig.…”

“…D29 gp10 Expresses Only One ORF That Lyses Mycobacterial Cells-It has been suggested previously that lysA of Ms6 contains more than one ORF (36). Interestingly, both the protein products were shown to have activity against mycobacteria.…”

“…Zymography-Zymograms were prepared to test the activity of the purified proteins using 0.2% Micrococcus lysodeikticus cells (Sigma) as substrate in a 12% SDS-polyacrylamide gel. The experiment was carried out as described (36) with some modifications. Briefly, 5 g of each of the proteins were boiled in SDS sample loading buffer and loaded on to the SDS-polyacrylamide gel containing the substrate.…”

“…Briefly, 5 g of each of the proteins were boiled in SDS sample loading buffer and loaded on to the SDS-polyacrylamide gel containing the substrate. After electrophoresis, the gel was washed twice with renaturation buffer containing 25 mM TrisCl, pH 8.0, 1% Triton X-100, and 5 mM EDTA and incubated in the same buffer at 25°C for 12 h. It was then developed essentially as described (36) and photographed. BSA and lysozyme were used as negative and positive controls, respectively.…”

Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a Useful Analytical Tool

“…The protein encoded by lysA in mycobacterial phage Ms6 causes Escherichia coli cells to lyse, after the addition of CHCl 3 , when expressed in the heterologous host (13,14). In addition to Ms6 LysA, four other mycobacteriophages generate cleared zones in zymograms where lyophilized Micrococcus luteus was incorporated into the gel matrix (15,(15)(16)(17), strongly suggesting that mycobacteriophage LysA proteins are murolytic. Thus, it seemed possible that LysA could be a useful reagent for generating peptidoglycan fragments from mycobacterial peptidoglycan for analytical purposes.…”