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.
Highlights
At the end of the replication cycle, bacteriophages must exit the host cell and disperse their newly formed progeny to infect new cells
Identification of two gene products from Ms6 lysA The 1155 bp lysA gene of mycobacteriophage Ms6 starts at a GTG codon that overlaps the gp1 TGA stop codon in a different reading frame, and is preceded in four nucleotides by a ribosome-binding site (RBS) sequence (59-GGGAGCA-39) (Fig. S1)
We provide evidence that two proteins (Lysin384 and Lysin241) with endolysin activity are produced from the mycobacteriophage Ms6 lysA gene
Summary
At the end of the replication cycle, bacteriophages must exit the host cell and disperse their newly formed progeny to infect new cells. The term endolysin is used to describe the dsDNA bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in phage-infected cells at the end of the multiplication cycle. They are characterized by the ability to directly target bonds in the peptidoglycan layer of the bacterial cell wall; the result of this activity is degradation of the rigid murein layer and release of newly assembled virions by way of lysis [7,8]. A survey of orthologous endolysins from other phages of Gram-positive hosts suggested that some of these have N-terminal sequences resembling secretory signals, in every case an adjacent holin gene was present [6,13]. Remarkable cases are the endolysins of E. coli phages P1 and 21, which feature an N-terminal signal-arrest-release (SAR) sequence that allows the enzyme to be exported to the membrane where it is arrested, and to be released as a soluble active enzyme in the periplasm [15,16,17]
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