Abstract
Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.
Highlights
Introduction published maps and institutional affilBacteriophages, known as phages, are viruses that are able to propagate by infecting bacteria
Previous studies have shown that LysB homologues encoded by mycobacteriophages belonging to diverse clusters, sharing the conserved pentapeptide G-X-S-XG, characteristic of enzymes with lipolytic activity, are highly diverse [20,22]
Since the Ms6 LysB PG-binding domain (PGBD) region is separated from the catalytic domain, we investigated the contribution of this N-terminal region to the phage lysis phenotype
Summary
Introduction published maps and institutional affilBacteriophages, known as phages, are viruses that are able to propagate by infecting bacteria. The infection process used by double-stranded DNA (dsDNA). Phages begins with the binding of the phage particle to a receptor present at the bacterial surface, followed by injection of the phage DNA into the bacterial host. Phages may undergo a lytic cycle and after replication of the phage DNA, and synthesis of the virion components, they are assembled to form new phage particles. Phages need to lyse the host to release the viral progeny that will start a new infection cycle [1]. The lytic system employed by dsDNA phages depends on at least two proteins: an endolysin and a holin [1]. Holins are membrane proteins that permeabilize the cytoplasmic membrane (CM), determining the timing of lysis. Endolysins are enzymes that cleave covalent bonds in the peptidoglycan (PG) compromising the integrity of the iations
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