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

Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins CO 80523, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 11/2012; 79(3). DOI: 10.1128/AEM.02263-12
Source: PubMed


Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found
on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan
from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant
protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial
cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of
isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

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    • ". M. smegmatis mc 2 155 (ATCC 700084) was grown in Middlebrook 7H9 media (Sigma) supplemented with ADC (BD biosciences) enrichment and 0.2% glycerol (Sigma) at 37 • C with aeration at 220 rpm [26] [27] "

    Full-text · Dataset · Dec 2014
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    • ". M. smegmatis mc 2 155 (ATCC 700084) was grown in Middlebrook 7H9 media (Sigma) supplemented with ADC (BD biosciences) enrichment and 0.2% glycerol (Sigma) at 37 • C with aeration at 220 rpm [26] [27] "
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    ABSTRACT: We report the ability of mycobacteriophage-derived endolysins to inhibit the growth of Mycobacterium smegmatis. We expressed and purified LysB from mycobacteriophage Bxz2 and compared its activity with that of a previously reported LysB from mycobacteriophage Ms6. The esterase activity of Bxz2 LysB with p-NP esters was 10-fold higher than that of the previously reported LysB but its lipolytic activity was significantly lower. The presence of surfactant–Tween 80 or Triton X-100–significantly increased the activity of LysB. Characterization of LysB-treated M. smegmatis cells and LysB-treated purified cell wall by mass spectroscopy confirmed the hydrolytic activity of the enzyme. Both enzymes were equally effective in inhibiting the growth of M. smegmatis, demonstrating their potential as bacteriostatic agents.
    Full-text · Article · Sep 2014 · Enzyme and Microbial Technology
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    ABSTRACT: We have been witnessing an increased interest in bacteriophage studies focused on their use as antibacterial agents to fight pathogenic bacteria. This interest is a consequence of the phages’ ability to lyse a bacterial host. Until recently, little was known about the mechanisms used by mycobacteriophages to induce lysis of their complex hosts. However, studies on Ms6-induced lysis have changed this scenario and provided new insights into the mechanisms of bacteriophage-induced lysis. Specific lysis protein genes have been identified in mycobacteriophage genomes, reflecting the particular mycobacterial cell envelope composition. These include enzymes that target mycolic acid–containing lipids and proteins that participate in the secretion of the phage endolysin, functioning as chaperone-like proteins. This chapter focuses on the current knowledge of mycobacteriophage-induced lysis, starting with an overview of phage lysis and basic features of the lysis players.
    Preview · Article · Jan 2014
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