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Mycobacteriophage Ms6 LysA is 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: 3.95). 11/2012; DOI: 10.1128/AEM.02263-12
Source: PubMed

ABSTRACT 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 analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified lysA, a protein with homology to known peptidoglycan degrading amidases, from the 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 isolated mycobacterial cell-wall. In contrast to lysozyme, which, in culture, inhibits growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis LysA had no effect on growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. Data indicates that the stem peptides from M. smegmatis are heavily amidated containing few free carboxylic acids regardless of 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] "
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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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