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

Author:

Mahapatra Sebabrata1,Piechota Charles1,Gil Filipa2,Ma Yufang13,Huang Hairong14,Scherman Michael S.1,Jones Victoria1,Pavelka Martin S.5,Moniz-Pereira Jose2,Pimentel Madalena2,McNeil Michael R.1,Crick Dean C.1

Affiliation:

1. Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

2. Centro de Patogénese Molecula, Unidade dos Retrovirus e Infecções Associadas, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal

3. Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, People's Republic of China

4. Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People's Republic of China

5. Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA

Abstract

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 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Reference31 articles.

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2. MahapatraS BasuJ BrennanPJ CrickDC. 2005. Structure, biosynthesis, and genetics of the mycolic acid-arabinogalactan-peptidoglycan complex, p 275–285. In ColeST EisenachKD McMurrayDN JacobsWRJr (ed), Tuberculosis and the tubercle bacillus. ASM Press, Washington, DC.

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