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Partial characterization of a major autolysin from Mycobacterium phlei

Zusheng S. Li

Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Immunopathology Unit, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage SG1 2NY, UK

Author for correspondence: Anthony J. Clarke. Tel: +1 519 824 4120 ext. 3361. Fax: +1 519 837 1802. e-mail: aclarke@micro.uoguelph.ca

ABSTRACT

Summary: Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorportated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5·5 and a pH optimum of pH 7·5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a β-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene.

The SWISS-PROT accession number for the Mycobacterium phlei autolysin reported in this paper is P81528.

Present address: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA.