A novel spore peptidoglycan hydrolase of Bacillus cereus: Biochemical characterization and nucleotide sequence of the corresponding gene, sleL

The exudate of germinated spores of B, cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has subs trate specificity for fragmented spore cortex from wild-type organisms (cor tical-fragment-lytic enzyme [CFLE]), in addition to a previously characteri zed germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R, Moriyama, S, Kudoh, S. Miyata, S, Nonobe, A. Hattori, and S, Makino, J, Bacteriol, 178:5330-5332, 1996), CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus s ubtilis ADD1, which lacks muramic acid delta-lactam, This suggests that CFL E cooperates with SCLE in cortex hydrolysis during germination. CFLE was pu rified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase, Immunochemical studies suggested that the m ature enzyme is localized on a rather peripheral region of the dormant spor e, probably the exterior of the cortex layer. A gene encoding the enzyme, s leL, was cloned in Escherichia coli, and the nucleotide sequence was determ ined. The gene encodes a protein of 430 amino acids with a deduced molecula r weight of 48,136, The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks sho wed no similarity of CFLE with N-acetylglucosaminidases found so far, sugge sting that CFLE is a novel type of N-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative prote ins showing similarity to SleL.