Characterization of new L,D-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK ( YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic L, D- endopeptidase ( PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a D- alanyl- D- alanine carboxypeptidase ( Pfam: http:// www. sanger. ac. uk/ Software/ Pfam/). The beta- galactosidase activity observed on cwlK- lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidinetag ( h- Delta CwlK) was produced in Escherichia coli and puriWed on a nickel column. The h- Delta CwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h- Delta CwlK were pH 6.5, 37 C, and 0 M, respectively. Interestingly, h- Delta CwlK could hydrolyze the linkage of L- alanine- D- glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of D- alanine- D- alanine, suggesting that CwlK is an L, D- endopeptidase not a D- carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the Wrst report describing the characterization of an L, D- endopeptidase in B. subtilis and also the Wrst report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA.