Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis
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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 β-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 histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK 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,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA.
KeywordsAutolysin l,d-endopeptidase Peptidoglycan hydrolase Cell wall Bacillus subtilis
We would like to thank K. Ozaki, Kao Corporation, for preparing the B. subtilis 168 cells for purification of the cell wall and H. Karasawa, Food Technology Research Institute of Nagano Prefecture, for determination of the MW by ESI-MS. This research was supported by Grants-in-Aid for Scientific Research (B) (grant 16380059) and the twenty-first century COE program (to J.S.), and Young Scientists (B) (grant 14760046) (to H.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
- Foster SJ, Popham DL (2002) Structure and synthesis of cell wall, spore cortex, teichoic acids, S-layers, and capsules. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington, DC, pp 21–41Google Scholar
- Fukushima T, Ishikawa S, Yamamoto H, Ogasawara N, Sekiguchi J (2003) Transcriptional, functional and cytochemical analyses of the veg gene in Bacillus subtilis. J Biochem (Tokyo) 133:475–483Google Scholar
- Fukushima T, Tanabe T, Yamamoto H, Hosoya S, Sato T, Yoshikawa H, Sekiguchi J (2004) Characterization of a polysaccharide deacetylase gene homologue (pdaB) on sporulation of Bacillus subtilis. J Biochem (Tokyo) 136:283–291Google Scholar
- Helmann JD, Moran CP Jr (2002) RNA polymerase and sigma factors. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington, DC, pp 289–312Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
- Shida T, Sekiguchi J (2005) Cell wall degradation and modification hydrolases in Bacillus subtilis. In: Yamada M (ed) Survival and death in bacteria. Research Signpost, Kerala, pp 117–142Google Scholar