Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Anagnostopoulos C, Spizizen J (1961) Requirement for transformation in Bacillus subtilis. J Bacteriol 81:741–746

    PubMed  CAS  Google Scholar 

  • Antelmann H, Yamamoto H, Sekiguchi J, Hecker M (2002) Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach. Proteomics 2:591–602

    Article  PubMed  CAS  Google Scholar 

  • Atrih A, Bacher G, Allmaier G, Williamson MP, Foster SJ (1999) Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation. J Bacteriol 181:3956–3966

    PubMed  CAS  Google Scholar 

  • Blackman SA, Smith TJ, Foster SJ (1998) The role of autolysins during vegetative growth of Bacillus subtilis 168. Microbiology 144:73–82

    PubMed  CAS  Google Scholar 

  • Fein JE, Rogers HJ (1976) Autolytic enzyme-deficient mutants of Bacillus subtilis 168. J Bacteriol 127:1427–1442

    PubMed  CAS  Google Scholar 

  • 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 21–41

    Google Scholar 

  • Fukushima T, Afkham A, Kurosawa S, Tanabe T, Yamamoto H, Sekiguchi J (2006) A new D,L-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis. J Bacteriol 188:5541–5550

    Article  PubMed  CAS  Google 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) 133:475–483

    CAS  Google Scholar 

  • Fukushima T, Kitajima T, Sekiguchi J (2005) A polysaccharide deacetylase homologue, PdaA, in Bacillus subtilis acts as an N-acetylmuramic acid deacetylase in vitro. J Bacteriol 187:1287–1292

    Article  PubMed  CAS  Google 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) 136:283–291

    CAS  Google Scholar 

  • Fukushima T, Yamamoto H, Atrih A, Foster SJ, Sekiguchi J (2002) A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic δ–lactam residues in the spore cortex of Bacillus subtillis. J Bacteriol 184:6007–6015

    Article  PubMed  CAS  Google Scholar 

  • Hara T, Aumayr A, Fujio Y, Ueda S (1982) Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange. Appl Environ Microbiol 44:1456–1458

    PubMed  CAS  Google 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 289–312

    Google Scholar 

  • Horsburgh G, Atrih A, Foster SJ (2003) Characterization of LytH, a differentiation-associated peptidoglycan hydrolase of Bacillus subtilis involved in endospore cortex maturation. J Bacteriol 185:3813–3820

    Article  PubMed  CAS  Google Scholar 

  • Horsburgh G, Atrih A, Williamson MP, Foster SJ (2003) LytG of Bacillus subtilis is a novel peptidoglycan hydrolase: the major active glucosaminidase. Biochemistry 42:257–264

    Article  PubMed  CAS  Google Scholar 

  • Ishikawa S, Hara Y, Ohnishi R, Sekiguchi J (1998) Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis. J Bacteriol 180:2549–2555

    PubMed  CAS  Google Scholar 

  • Kuroda A, Sekiguchi J (1991) Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene. J Bacteriol 173:7304–7312

    PubMed  CAS  Google Scholar 

  • Kuroda A, Sekiguchi J (1993) High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation. J Bacteriol 175:795–801

    PubMed  CAS  Google Scholar 

  • Lazarevic V, Margot P, Soldo B, Karamata D (1992) Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. J Gen Microbiol 138:1949–1961

    PubMed  CAS  Google Scholar 

  • Leclerc D, Asselin A (1989) Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can J Microbiol 35:749–753

    Article  PubMed  CAS  Google Scholar 

  • Loessner MJ, Kramer K, Ebel F, Scherer S (2002) C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol Microbiol 44:335–349

    Article  PubMed  CAS  Google Scholar 

  • Loessner MJ, Wendlinger G, Scherer S (1995) Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. Mol Microbiol 16:1231–1241

    Article  PubMed  CAS  Google Scholar 

  • Margot P, Mauël C, Karamata D (1994) The gene of the N-acetylglucosaminidase, a Bacillus subtilis 168 cell wall hydrolase not involved in vegetative cell autolysis. Mol Microbiol 12:535–545

    Article  PubMed  CAS  Google Scholar 

  • Margot P, Pagni M, Karamata D (1999) Bacillus subtilis 168 gene lytF encodes a gamma-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, sigmaD. Microbiology 145:57–65

    PubMed  CAS  Google Scholar 

  • Margot P, Wahlen M, Gholamhoseinian A, Piggot P, Karamata D (1998) The lytE gene of Bacillus subtilis 168 encodes a cell wall hydrolase. J Bacteriol 180:749–752

    PubMed  CAS  Google Scholar 

  • Ohnishi R, Ishikawa S, Sekiguchi J (1999) Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis. J Bacteriol 181:3178–3184

    PubMed  CAS  Google Scholar 

  • Rashid MH, Mori M, Sekiguchi J (1995) Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization. Microbiology 141:2391–2404

    Article  PubMed  CAS  Google Scholar 

  • Reynolds PE, Ambur OH, Casadewall B, Courvalin P (2001) The VanY(D) DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein. Microbiology 147:2571–2578

    PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Schaeffer P, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci USA 54:704–711

    Article  PubMed  CAS  Google Scholar 

  • Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477

    PubMed  CAS  Google Scholar 

  • Serizawa M, Yamamoto H, Yamaguchi H, Fujita Y, Kobayashi K, Ogasawara N, Sekiguchi J (2004) Systematic analysis of SigD-regulated genes in Bacillus subtilis by DNA microarray and Northern blotting analyses. Gene 329:125–136

    Article  PubMed  CAS  Google 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 117–142

    Google Scholar 

  • Shimotsu H, Henner DJ (1986) Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. J Bacteriol 168:380–388

    PubMed  CAS  Google Scholar 

  • Smith TJ, Blackman SA, Foster SJ (2000) Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146:249–262

    PubMed  CAS  Google Scholar 

  • Suzuki T, Tahara Y (2003) Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation. J Bacteriol 185:2379–2382

    Article  PubMed  CAS  Google Scholar 

  • Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, van Dijl JM (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515–547

    Article  PubMed  CAS  Google Scholar 

  • Urushibata Y, Tokuyama S, Tahara Y (2002) Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production. J Bacteriol 184:337–343

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi H, Furuhata K, Fukushima T, Yamamoto H, Sekiguchi J (2004) Characterization of a new Bacillus subtilis peptidoglycan hydrolase gene, yvcE (named cwlO), and the enzymatic properties of its encoded protein. J Biosci Bioeng 98:174–181

    PubMed  CAS  Google Scholar 

  • Yamamoto H, Kurosawa S, Sekiguchi J (2003) Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases. J Bacteriol 185:6666–6677

    Article  PubMed  CAS  Google Scholar 

  • Zwartouw HT, Smith H (1956) Polyglutamic acid from Bacillus anthracis grown in vivo; structure and aggressin activity. Biochem J 63:437–442

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano, 386-8567, Japan

    Tatsuya Fukushima, Yang Yao, Toshihiko Kitajima, Hiroki Yamamoto & Junichi Sekiguchi

  2. Division of Gene Research, Department of Life Sciences, Research Center for Human and Environmental Sciences, Shinshu University, Ueda-shi, Nagano, 386-8567, Japan

    Junichi Sekiguchi

Authors
  1. Tatsuya Fukushima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yang Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Toshihiko Kitajima
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroki Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Junichi Sekiguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Junichi Sekiguchi.

Additional information

Communicated by M. Hecker.

Tatsuya Fukushima and Yang Yao contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fukushima, T., Yao, Y., Kitajima, T. et al. Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis . Mol Genet Genomics 278, 371–383 (2007). https://doi.org/10.1007/s00438-007-0255-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-007-0255-8

Keywords

Search

Navigation