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Applied Microbiology and Biotechnology

, Volume 69, Issue 6, pp 722–730 | Cite as

Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor

  • Niu Qiuhong
  • Huang Xiaowei
  • Tian Baoyu
  • Yang Jinkui
  • Liu Jiang
  • Zhang Lin
  • Zhang Keqin
Applied Microbial and Cell Physiology

Abstract

An endospore-forming bacterium, strain B16, was isolated from a soil sample and identified as a Bacillus sp. The strain presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The crude extracellular protein extract from culture supernatant of the bacteria killed about 80% of the tested nematodes within 24 h, suggesting the involvement of extracellular proteases. A homogeneous extracellular protease was purified by chromatography, and the hypothesis of proteinaceous pathogeny in the infection of B16 strain was confirmed by the experiments of killing living nematodes and by the degradation of purified nematode cuticle when treated with the homogenous protease. The gene for the virulence protease was cloned, and the nucleotide sequence was determined. The deduced amino acid sequence showed significant similarity with subtilisin BPN' but low homology with the other cuticle-degrading proteases previously reported in fungi. Characterization of the purified protease revealed the molecular mass of 28 kDa and the optimum activity at pH 10, 50°C. The purified protease can hydrolyze several native proteinaceous substrates, including collagen and nematode cuticle. To our knowledge, this is the first report of a serine protease from a Bacillus genus of bacteria that serves as a pathogenic factor against nematodes, an important step in understanding the relationship between bacterial pathogen and host and in improving the nematocidal activity in biological control.

Keywords

Serine Protease Extracellular Protease Nematophagous Fungus Nematocide Nematode Cuticle 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We express our gratitude to Dilantha Fernando and Zhang L.M. for their invaluable discussions and assistance in preparing the manuscript. We are also grateful to Zhou W. for her invaluable help in facilitating the work and to Wang M., Sun H., Xu J., Wang R.B., Lin C., and Li J. for their helpful advices in the study. We thank Li W.J. and Zhang Y.Q. for helping identify the bacterium strain B16.

This work was supported by the projects from National Natural Science Foundation Program of P. R. China (30470067), Ministry of Science and Technology of P. R. China (2002BA901A21), and Department of Science and Technology of Yunnan Province, P. R. China (No. 2004C0004Q and 2004C0001Z).

References

  1. Åhman J (2000) Extracellular serine proteases as virulence factors in nematophagous fungi: molecular characterization and functional analysis of the PII protease in Arthrobotrys oligospora. Doctoral dissertation, Lund UniversityGoogle Scholar
  2. Åhman J, Johansson T, Olsson M, Punt PJ, van den Hondel CAMJJ, Tunlid AS (2002) Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Appl Environ Microbiol 689:3408–3415CrossRefGoogle Scholar
  3. Atibalentja N, Noel GR, Domier LL (2000) Phylogenetic position of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, as inferred from 16S rDNA sequence analysis. Int J Syst Evol Microbiol 50:605–613Google Scholar
  4. Balaji V, Jesudason MV, Sriclharan G, Subramanian K (2002) Detection of virulence attributes of Burkholderia pseudomallei. Indian J Med Res 119:101–106Google Scholar
  5. Beg QK, Gupta R (2003) Purification and characterization of an oxidation stable, thiol-dependent serine alkaline protease from Bacillus mojavavensis. Enzyme Microb Technol 32:294–304CrossRefGoogle Scholar
  6. Cox GN, Kusch M, Edgar RS (1981) Cuticle of Caenorhabditis elegans: its isolation and partial characterization. J Cell Biol 90:7–17CrossRefGoogle Scholar
  7. Day RM, Thalhauser CJ, Sudmeier JL, Vincent MP, Torchilin EV, Sanford DG, Bachovchin CW, Bachovchin WW (2003) Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR. Protein Sci 12:794–810CrossRefGoogle Scholar
  8. Duncon LW (1991) Current options for nematode management. Annu Rev Phytopathol 29:469–490CrossRefGoogle Scholar
  9. Gray NF (1984) Ecology of nematophagous fungi: comparison of the soil sprinkling method with the Baerman funnel technique in the isolation of endoparasites. Soil Biol Biochem 16:81–83CrossRefGoogle Scholar
  10. Huang HW, Chen WC, Wu CY, Yu HC, Lin WY, Chen ST, Wang KT (1997) Kinetic studies of the inhibitory effects of propeptides subtilisin BPN' and carlsberg to bacterial serine proteases. Protein Eng 10:1227–1233CrossRefGoogle Scholar
  11. Huang XW, Zhao NH, Zhang KQ (2004) Extracellular enzymes serving as virulence factors in nematophageous fungi involved in infection of the host. Res Microbiol 155:811–816CrossRefGoogle Scholar
  12. Huang XW, Niu QH, Zhou W, Zhang KQ (2005) Bacillus nematocida sp. nov., a novel bacterial strain with nematotoxic activity isolated from soil in Yunnan, China. Syst Appl Microbiol (In press)Google Scholar
  13. Jacobs M, Eliasson M, Uhlen M, Flock JI (1985) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res 13:8913–8926CrossRefGoogle Scholar
  14. Jansson HB, Nordbring-Hertz B (1988). Infection mechanisms in the fungus-nematode system. In: Poinar GO Jr, Jansson HB (eds) Diseases of nematodes, vol. 2. CRC, Boca Raton, FL, pp 59–72Google Scholar
  15. Laemmli UK (1970) Cleaving of the structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685CrossRefGoogle Scholar
  16. Leger RJ, Richard MC, Charnley AK (1987) Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vomitoria and Manduca sexta. J Gen Microbiol 133:1371–1382Google Scholar
  17. Leger RJ, Nelson JO, Screen SE (1999) The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production. Microbiology 145:2691–2699Google Scholar
  18. Singh J, Batra N, Sobti RC (2001) Serine alkaline protease from a newly isolated Bacillus sp. SSR1. Proc Biochem 36:781–785CrossRefGoogle Scholar
  19. Tikhonov VE, Lopez-Llorca LV, Salinas J, Jansson HB (2002) Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium. Fungal Genet Biol 35:67–78CrossRefGoogle Scholar
  20. Tunlid A, Rosen S, Ek B, Rask L (1994) Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora. Microbiology 140:1687–1695CrossRefGoogle Scholar
  21. Wei J, Hale K, Carta L, Platzer E, Wong C, Fang S, Aroian V (2003) Bacillus thurigiensis crystal proteins that target nematodes. Microbiology 100:2760–2765Google Scholar
  22. Yoon JH, Kim IG, Kang KH, Oh TK, Park YH (2003) Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 53:1297–1303CrossRefGoogle Scholar
  23. Zhou XS, Kaya HK, Heungens K, Heidi GB (2002) Response of ants to a deterrent factor(s) produced by the symbiotic bacteria of entomopathogenic nematodes. Appl Environ Microbiol 68:6202–6209CrossRefGoogle Scholar
  24. Zuckerman (1984) Nematode chemotaxis and possible mechanisms of host/prey recognition. Annu Rev Phytopathol 2295–22113Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Niu Qiuhong
    • 1
  • Huang Xiaowei
    • 1
  • Tian Baoyu
    • 1
  • Yang Jinkui
    • 1
  • Liu Jiang
    • 1
  • Zhang Lin
    • 1
  • Zhang Keqin
    • 1
  1. 1.Laboratory for Conservation and Utilization of Bio-resourcesYunnan UniversityKunmingPeople' Republic of China

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