Two- Dimensional Electrophoresis Analysis of Proteins; Bacillus subtilis LTD and Its Antifungal Activity Deficient Mutant

  • Lee, Young-Keun (Radiation Application Research Division, Korea Atomic Energy Research Institute) ;
  • Dinh, Le Thi (Institute for Nuclear Science and Techniques) ;
  • Jang, Yu-Sin (Radiation Application Research Division, Korea Atomic Energy Research Institute) ;
  • Chung, Hye-Young (Radiation Application Research Division, Korea Atomic Energy Research Institute) ;
  • Chang, Hwa-Hyoung (Radiation Application Research Division, Korea Atomic Energy Research Institute)
  • Published : 2004.12.01


To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA sequencing. It has an antifungal activity against 9 plant pathogenic fungi in a paper disc assay. The antifungal activity- deficient mutant, B. subtilis mLTD was induced at a 5 kGy dose of $^{60}Co$ gamma radiation. Using the two-dimensional electrophoresis and the matrix assisted laser desorption ionization time-of-flight mass spectrometry, the comparison analysis of proteins between the wild and mutant were performed. A major intracellular serine proteinase IspA (MW: 32.5 kDa), a NAD (P) H dehydrogenase (MW: 20.0 kDa), and a stage II sporulation protein AA, SpoIIAA (MW: 14.3kDa) were detected only in the B. subtilis LTD. These results suggested that the functions of these proteins found only in the B. subtilis LTD could. be closely related to the antifungal activity against plant pathogenic fungi.


  1. Cohen AM, K Rumpel, GH Coombs and JM Wastling. 2002. Characterisation of global protein expression by twodimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii. Int. J. Parasitol. 32: 39-51
  2. Emmert EAB and J Handelsman. 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS MicrobioI. Lett. 171:1-9
  3. Glick BR. 1995. The enhancement of plant growth by free living bacteria. Can. J. Microbiol. 41:109-117
  4. Herish S, K Manjula and AR Podile. 1998. Furarium udum is resistant to the mycolytic activity of a biocontrol strain of Bacillus subtilis AF 1. FEMS Microbiol. Ecol. 25:385-390
  5. Kim ST, KS Cho, YS Jang and KY Kang. 2001. Twodimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays. Electrophoresis 22:2103-2109<2103::AID-ELPS2103>3.0.CO;2-W
  6. Laemmli UK. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277:680-685
  7. Nordhoff E, V Egelhofer, P Giavalosco, H Eickhoff, M Horn, T Przewieslik, D Theiss, U Schneider, H Lehrach and J Gobom. 2001. Large-gel two-dimensional electrophoresis-matrix assisted laser desorption lionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures. Electrophoresis 22:2844-2855<2844::AID-ELPS2844>3.0.CO;2-7
  8. O'Farrell PH. 1975. High resolution two-dimensional electrophoresis of proteins. J. BioI. Chern. 250:4007-402l
  9. Phae CG and M Shoda. 1991. Investigation of optimal condition for foam separation of iturin, an antifungal peptide produced by Bacillus subtilis. J. Ferment. Bioeng. 71:118-121
  10. Thacker J. 1999. Repair of ionizing radiation damage in mammalian cells. Alternative pathways and their fidelity. C. R. Acad. Sci. 322:103-108
  11. Hutchinson F. 1985. Chemical changes induced in DNA by ionizing radiation. Prog. Nucleic Acid Re. 32:115-154.
  12. Lee Y-K, J-S Kim, H-Y Chung, Y-S Jang and B-1 Jang. 2003a. Two-dimensional electrophoresis analysis of proteins between Bacillus licheniformis DM3 and its antifungal deficient mutant. Kor. J. Environ. Agric. 22:203-209
  13. Baker KF. 1987. Evolving concepts of biological control of plant pathogens. Ann. Rev. Phytopathol. 25:67 -85
  14. Lee Y-K, J-S Kim, Y-S Jang, K-S Cho and H-H Chang. 2003b. DNA microarray analysis of gene expression in antifungal bacterium of Bacillus lentimorbus WJ5. Kor. J. Microbiol. 39:141-147
  15. Perkins DN, DJ Pappin, DM Ceasy and JS Cotrell. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551-3567<3551::AID-ELPS3551>3.0.CO;2-2
  16. Strauch MA. 1993. Regulation of Bacillus subtilis gene expression during the transition from exponential growth to stationary phase. Prog. Nucleic Acid Re. 46:121-153
  17. Fravel DR. 1988. Role of antibiosis in the biocontrol of diseases. Ann. Rev. Phytopathol. 26:75-91
  18. Tang WH. 1994. Yield-increasing bacteria (YIB) and biocontrol of sheath blight of rice. pp. 267-278. In MH Ryder, PM Stephens, and GD Bowen (eds.), Improving plant productivity with rhizosphere bacteria. Common Wealth Scientific and Industrial Research Organization, Adelaide
  19. Leong J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogen. Ann. Rev. Phytopathol. 24:187-209
  20. Cho SJ, SK Lee, BJ Cha, YH Kim and KS Shin. 2003. Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03. FEMS Microbiol. Lett. 223:47- 51
  21. Shoda M. 2000. Bacterial control of plant diseases. J. Biosci. Bioeng. 89:515-52l
  22. Chen FC, LF Shen, MC Tsai and KF Chak. 2003. The IspA protease's involvement in the regulation of the sporulation process of Bacillus thuringiensis is revealed by proteomic analysis. Biochem. Biophys. Res. Comm. 312:708-715
  23. Kloepper JW, R Lifshitz and RM Zablotowicz. 1989. Free living bacterial inocula for enhancing crop productivity. Trends Biotechnol. 7:9-43
  24. Lee YK, HH Chang, JS Kim, JK Kim and KS Lee. 2000. Lignocellulolytic mutants of Pleurotus ostreatus induced by gamma-ray radJation and their genetic similarities. Rad. Phys. Chern. 57:145-150
  25. Phillips ZEV and MA Strauch. 2002. Bacillus subtilis sporulation and stationary phase gene expression. Cell. Mol. Life Sci. 59:392-402
  26. Woods RJ and AK Pikaev. 1994. Radiation dosimetry. pp.106-120. In Applied Radiation Chemistry: Radiation Processing. John Wiley & Sons, Inc., New York
  27. Baum JA and T Malvar. 1995. Regulation of insecticidal crystal protein production in Bacillus thuringiensis. Mol. Microbiol. 18:1-12
  28. Kerscher SJ. 2000. Diversity and origin of alternative NADH: ubiquinone oxidoreductases. Biochim. Biophys. Acta 1459:274-283