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Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24

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Abstract

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma–Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4′-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.

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References

  1. Asaka O, Shoda M (1996) Biocontrol of Rhizoctonia solani damping off of tomato with Bacillus subtilis RB14. Appl Environ Microbiol 62:4081–4085

    CAS  PubMed  Google Scholar 

  2. Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitates by biofilm formation and surfactin production. Plant Physiol 134:307–319

    Article  CAS  PubMed  Google Scholar 

  3. Besson F, Peypoux F, Quentin MJ, Michel G (1984) Action of antifungal peptidolipids from Bacillus subtilis on the cell membrane of Saccharomyces cerevisiae. J Antibiot (Tokyo) 37:172–177

    CAS  Google Scholar 

  4. Besson F, Chevenet C, Michel G (1987) Influence of the general culture medium on the production of iturin A by Bacillus subtilis. J Gen Microbiol 13:767–772

    Google Scholar 

  5. Hiraoka H, Ano T, Shoda M (1992) Characterization of Bacillus subtilis RB14, coproducer of peptide antibiotic iturin A and surfactin. J Gen Appl Microbiol 38:635–640

    Article  CAS  Google Scholar 

  6. Hsieh FC, Lin TC, Meng M, Kao SS (2008) Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Curr Microbiol 56:1–5

    Article  CAS  PubMed  Google Scholar 

  7. Huang CC, Ano T, Shoda M (1993) Nucleutide sequence and characteristics of the gene, lpa-14, responsible for biosynthesis of the lipopeptide antibiotic iturin A and surfactin from Bacillus subtilis RB14. J Ferment Bioeng 76:445–450

    Article  CAS  Google Scholar 

  8. Kunst F, Ogasawara N, Moszer I (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256

    Article  CAS  PubMed  Google Scholar 

  9. Loeffler W, Tschen JSM, Vanittanatkcom N et al (1986) Antifungal effects of bacilysin and fengymycin from Bacillus subtilis F-29-3. A comparision with activities of other Bacillus antibiotics. J Phytopathol 78:1075–1078

    Google Scholar 

  10. Maget-dana R, Peypoux F (1994) Iturin, a special class of pore-forming lipopeptides: biological and physiochemical properties. Toxicology 87:151–174

    Article  CAS  PubMed  Google Scholar 

  11. Marahiel MA, Stachelhaus T, Mootz HD (1997) Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 97:2651–2673

    Article  CAS  PubMed  Google Scholar 

  12. McKeen CD, Reilly CC, Pusey PL (1986) Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopathology 76:136–139

    Article  CAS  Google Scholar 

  13. Minakshi (2002) Coinoculation effects of plant growth promoting rhizobacteria and Bradyrhizobium on growth and nodulation of pigeonpea [Cajanus cajan (L) Millsp.]. Ph.D thesis submitted to Kurukshetra University, India

  14. Grover M, Saxena AK, Matta NK (2005) Selection of culturable PGPR from diverse pool of pigeonpea rhizosphere. Ind J Microbiol 45:21–26

    Google Scholar 

  15. Grover M, Nain L, Saxena AK (2009) Comparision between Bacillus subtilis RP24 and its antibiotic-defective mutants. World J Microbiol Biotechnol 25:1329–1335

    Article  CAS  Google Scholar 

  16. Nakano MM, Corbell N, Besson J, Zuber P (1992) Isolation and characterization of sfp: a gene that functions in the production of lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol Gen Genet 232:313–321

    CAS  PubMed  Google Scholar 

  17. Romero D, Vivente AD, Rakotoaly RH, Dufour SE, Veening J, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Perez-Garcia A (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis towards Podosphaera fusca. Mol Plant Microbe Interact 20:430–440

    Article  CAS  PubMed  Google Scholar 

  18. Sandrin C, Peypoux F, Michel G (1990) Coproductin of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis. Biotechnol Appl Biochem 12:370–375

    CAS  PubMed  Google Scholar 

  19. Souto GI, Correa OS, Montecchia MS et al (2004) Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds. J Appl Microbiol 97:1247–1256

    Article  CAS  PubMed  Google Scholar 

  20. Stein T (2005) Bacillus subtilis antibiotics:Atructures, synthesis and specific functions. Mol Microbiol 56:845–857

    Article  CAS  PubMed  Google Scholar 

  21. Sten T, Vater J, Kruft V, Otto A et al (1996) The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J Biol Chem 271:15428–15435

    Article  Google Scholar 

  22. Tendulkar SR, Siakumar YK, Patel V, Raghotama S, Munshi TK, Balaram P, Chattoo BB (2007) Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98, and its effect on phytopathogen Magnaporthe grisea. J Appl Microbiol 103:2331–2339

    Article  CAS  PubMed  Google Scholar 

  23. Thimon L, Peypoux F, Maget-Dana R, Roux Band Michel G (1992) Interaction of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis. Biotechnol Appl Biochem 16:144–151

    CAS  PubMed  Google Scholar 

  24. Toure Y, Ongena M, Jacques P, Guiro A, Thonart P (2004) Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J Appl Microbiol 96:1151–1160

    Article  CAS  PubMed  Google Scholar 

  25. Tsuge K, Akiyama T, Shoda M (2001) Cloning sequencing and characterization of the iturin A operon. J Bacteriol 183:6265–6273

    Article  CAS  PubMed  Google Scholar 

  26. Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M (1999) The genes degQ, pps and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Chemother 43:2183–2192

    CAS  PubMed  Google Scholar 

  27. Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68:6210–6219

    Article  CAS  PubMed  Google Scholar 

  28. von Döhren H, Keller U, Vater J, Zocher R (1997) Multifunctional peptide synthetases. Chem Rev 97:2675–2705

    Article  Google Scholar 

  29. Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL (2002) Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol Biochem 34:955–963

    Article  CAS  Google Scholar 

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Acknowledgments

Grant provided by Department of Science and Technology, Ministry of Science and Technology, Government of India, for this project under “Women Scientist Scheme, WOS-A” is highly acknowledged.

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Authors and Affiliations

  1. Division of Crop Sciences, Central Research Institute for Dryland Agriculture, Hyderabad, 50059, India

    Minakshi Grover

  2. Division of Microbiology, Indian Agricultural Research Institute, New-Delhi, 110012, India

    Lata Nain & Anil Kumar Saxena

  3. Division of Agricultural Chemicals, Indian Agricultural Research Institute, New-Delhi, 110012, India

    Shashi Bala Singh

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  1. Minakshi Grover
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  2. Lata Nain
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  3. Shashi Bala Singh
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  4. Anil Kumar Saxena
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Correspondence to Minakshi Grover.

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Grover, M., Nain, L., Singh, S.B. et al. Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24. Curr Microbiol 60, 99–106 (2010). https://doi.org/10.1007/s00284-009-9508-6

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  • DOI: https://doi.org/10.1007/s00284-009-9508-6

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