Applied Microbiology and Biotechnology

, Volume 92, Issue 3, pp 571–581 | Cite as

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from Bacillus thuringiensis

  • Ahmed Abderrahmani
  • Arthur Tapi
  • Farida Nateche
  • Marlène Chollet
  • Valérie Leclère
  • Bernard Wathelet
  • Hocine Hacene
  • Philippe Jacques
Applied Genetics and Molecular Biotechnology

Abstract

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.

Keywords

Bacillus thuringiensis MALDI-ToF PCR Nonribosomal lipopeptides Kurstakins Spreading 

References

  1. Ansari MZ, Yadav G, Gokhale RS, Mohanty D (2004) NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases. Nucleic Acids Res 32:405–413CrossRefGoogle Scholar
  2. Bachmann BO, Ravel J (2009) Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. Methods Enzymol 458:181–217CrossRefGoogle Scholar
  3. Bumpus SB, Evans BS, Thomas PM, Ntai I, Kelleher NL (2009) A proteomics approach to discovering natural products and their biosynthetic pathways. Nat Biotechnol 27:951–956CrossRefGoogle Scholar
  4. Caboche S, Pupin M, Leclère V, Fontaine A, Jacques P, Kucherov G (2008) NORINE: a database of nonribosomal peptides. Nucleic Acids Res 36:D326–D331CrossRefGoogle Scholar
  5. Caboche S, Pupin M, Leclère V, Jacques P, Kucherov G (2009) Structural pattern matching of nonribosomal peptides. BMC Struct Biol 18:9–15Google Scholar
  6. Chauffaux J, Marchal M, Gilois N, Jehanno I, Buisson C (1997) Recherche de souches de Bacillus thuringiensis dans différents biotopes à travers le monde. Can J Microbiol 43:337–343CrossRefGoogle Scholar
  7. Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuth R, Piel J, Borriss R (2009) Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol 140:27–37CrossRefGoogle Scholar
  8. De Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199CrossRefGoogle Scholar
  9. Hathout Y, Ho YP, Ryzhov V, Demirev P, Fenselau C (2000) Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J Nat Prod 63:1492–1496CrossRefGoogle Scholar
  10. Hofte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255Google Scholar
  11. Höfte M, Mergeay M, Verstraete W (1990) Making the rhizopseudomonas strain 7NSK2 with a Mu d(lac) element for ecological studies. Appl Environ Microbiol 56:1046–1052Google Scholar
  12. Kim PI, Bai H, Bai D, Chae H, Chung S, Kim Y, Park R, Chi YT (2004) Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. J Appl Microbiol 97:942–949CrossRefGoogle Scholar
  13. Kotoujansky A, Lemattre M, Boistard P (1982) Utilization of a thermosensitive episome bearing transposon Tn10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi. J Bacteriol 150:122–131Google Scholar
  14. Leclère V, Béchet M, Adam A, Guez JS, Wathelet B, Ongena M, Thonart P, Gancel F, Chollet-Imbert M, Jacques P (2005) Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities. Appl Environ Microbiol 71:4577–4584CrossRefGoogle Scholar
  15. Leclère V, Marti R, Béchet M, Fickers P, Jacques P (2006) The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties. Arch Microbiol 186:475–483CrossRefGoogle Scholar
  16. Lee YK, Kim SB, Park CS, Kim JG, Oh HM, Yoon BD, Kim HS (2005) Chromosomal integration of sfp gene in Bacillus subtilis to enhance bioavailability of hydrophobic liquids. Appl Microbiol Biotechnol 67:789–794CrossRefGoogle Scholar
  17. Leenders F, Stein TH, Kablitz B, Franke P, Vater J (1999) Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix-assisted laser desorption/ionization mass spectrometry of intact cells. Rapid Commun Mass Spectrom 13:943–949CrossRefGoogle Scholar
  18. Marahiel MA, Essen LO (2009) Nonribosomal peptide synthetases mechanistic and structural aspects of essential domains. Methods Enzymol 458:337–351CrossRefGoogle Scholar
  19. May JJ, Wendrich TM, Marahiel MA (2001) The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J Biol Chem 276:7209–7217CrossRefGoogle Scholar
  20. Moyne AL, Cleveland TE, Tuzun S (2004) Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol Lett 234:43–49CrossRefGoogle Scholar
  21. Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48:443–453CrossRefGoogle Scholar
  22. Nihorimbere V, Fickers P, Thonart P, Ongena M (2009) Ecological fitness of Bacillus subtilis BGS3 regarding production of the surfactin lipopeptide in the rhizosphere. Env Microbiol Reports 1:124–130CrossRefGoogle Scholar
  23. Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16:116–125CrossRefGoogle Scholar
  24. Ongena M, Jourdan E, Adam A, Paquot M, Brans A, Joris B, Arpigny JL, Thonart P (2007) Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ Microbiol 9:1084–1090CrossRefGoogle Scholar
  25. Price NP, Rooney AP, Swezey JL, Perry E, Cohan FM (2007) Mass spectrometric analyses of lipopeptides from Bacillus strains isolated from diverse geographical locations. FEMS Microbiol Lett 271:83–89CrossRefGoogle Scholar
  26. Rausch C, Weber T, Kohlbacher O, Wohlleben W, Huson DH (2005) Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVM). Nucleic Acids Res 33:5799–5808CrossRefGoogle Scholar
  27. Read TD, Akmal A, Bishop-Lilly K, Chen PE, Cook C, Kiley MP, Lentz S, Mateczun A, Nagarajan N, Nolan N, Osborne BI, Pop M, Sozhamannan S, Stewart AC, Sulakvelidze A, Thomason B, Willner K, Zwick ME (2009) Annotation of the Bacillus thuringiensis serovar pondicheriensis BGSC 4BA1 genome. Direct Submission in Genbank databases, NCBI, USAGoogle Scholar
  28. Tapi A, Chollet-Imbert M, Scherens B, Jacques P (2010) New approach for the detection of non ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl Microbiol Biotechnol 85:1521–1531CrossRefGoogle Scholar
  29. Tseng CC, Bruner SD, Kohli RM, Marahiel MA, Walsh CT, Sieber SA (2002) Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase. Biochem 41:13350–13351CrossRefGoogle Scholar
  30. Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29–3. J Antibiot 39:888–901Google Scholar
  31. 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 68:6210–6219CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Ahmed Abderrahmani
    • 1
    • 2
  • Arthur Tapi
    • 1
  • Farida Nateche
    • 2
  • Marlène Chollet
    • 1
  • Valérie Leclère
    • 1
  • Bernard Wathelet
    • 3
  • Hocine Hacene
    • 2
  • Philippe Jacques
    • 1
  1. 1.Laboratoire des Procédés Biologiques, Génie Enzymatique et Microbien, ProBioGEM, UPRES-EA 1026, Polytech’Lille/IUT AUniversité Lille Nord de France-Sciences et Technologies, USTLVilleneuve d’Ascq CedexFrance
  2. 2.Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences BiologiquesUniversité des Sciences et de la Technologie Houari BoumedieneEl Alia. AlgerAlgeria
  3. 3.Unité de Chimie Biologique IndustrielleUniversité de Liège, Gembloux Agro-Bio TechGemblouxBelgium

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