Advertisement

Applied Microbiology and Biotechnology

, Volume 85, Issue 4, pp 1201–1209 | Cite as

A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains

  • Natalia P. Zakataeva
  • Oksana V. Nikitina
  • Sergey V. Gronskiy
  • Dmitriy V. Romanenkov
  • Vitaliy A. Livshits
Methods

Abstract

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening. Using PCR primers with mismatches at the 3′ end enables the selection of strains that contain a single nucleotide substitution in the target gene. This approach can be used as a routine method for the investigation of complex physiological pathways and for the metabolic engineering of food-grade industrial B. amyloliquefaciens and other Bacillus strains.

Keywords

Naturally nontransformable Bacillus amyloliquefaciens Thermosensitive rolling-circle plasmid Replacement recombination Marker-free genetic modifications Point mutation Colony PCR analysis 

Notes

Acknowledgments

We are very grateful to J. Jomantas for the gift of the E40 bacteriophage and for his help with its use. We would also like to thank A. S. Mironov for providing the pKS1 plasmid and K. Matsuno for a critical reading of the manuscript.

References

  1. Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:741–746Google Scholar
  2. Arnaud M, Chastanet A, Débarbouillé M (2004) New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol 70:6887–6891CrossRefGoogle Scholar
  3. Bhowmik T, Fernández L, Steele JL (1993) Gene replacement in Lactobacillus helveticus. J Bacteriol 175:6341–6344Google Scholar
  4. Biswas I, Gruss A, Ehrlich SD, Maguin E (1993) High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175:3628–3635Google Scholar
  5. Bloor AE, Cranenburgh RM (2006) An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 72:2520–2525CrossRefGoogle Scholar
  6. Brans A, Filée P, Chevigné A, Claessens A, Joris B (2004) New integrative method to generate Bacillus subtilis recombinant strains free of selection markers. Appl Environ Microbiol 70:7241–7250CrossRefGoogle Scholar
  7. Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut PR, Vater J, Süssmuth R, Liesegang H, Strittmatter A, Gottschalk G, Borriss R (2007) Comparative analysis of the complete genome sequence of the plant growth promoting Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25:1007–1014CrossRefGoogle Scholar
  8. Coukoulis H, Campbell LL (1971) Transformation in Bacillus amyloliquefaciens. J Bacteriol 105:319–322Google Scholar
  9. Fabret C, Ehrlich SD, Noirot P (2002) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46:25–36CrossRefGoogle Scholar
  10. Huang MM, Arnheim N, Goodman MF (1992) Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res 20:4567–4573CrossRefGoogle Scholar
  11. Janes BK, Stibitz S (2006) Routine markerless gene replacement in Bacillus anthracis. Infect Immun 74:1949–1953CrossRefGoogle Scholar
  12. Jomantas JAV, Fiodorova JA, Abalakina EG, Kozlov YI (1991) Genetics of Bacillus amyloliquefaciens. 6th International Conference on Bacilli, Stanford, Calif., abstr. no T7Google Scholar
  13. Liu S, Endo K, Ara K, Ozaki K, Ogasawara N (2008) Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter. Microbiology 154:2562–2570CrossRefGoogle Scholar
  14. Maguin E, Prevost H, Ehrlich SD, Gruss A (1996) Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. J Bacteriol 178:931–935Google Scholar
  15. Mandal M, Breaker RR (2004) Adenine riboswitches and gene activation by disruption of a transcription terminator. Nat Struct Mol Biol 11:29–35CrossRefGoogle Scholar
  16. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  17. Noirot P, Petit MA, Ehrlich SD (1987) Plasmid replication stimulates DNA recombination in Bacillus subtilis. J Mol Biol 196:39–48CrossRefGoogle Scholar
  18. Nygaard P, Saxild HH (2005) The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size. J Bacteriol 187:791–794CrossRefGoogle Scholar
  19. Ramaley RF, Vasantha N (1983) Glycerol protection and purification of Bacillus subtilis glucose dehydrogenase. J Biol Chem 258:12558–12565Google Scholar
  20. Razer PN, Moran CP Jr (1988) Compartment-specific transcription in Bacillus subtilis: identification of the promoter for gdh. J Bacteriol 170:5086–5092Google Scholar
  21. Sambrook J, Russell DW (2001) Molecular cloning: laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  22. Shatalin KY, Neyfakh AA (2005) Efficient gene inactivation in Bacillus anthracis. FEMS Microbiol Lett 15:315–319CrossRefGoogle Scholar
  23. Sommer SS, Cassady JD, Sobell JL, Bottema CD (1989) A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria. Mayo Clin Proc 64:1361–1372Google Scholar
  24. Vehmaanperä J (1988) Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA. FEMS Microbiol Lett 49:101–105CrossRefGoogle Scholar
  25. Vehmaanperä J (1989) Transformation of Bacillus amyloliquefaciens by electroporation. FEMS Microbiol Lett 52:165–169CrossRefGoogle Scholar
  26. Vehmaanperä J, Steinborn G, Hofemeister J (1991) Genetic manipulation of Bacillus amyloliquefaciens. J Biotechnol 19:221–240CrossRefGoogle Scholar
  27. Waschkau B, Waldeck J, Wieland S, Eichstädt R, Meinhardt F (2008) Generation of readily transformable Bacillus licheniformis mutants. Appl Microbiol Biotechnol 78:181–188CrossRefGoogle Scholar
  28. Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86:2757–2760CrossRefGoogle Scholar
  29. Yan X, Yu HJ, Hong Q, Li SP (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microbiol 74:5556–5562CrossRefGoogle Scholar
  30. Zakataeva NP, Gronskiy SV, Sheremet AS, Kutukova EA, Novikova AE, Livshits VA (2007) A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides. Res Microbiol 158:659–665CrossRefGoogle Scholar
  31. Zhang XZ, Yan X, Cui ZL, Hong Q, Li SP (2006) mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res 34:e71CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Natalia P. Zakataeva
    • 1
  • Oksana V. Nikitina
    • 1
  • Sergey V. Gronskiy
    • 1
  • Dmitriy V. Romanenkov
    • 1
  • Vitaliy A. Livshits
    • 1
  1. 1.Ajinomoto–Genetika Research InstituteMoscowRussia

Personalised recommendations