High-Throughput Allelic Replacement Screening in Bacillus subtilis

  • Marie-Laure Diebold-Durand
  • Frank Bürmann
  • Stephan GruberEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)


Site-directed mutagenesis is a key tool in the analysis of biological mechanisms. We have established an efficient and systematic gene targeting strategy for Bacillus subtilis based on the Golden Gate cloning methodology. Our approach permits the introduction of single or multiple point mutations or of heavily engineered alleles into the endogenous gene locus in a single step using a 96-well microtiter plate format. We have successfully applied this system for high-throughput functional screening of resized variants of the Structural Maintenance of Chromosome (Smc) protein and for exhaustive cysteine cross-linking mutagenesis. Here we describe, in detail, the experimental setup for high-throughput introduction of modifications into the B. subtilis chromosome. With minor modifications, the approach should be applicable to other bacteria and yeast.

Key words

Golden Gate assembly Bacillus subtilis Gene targeting High-throughput screening Cysteine scanning 



  1. 1.
    Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus Subtilis by deoxyribonucleate. Proc Natl Acad Sci U S A 44(10):1072–1078CrossRefGoogle Scholar
  2. 2.
    Hamoen LW, Smits WK, Ad J, Holsappel S, Kuipers OP (2002) Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach. Nucleic Acids Res 30(24):5517–5528CrossRefGoogle Scholar
  3. 3.
    Tomasz A, Hotchkiss RD (1964) Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc Natl Acad Sci U S A 51(3):480–487CrossRefGoogle Scholar
  4. 4.
    Alexander HE, Leidy G (1950) Transformation of type specificity of Haemophilus influenzae. Proc Soc Exp Biol Med 73:485–487CrossRefGoogle Scholar
  5. 5.
    Meibom KL, Blokesch M, Dolganov NA, Wu C-Y, Schoolnik GK (2005) Chitin induces natural competence in Vibrio cholerae. Science 310(5755):1824CrossRefGoogle Scholar
  6. 6.
    Mell JC, Redfield RJ (2014) Natural competence and the evolution of DNA uptake specificity. J Bacteriol 196(8):1471CrossRefGoogle Scholar
  7. 7.
    Blokesch M (2016) Natural competence for transformation. Curr Biol 26(21):R1126–R1130. CrossRefPubMedGoogle Scholar
  8. 8.
    Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3(11):e3647. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4(5):e5553. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Diebold-Durand ML, Lee H, Ruiz Avila LB, Noh H, Shin HC, Im H, Bock FP, Burmann F, Durand A, Basfeld A, Ham S, Basquin J, Oh BH, Gruber S (2017) Structure of full-length SMC and rearrangements required for chromosome organization. Mol Cell 67(2):334–347.e335. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Burmann F, Basfeld A, Vazquez Nunez R, Diebold-Durand ML, Wilhelm L, Gruber S (2017) Tuned SMC arms drive chromosomal loading of prokaryotic condensin. Mol Cell 65(5):861–872.e869. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Aizenman E, Engelberg-Kulka H, Glaser G (1996) An Escherichia coli chromosomal “addiction module” regulated by 3′,5′-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci 93(12):6059CrossRefGoogle Scholar
  13. 13.
    Gerdes K, Christensen SK, Løbner-Olesen A (2005) Prokaryotic toxin–antitoxin stress response loci. Nat Rev Microbiol 3:371. CrossRefPubMedGoogle Scholar
  14. 14.
    Cegłowski P, Boitsov A, Chai S, Alonso JC (1993) Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis. Gene 136(1):1–12. CrossRefPubMedGoogle Scholar
  15. 15.
    Lioy VS, Machon C, Tabone M, Gonzalez-Pastor JE, Daugelavicius R, Ayora S, Alonso JC (2012) The ζ toxin induces a set of protective responses and dormancy. PLoS One 7(1):e30282. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bieniossek C, Imasaki T, Takagi Y, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37(2):49–57. CrossRefPubMedGoogle Scholar
  17. 17.
    Benoit RM, Ostermeier C, Geiser M, Li JSZ, Widmer H, Auer M (2016) Seamless insert-plasmid assembly at high efficiency and low cost. PLoS One 11(4):e0153158. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marie-Laure Diebold-Durand
    • 1
  • Frank Bürmann
    • 2
  • Stephan Gruber
    • 1
    Email author
  1. 1.Department of Fundamental MicrobiologyUniversity of LausanneLausanneSwitzerland
  2. 2.Structural StudiesMRC Laboratory of Molecular BiologyCambridgeUK

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