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Natural Genetic Transformation: A Direct Route to Easy Insertion of Chimeric Genes into the Pneumococcal Chromosome

  • Isabelle Mortier-BarrièreEmail author
  • Nathalie Campo
  • Mathieu A. Bergé
  • Marc Prudhomme
  • Patrice Polard
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1968)

Abstract

The ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.

Key words

Natural genetic transformation Gene transfer Markerless integration/mutagenesis Overlapping PCR 

Notes

Acknowledgments

We warmly thank Jean-Pierre Claverys and Bernard Martin for their prominent contribution to development of pneumococcal genetics. We thank Dave Lane and Calum Johnston for critical reading of the manuscript. We also thank all past members of the Claverys lab, past and present members of the Polard lab who participated in development of the method. This work was funded by the Centre National de la Recherche Scientifique, Université Paul Sabatier and Agence Nationale de la Recherche (Grant ANR-13-BSV8-0022 and ANR-17-CE13-0031).

References

  1. 1.
    Sung CK, Li H, Claverys JP, Morrison DA (2001) An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67:5190–5196.  https://doi.org/10.1128/AEM.67.11.5190-5196.2001CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Weng L, Biswas I, Morrison DA (2009) A self-deleting Cre-lox-ermAM cassette, Cheshire, for marker-less gene deletion in Streptococcus pneumoniae. J Microbiol Methods 79:353–357.  https://doi.org/10.1016/j.mimet.2009.10.007CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Iannelli F, Pozzi G (2004) Method for introducing specific and unmarked mutations into the chromosome of Streptococcus pneumoniae. Mol Biotechnol 26:81–86.  https://doi.org/10.1385/MB:26:1:81CrossRefPubMedGoogle Scholar
  4. 4.
    Junges R, Khan R, Tovpeko Y et al (2017) Markerless genome editing in competent streptococci. Methods Mol Biol 1537:233–247.  https://doi.org/10.1007/978-1-4939-6685-1_14CrossRefPubMedGoogle Scholar
  5. 5.
    Bergé MJ, Mercy C, Mortier-Barrière I et al (2017) A programmed cell division delay preserves genome integrity during natural genetic transformation in Streptococcus pneumoniae. Nat Commun 8:1621.  https://doi.org/10.1038/s41467-017-01716-9CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Mortier-Barrière I, de Saizieu A, Claverys JP, Martin B (1998) Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae. Mol Microbiol 27:159–170CrossRefGoogle Scholar
  7. 7.
    Dagkessamanskaia A, Moscoso M, Hénard V et al (2004) Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells. Mol Microbiol 51:1071–1086CrossRefGoogle Scholar
  8. 8.
    Prudhomme M, Attaiech L, Sanchez G et al (2006) Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae. Science 313:89–92.  https://doi.org/10.1126/science.1127912CrossRefPubMedGoogle Scholar
  9. 9.
    Lau PCY, Sung CK, Lee JH et al (2002) PCR ligation mutagenesis in transformable streptococci: application and efficiency. J Microbiol Methods 49:193–205CrossRefGoogle Scholar
  10. 10.
    Tomasz A (1967) Choline in the cell wall of a bacterium: novel type of polymer-linked choline in pneumococcus. Science 157:694–697CrossRefGoogle Scholar
  11. 11.
    Lefevre JC, Claverys JP, Sicard AM (1979) Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation. J Bacteriol 138:80–86PubMedPubMedCentralGoogle Scholar
  12. 12.
    Mérola F, Fredj A, Betolngar D-B et al (2014) Newly engineered cyan fluorescent proteins with enhanced performances for live cell FRET imaging. Biotechnol J 9:180–191.  https://doi.org/10.1002/biot.201300198CrossRefPubMedGoogle Scholar
  13. 13.
    Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916.  https://doi.org/10.1126/science.1068539CrossRefPubMedGoogle Scholar
  14. 14.
    Bi EF, Lutkenhaus J (1991) FtsZ ring structure associated with division in Escherichia coli. Nature 354:161–164.  https://doi.org/10.1038/354161a0CrossRefPubMedGoogle Scholar
  15. 15.
    Jacq M, Adam V, Bourgeois D et al (2015) Remodeling of the Z-ring nanostructure during the Streptococcus pneumoniae cell cycle revealed by Photoactivated localization microscopy. MBio 6:e01108-15.  https://doi.org/10.1128/mBio.01108-15CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bergé MJ, Kamgoué A, Martin B et al (2013) Midcell recruitment of the DNA uptake and virulence nuclease, EndA, for pneumococcal transformation. PLoS Pathog 9:e1003596.  https://doi.org/10.1371/journal.ppat.1003596CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Morrison DA, Khan R, Junges R et al (2015) Genome editing by natural genetic transformation in Streptococcus mutans. J Microbiol Methods 119:134–141.  https://doi.org/10.1016/j.mimet.2015.09.023CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Isabelle Mortier-Barrière
    • 1
    • 2
    Email author
  • Nathalie Campo
    • 1
    • 2
  • Mathieu A. Bergé
    • 1
    • 2
  • Marc Prudhomme
    • 1
    • 2
  • Patrice Polard
    • 1
    • 2
  1. 1.Laboratoire de Microbiologie et Génétique Moléculaires (LMGM)Centre de Biologie Intégrative (CBI)ToulouseFrance
  2. 2.Centre National de la Recherche Scientifique (CNRS)Université de Toulouse, Université Paul Sabatier (UPS)ToulouseFrance

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