Protocol | Published:

Genetic engineering of untransformable coagulase-negative staphylococcal pathogens

Nature Protocols volume 11, pages 949959 (2016) | Download Citation

Abstract

Coagulase-negative staphylococci (CoNS) are recognized as significant opportunistic pathogens. However, current knowledge of virulence mechanisms is very limited because a significant proportion of CoNS are refractory to available techniques for DNA transformation. We describe an efficient protocol for plasmid transfer using bacteriophage Φ187, which can transduce plasmid DNA to a wide range of CoNS from a unique, engineered Staphylococcus aureus strain. The use of a restriction-deficient, modification-proficient S. aureus PS187 mutant, which has a CoNS-type bacteriophage surface receptor, allows plasmid transfer to CoNS even when they are refractory to electroporation. Once the Φ187 titer reaches 109 plaque-forming units per milliliter, plasmid transfer can be accomplished within 1–2 d. Thus, our protocol is a major technical advance offering attractive opportunities for research on CoNS-mediated infections.

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References

  1. 1.

    & Genetic manipulation of Staphylococcus aureus. Curr. Protoc. Microbiol. 32, 9C.3.1–9C.3.19 (2014).

  2. 2.

    & Genetic manipulation of staphylococci-breaking through the barrier. Front. Cell. Infect. Microbiol. 2, 49 (2012).

  3. 3.

    , & An update on the molecular genetics toolbox for staphylococci. Microbiology 159, 421–435 (2013).

  4. 4.

    , , & Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37, 1.8.1–1.8.10 (2001).

  5. 5.

    & Plasmid uptake by bacteria: a comparison of methods and efficiencies. Appl. Microbiol. Biotechnol. 83, 791–798 (2009).

  6. 6.

    , & Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector. J. Bacteriol. 165, 831–836 (1986).

  7. 7.

    , & Plasmid transfer and genetic recombination by protoplast fusion in staphylococci. J. Bacteriol. 145, 74–81 (1981).

  8. 8.

    , & Genetic manipulation of Clostridium difficile. Curr. Protoc. Microbiol. 20, 9A.2.1–9A.2.17 (2011).

  9. 9.

    , & E. coli genome manipulation by P1 transduction. Curr. Protoc. Mol. Biol. 79, 1.17.1–1.17.8 (2007).

  10. 10.

    , , , & Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage. Appl. Environ. Microbiol. 81, 2481–2488 (2015).

  11. 11.

    & Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol. Lett. 54, 203–207 (1990).

  12. 12.

    & High-frequency transformation of Staphylococcus aureus by electroporation. Curr. Microbiol. 21, 373–376 (1990).

  13. 13.

    & Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol. Lett. 73, 133–138 (1992).

  14. 14.

    , , , & Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism. J. Appl. Microbiol. 102, 736–747 (2007).

  15. 15.

    , , , & Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3, e00277–00211 (2012).

  16. 16.

    , , , & Complete bypass of restriction systems for major Staphylococcus aureus lineages. MBio 6, e00308–00315 (2015).

  17. 17.

    Staphylococcus epidermidis–the 'accidental' pathogen. Nat. Rev. Microbiol. 7, 555–567 (2009).

  18. 18.

    , & Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect. Dis. 2, 677–685 (2002).

  19. 19.

    & CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11, 181–190 (2010).

  20. 20.

    & Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3, 711–721 (2005).

  21. 21.

    , , , & Sortase A promotes virulence in experimental Staphylococcus lugdunensis endocarditis. Microbiology 159, 2141–2152 (2013).

  22. 22.

    & CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

  23. 23.

    et al. Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens. Nat. Commun. 4, 2345 (2013).

  24. 24.

    & Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58–63 (2006).

  25. 25.

    et al. The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression. PLoS Pathog. 8, e1003016 (2012).

  26. 26.

    , & Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system. FEMS Microbiol. Lett. 137, 279–284 (1996).

  27. 27.

    A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122, 187–192 (1992).

  28. 28.

    et al. Staphylococcus aureus mutant screen reveals interaction of the human antimicrobial peptide dermcidin with membrane phospholipids. Antimicrob. Agents Chemother. 53, 4200–4210 (2009).

  29. 29.

    & Use of electroporation and conjugative mobilization for genetic manipulation of Staphylococcus epidermidis. Methods Mol. Biol. 1106, 125–134 (2014).

  30. 30.

    Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol. Mol. Biol. Rev. 64, 412–434 (2000).

  31. 31.

    & Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages. J. Bacteriol. 188, 5578–5585 (2006).

  32. 32.

    , , , & The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc. Natl. Acad. Sci. USA 102, 5174–5179 (2005).

  33. 33.

    & Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages. Mol. Gen. Genet. 210, 347–351 (1987).

  34. 34.

    , , & Packaging of the bacteriophage lambda chromosome: effect of chromosome length. Virology 77, 281–293 (1977).

  35. 35.

    , , , & Challenging packaging limits and infectivity of phage lambda. J. Mol. Biol. 415, 263–273 (2012).

  36. 36.

    , , & Agarose gel electrophoresis for the separation of DNA fragments. J. Vis. Exp. 10.3791/3923 (2012).

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Acknowledgements

This work was supported by German Research Council grants TRR34 and SFB766 to A.P. and Ro2413/4-1 to H.R., and by German Center for Infection Research (DZIF) grants to H.R. and A.P.

Author information

Author notes

    • Volker Winstel

    Present address: Department of Microbiology, University of Chicago, Chicago, Illinois, USA.

Affiliations

  1. Infection Biology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.

    • Volker Winstel
    • , Petra Kühner
    •  & Andreas Peschel
  2. German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.

    • Volker Winstel
    • , Petra Kühner
    •  & Andreas Peschel
  3. Institute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.

    • Holger Rohde

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Contributions

V.W., H.R. and A.P. designed the study. V.W. and P.K. performed the experiments. V.W., P.K. and A.P. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andreas Peschel.

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DOI

https://doi.org/10.1038/nprot.2016.058

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