Genetic engineering of untransformable coagulase-negative staphylococcal pathogens

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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Vis. Exp._ 10.3791/3923 (2012). Download references 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: Present address: Department of Microbiology, University of

Chicago, Chicago, Illinois, USA., AUTHORS AND AFFILIATIONS * Infection Biology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany

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

* Institute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany Holger Rohde Authors * Volker Winstel View author publications You

can also search for this author inPubMed Google Scholar * Petra Kühner View author publications You can also search for this author inPubMed Google Scholar * Holger Rohde View author

publications You can also search for this author inPubMed Google Scholar * Andreas Peschel View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Andreas Peschel. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Winstel, V.,

Kühner, P., Rohde, H. _et al._ Genetic engineering of untransformable coagulase-negative staphylococcal pathogens. _Nat Protoc_ 11, 949–959 (2016). https://doi.org/10.1038/nprot.2016.058

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