title: Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage.
abstract: Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.
authors.affiliation: Cellular and Molecular Microbiology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany volker.winstel@med.uni-tuebingen.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Winstel
authors.affiliation: Cellular and Molecular Microbiology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Kühner
authors.affiliation: Cellular and Molecular Microbiology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Krismer
authors.affiliation: Cellular and Molecular Microbiology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Peschel
authors.affiliation: Institute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Rohde
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D022761:Gene Transfer, Horizontal; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D010957:Plasmids; D012808:Sigma Factor / Q000235:genetics; D013204:Staphylococcus Phages / Q000235:genetics*; D013211:Staphylococcus aureus / Q000235:genetics*; D013212:Staphylococcus epidermidis / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D001426:Bacterial Proteins; C079011:SigB protein, Bacteria; D012808:Sigma Factor