Genetic engineering of untransformable coagulase-negative staphylococcal pathogens.
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 10(9) 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.
Infection Biology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.|German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
Volker
V
Winstel
Infection Biology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.|German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
Petra
P
Kühner
Institute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
Holger
H
Rohde
Infection Biology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.|German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
Andreas
A
Peschel
D001435:Bacteriophages / Q000235:genetics; D003030:Coagulase / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics* / Q000472:pathogenicity; D014161:Transduction, Genetic; D057188:Workflow
D003030:Coagulase