Optimization of Methods for Transfecting Spiroplasma citri Strain R8A2 HP with the Spiroplasma Virus SpV1 Replicative Form

Abstract

Seven methods for the transfection of bacteria were compared and optimized for use in Spiroplasma citri strain HP using the spiroplasma virus SpV1 R8A2 B replicative form (RF). These methods included both chemical-mediated protocols [CaCl2, RbCl/CaCl2, polyethylene glycol (PEG)], liposome-mediated transfection, electroporation, freeze/thaw cycling, and natural competence. The best protocols were those which utilized PEG or electroporation, yielding transfection frequencies of 1.4 × 10-4 and 9.1 × 10-4 transfectants/colony-forming unit (CFU), respectively. For both of these protocols, transfection frequencies were higher using CsCl-purified, covalently closed, circular DNA. In the PEG-mediated protocol, Sigma 8000 brand PEG at a final concentration of 44%, and the presence of carrier DNA proved to be optimal with a PEG exposure time of 2 min. Using electroporation, a 1-2 ms pulse of a 6.5 kV/cm electric field was best; washing the host cell pellet prior to electroporation enhanced efficiencies by 50%. Linearization of the DNA resulted in lower transfection efficiencies by either method.