A Simple and Rapid Method of Transformation of Streptomyces rimosus R6 and Other Streptomycetes by Electroporation.
Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-h-old cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at -70 deg C. The electric field strength of 10 kV/cm at 400 (Omega) and a capacitance of 25 (mu)F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10(sup5) to 10(sup6) transformants per (mu)g of plasmid DNA were reproducibly achieved for S. rimosus R6 and its mutants, and these numbers were 10(sup2) to 10(sup3) higher than those attained by polyethylene glycol-assisted transformation of protoplasts. In addition, we show that electroporation can be applied to other Streptomyces species, such as S. lividans 66, S. coelicolor A3(2), and an S. venezuelae strain. This last one could not be transformed by the standard protoplast procedure. Our data suggest that, because of the diversity of streptomycetes, the conditions have to be optimized for each strain.
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Pigac
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Schrempf