title: Development of an efficient spheroplast transformation procedure for S. thermophilus: the use of transfection to define a regeneration medium.
abstract: The conditions for the efficient polyethylene glycol (PEG)-induced spheroplast transformation of three strains of Streptococcus thermophilus have been established. This required the careful optimization of various experimental parameters, the most important being the choice of the lytic enzyme (lysozyme versus mutanolysin), the extent of cell wall digestion and the conditions for the PEG shock which were found to be strain-specific. The transfection assay we had previously developed for S. thermophilus represented a key step and powerful tool in our transformation studies. It allowed individual and stepwise adjustment of the above mentioned factors, but was also compulsory for the establishment of an effective regeneration medium for the strains we examined. Among various potential osmotic protectors tested, raffinose in combination with CaCl2 and MgCl2 was most efficient and routinely supported regeneration with up to 10% efficiency, after PEG treatment. With the spheroplast transformation procedure described in this paper, shuttle vectors and recombinant plasmids could be introduced into three industrial yogurt starters, with maximal efficiencies of 7.5 x 10(4) transformants/micrograms of liposome encapsulated, covalently closed circular DNA. A striking, yet unexplained, reduction in transformation rates was observed when erythromycin rather than chloramphenicol was used as the selecting agent.
mesh_terms: D002122:Calcium Chloride / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D010450:Endopeptidases / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009113:Muramidase / Q000378:metabolism; D010957:Plasmids; D011092:Polyethylene Glycols / Q000494:pharmacology; D011887:Raffinose / Q000494:pharmacology; D013104:Spheroplasts / Q000187:drug effects; D013291:Streptococcus / Q000235:genetics*; D014162:Transfection; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D004274:DNA, Recombinant; D015636:Magnesium Chloride; D011092:Polyethylene Glycols; D009113:Muramidase; D010450:Endopeptidases; C021816:mutanolysin; D008274:Magnesium; D002122:Calcium Chloride; D011887:Raffinose