A genetic transformation system for Streptococcus pyogenes.

Abstract

An efficient protoplast transformation system for Streptococcus pyogenes has been developed. Efficiencies of up to 7.1 x 10(6) transformants/micrograms DNA were achieved, with transformants recovered on selective media in 24-48 h. The system was characterized as to optimal protoplasting conditions, effective facilitators, dependency on concentration of transforming DNA, plasmid copy number in transformants and stability of transformants. Three isolates of S. pyogenes were used as recipients, and four plasmids were used as the transforming DNA. Growth of S. pyogenes in glycine followed by lysozyme treatment was necessary for optimal protoplast formation. The exact concentrations of these protoplasting agents which were used varied with each isolate tested. Both polyethylene glycol and dextran sulphate were efficient facilitators of transformation, at final concentrations of 10%. An inverse relationship between DNA concentration and efficiency of transformation was shown. The copy number of the AC-1 plasmid in the transformants was shown to be equivalent to that of the wild type S. pyogenes (AC-1) (one or two copies per chromosomal equivalent). Approximately 50% of the AC-1 transformants were stable after one passage on non-selective media, and 100% of those that retained the plasmid were stable for an additional twenty generations. Erythromycin resistance encoded on the AC-1 plasmid was inducible, and transformants with a constitutive mutant of the AC-1 plasmid were detected by growth on selective media. This plasmid may prove useful as a vector as it is readily transformed, expressed, and contains at least three unique restriction sites which could serve as insertion points for cloned DNA.