Efficient electrotransformation system and gene targeting in pyogenic streptococci.
Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1 x 10(7) transformants per mug of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo- or sagB- mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.
Kimoto
Hisashi
H
Department of Biochemistry I, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. hisashi@fmsrsa.fukui-med.ac.jp
Taketo
Akira
A
D018274:Electroporation; D020868:Gene Silencing / Q000187:drug effects; D018390:Gene Targeting / Q000379:methods*; D010957:Plasmids / Q000494:pharmacology; D013291:Streptococcus / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D014170:Transformation, Genetic