NEW GENETIC TECHNIQUES FOR GROUP-B STREPTOCOCCI - HIGH-EFFICIENCY TRANSFORMATION, MAINTENANCE OF TEMPERATURE-SENSITIVE PWV01 PLASMIDS, AND MUTAGENESIS WITH TN917

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transfo rmation of GBS by electroporation, which provided transformation effic iencies of 10(5) CFU/mu g. Variables that influenced the transformatio n efficiency were the glycine content of the competent cell growth med ia, the electric field strength during electroporation, the electropor ation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. O ur transformation protocol provides an efficiency sufficient for cloni ng from ligation reactions directly into GBS, obviating an intermediat e host such as Escherichia coli. Second, temperature-sensitive plasmid s of the pWV01 lineage were shown to transform GBS, and their temperat ure-sensitive replication was confirmed. Lastly, the temperature-sensi tive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transpo son delivery vector for the construction of genomic Tn917 mutant libra ries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, represen tative clones from a Tn917 library contained single transposon inserti ons that were randomly located throughout the chromosome. These techni ques should provide useful methods for cloning, mutagenesis, and chara cterization of genes from GBS.