Insertion-duplication mutagenesis in Streptococcus pneumoniae: Targeting fragment length is a critical parameter in use as a random insertion tool

To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool fur genomic analysis in Streptococcus pneumoniae, we studied the transformation effici ency and targeting specificity of the process by using a nonreplicative vec tor with homologous targeting inserts of various sizes. insertional recombi nation was very specific in targeting homologous sites. While the recombina tion rate did not depend on which site or region was targeted, it did depen d strongly on the size of the targeting insert in the donor plasmid, in pro portion to the fifth power of its length for inserts of 100 to 500 bp, The dependence of insertion-duplication events on the length of the targeting h omology was quite different from that for linear allele replacement and pla ces certain limits an the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to kn ock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results shaw that efficient and: thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.