The role of pneumolysin in the pneumococcal infection was studied by comparing the pathological effects of wild-type and its isogenic strain of pneumolysin-mutant S. pneumoniae. The erythromycin resistance gene marker was inserted into type 2 pneumolysin (ply) gene DNA to form a ply-mutant gene. This ply-mutant gene was transformed into the pneumococcal cells. A DNA fragment encoding erythromycin resistance gene was isolated from the Gram-positive plasmid PNG2 and ligated into a vector PUC18. This 1.2 kb DNA fragment was inserted into the BamHI site of pWJ208 type 2 pneumolysin gene to form pJWE89 ply-mutant gene. This mutant gene was transformed into the rough pneumococcal strain R61, by the method of insertion-duplication mutagenesis. Competence in strain R61 developed at a point in growth having a cell density of 0.3-0.5 O.D. at 600 nm. The genetic transformation was performed by adding 1 mg pJWE89 gene DNA into R61 cells. Some transformed colonies were formed on the erythromycin-containing plates during the incubation period of 80-110 min. None of these colonies produced pneumolysin.
