This work describes a procedure for the generation of site-specific mutations into the chromosome ofStreptococcus pneumoniaethat does not involve the use of an antibiotic resistance marker. A linear fragment of transforming deoxyribonucleic acid (DNA) is constructed by polymerase chain reaction (PCR) (gene splicing by overlap extension) and used to transform competent cells ofS. pneumoniae. Selection of transformants is performed by PCR, and typically, 1% of the transformed cells show the expected mutation. By this protocol it is possible to change a single base pair into the pneumococcal genome, as well as obtaining in-frame deletions and insertions.
