Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision.

A method for the rapid cloning of specific Streptococcus pneumoniae DNA fragments depends on the integration by homologous recombination into the bacterial chromosome of a plasmid which carries an insert of S. pneumoniae DNA, but which cannot be autonomously maintained in S. pneumoniae. Selection for plasmid integration employs aminopterin or erythromycin resistance. Host sequences adjacent to the site of insertion are easily cloned by enzymatic excision and recircularization of the plasmid, followed by propagation in Escherichia coli. This is particularly useful for repeated cloning of a given fragment that carries various mutations.