An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae

Natural genetic transformation offers a direct route by which synthetic gen e constructs can be placed into the single circular chromosome of Streptoco ccus pneumoniae. However, the lack of a general negative-selection marker h as hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) re sistance marker (kan) and a counterselectable rpsL(+) marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant backgro und in S. pneumoniae. It was demonstrated that it could be used in a two-st ep transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Repl acement of the cassette by an arbitrary segment of DNA during a second tran sformation restored Sin resistance (and Kn sensitivity), allowing construct ion of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred be tween the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.