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Appl Environ Microbiol. 2001 Nov;67(11):5190-6.

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

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  • 1Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.


Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL(+) marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step 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. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.

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