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An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae

By C. K. Sung, H. Li, J. P. Claverys and D. A. Morrison


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

Topics: Genetics and Molecular Biology
Publisher: American Society for Microbiology
Year: 2001
DOI identifier: 10.1128/AEM.67.11.5190-5196.2001
OAI identifier:
Provided by: PubMed Central
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