An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae.
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.
Sung
C K
CK
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Li
H
H
Claverys
J P
JP
Morrison
D A
DA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005785:Gene Conversion; D005819:Genetic Markers; D007613:Kanamycin Resistance / Q000235:genetics; D011995:Recombination, Genetic; D012269:Ribosomal Proteins / Q000235:genetics*; D012641:Selection, Genetic; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000235:genetics*; D013307:Streptomycin / Q000494:pharmacology; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D005819:Genetic Markers; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; D013307:Streptomycin