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Applied and Environmental Microbiology, November 2001, p. 5190-5196, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.5190-5196.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

C. K. Sung,1 H. Li,1 J. P. Claverys,2 and D. A. Morrison1,*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois,1 and Laboratoire de Microbiologie et Génétique Moléculaire, UMR5100 CNRS-Université Paul Sabatier, Toulouse, France2

Received 3 July 2001/Accepted 27 August 2001

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.

* Corresponding author. Mailing address: University of Illinois at Chicago, 900 South Ashland Ave., Room 4110, Chicago, IL 60607. Phone: (312) 996-6839. Fax: (312) 413-2691. E-mail: DAMorris{at}uic.edu.

Applied and Environmental Microbiology, November 2001, p. 5190-5196, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.5190-5196.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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