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Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing

¹ Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, The University of Texas at Austin, Austin, Texas 78712, USA
² Skirball Institute for Biomolecular Medicine, Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, New York, New York 10016, USA

We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivatives of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a derivative of pCN37 with a temperature-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%–100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the production of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32°C but not 43°C. The temperature sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temperature sensitivity of group II intron splicing could limit the temperature range of an organism containing a group II intron inserted in an essential gene.

Keywords: functional genomics; gene targeting; retrotransposon; reverse transcriptase; ribozyme

Reprint requests to: Alan M. Lambowitz, Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX 78712, USA; e-mail: lambowitz@mail.utexas.edu; fax: (512) 232-3420.

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.68706.

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