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Applied and Environmental Microbiology, October 2004, p. 6076-6085, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6076-6085.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria

Emmanuelle Charpentier,* Ana I. Anton, Peter Barry, Berenice Alfonso, Yuan Fang, and Richard P. Novick*

Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York

Received 24 November 2003/ Accepted 30 April 2004

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible Pcad-cadC and constitutive PblaZ promoters were designed and analyzed in transcriptional fusions to the staphylococcal ß-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

* Corresponding author. Mailing address for Emmanuelle Charpentier: Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9/4, A-1030 Vienna, Austria. Phone: 43-1-4277-54606. Fax: 43-1-4277-9546. E-mail: emmanuelle.charpentier{at}univie.ac.at. Mailing address for Richard P. Novick: Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, 540 First Ave., New York, NY 10016. Phone: (212) 263-6294. Fax: (212) 263-5711. E-mail: novick{at}saturn.med.nyu.edu.

Applied and Environmental Microbiology, October 2004, p. 6076-6085, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6076-6085.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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