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Applied and Environmental Microbiology, June 2008, p. 3419-3425, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.00476-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro Mutagenesis of Bacillus subtilis by Using a Modified Tn7 Transposon with an Outward-Facing Inducible Promoter{triangledown}

Christophe Bordi,{dagger} Bronwyn G. Butcher, Qiaojuan Shi, Anna-Barbara Hachmann, Joseph E. Peters, and John D. Helmann*

Department of Microbiology, Cornell University, Ithaca, New York 14853-8101

Received 27 February 2008/ Accepted 4 April 2008

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.

* Corresponding author. Mailing address: Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101. Phone: (607) 255-6570. Fax: (607) 255-3904. E-mail: jdh9{at}cornell.edu

{triangledown} Published ahead of print on 11 April 2008.

{dagger} Present address: Département de Biologie, Faculté des Sciences de Luminy, Université de la Méditerranée, 13402 Marseille Cedex 20, France.

Applied and Environmental Microbiology, June 2008, p. 3419-3425, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.00476-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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