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Applied and Environmental Microbiology, April 2007, p. 2735-2743, Vol. 73, No. 8
0099-2240/07/$08.00+0     doi:10.1128/AEM.02829-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron ("Targetron") Expressed from a Broad-Host-Range Vector{triangledown}

Jun Yao and Alan M. Lambowitz*

Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 78712

Received 5 December 2006/ Accepted 16 February 2007

Mobile group II introns ("targetrons") can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.

* Corresponding author. Mailing address: Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, 2500 Speedway, Austin, TX 78712. Phone: (512) 232-3418. Fax: (512) 232-3420. E-mail: lambowitz{at}mail.utexas.edu

{triangledown} Published ahead of print on 23 February 2007.

Applied and Environmental Microbiology, April 2007, p. 2735-2743, Vol. 73, No. 8
0099-2240/07/$08.00+0     doi:10.1128/AEM.02829-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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