Gene Targeting in Gram-Negative Bacteria Using a Mobile Group II Intron ("Targetron") Expressed from a Broad-Host-Range Vector

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

^* To whom correspondence should be addressed. Email: lambowitz{at}mail.utexas.edu.

	Abstract

Mobile group II introns ("targetrons") can be programmed to insert 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 mini-replicon can be used for efficient gene targeting in a variety 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 1-58% 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.