title: Gene targeting in gram-negative bacteria by use of a mobile group II intron ("Targetron") expressed from a broad-host-range vector.
abstract: 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.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, 2500 Speedway, Austin, TX 78712, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.forename: Alan M
authors.initials: AM
authors.lastname: Lambowitz
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D001565:Benzoates / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics
chemical_list: D001565:Benzoates; C009503:3-toluic acid