Gene targeting in gram-negative bacteria by use of a mobile group II intron ("Targetron") expressed from a broad-host-range vector.
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.
Yao
Jun
J
Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, 2500 Speedway, Austin, TX 78712, USA.
Lambowitz
Alan M
AM
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
D001565:Benzoates; C009503:3-toluic acid