Gene Targeting in Gram - Negative Bacteria Using a Mobile Group II Intron 1 ( “ Targetron ” ) Expressed from a Broad - Host - Range Vector

26 Mobile group II introns (" targetrons ") can be programmed to insert into virtually any desired 27 DNA target with high frequency and specificity. Here, we show that targetrons expressed via an 28 m-toluic acid-inducible promoter from a broad host-range vector containing an RK2 mini-replicon 29 can be used for efficient gene targeting in a variety Gram-negative bacteria, including Escherichia 30 coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor 31 plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies 32 of 1-58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. 33 tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron 34 expression should facilitate gene targeting in many bacteria. 35