Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in Gram-negative bacteria

Abstract

To combine the features of the Ω interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposen, called Omegon-Km. The Omegon-Km transposen is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS 7. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the β interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene ( aphA) which is expressed well in many Gram-negative bacteria. The IS 7 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain m which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gramnegative soil and water bacteria ( Rhizobium leguminosarum, Paracoccus denitrifians) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.