Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria.
To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon 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 IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 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 in 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 Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) 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.
Fellay
R
R
Département de Biochimie, Université de Genève, Switzerland.
Krisch
H M
HM
Prentki
P
P
Frey
J
J
D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D005787:Gene Frequency; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007613:Kanamycin Resistance; D009154:Mutation; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics; D015183:Restriction Mapping
D004251:DNA Transposable Elements; D004274:DNA, Recombinant