Vector for IS element entrapment and functional characterization based on turning on expression of distal promoterless genes.

We constructed and characterized a novel trap vector for rapid isolation of insertion sequences. The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion. The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance genes resulting in selectable phenotypes. The trap vector pAW1326 is based on a pBR322 replicon, it carries ampicillin and streptomycin resistance genes, and also silenced genes that confer chloramphenicol and kanamycin resistance once activated. The trap vector pAW1326 proved to be efficient and 85 percent of all isolated mutations were insertions. The majority of IS elements resident in the studied Escherichia coli strains tested became trapped, namely IS2, IS3, IS5, IS150, IS186 and Tn1000. We also encountered an insertion sequence, called IS10L/R-2, which is a hybrid of the two IS variants IS10L and IS10R. IS10L/R-2 is absent from most E. coli strains, but it is detectable in some strains such as JM109 which had been submitted to Tn10 mutagenesis. The distribution of the insertion sequences within the trap region was not random. Rather, the integration of chromosomal mobile genetic elements into the offered target sequence occurred in element-specific clusters. This is explained both by the target specificity and by the specific requirements for the activation of gene transcription by the DNA rearrangement. The employed trap vector pAW1326 proved to be useful for the isolation of mobile genetic elements, for a demonstration of their transposition activity as well as for the further characterization of some of the functional parameters of transposition.

Isolation, characterization and transposition of an (IS2)2 intermediate.

We have isolated and characterized a dimer derivative of the extensively studied Escherichia coli insertion sequence IS2. The dimer structure--called (IS2)2--consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)2 dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)2 into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)2 dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)2 indicates that this is an active transposition intermediate. The mode of transposition of (IS2)2 is analogous to the joined dimer model described in the case of (IS21)2 and (IS30)2.