Plasmid cointegrates and their resolution mediated by transposon Tn3 mutants

We have generated recombinant plasmids in vivo between pHS1, a temperature-sensitive replication mutant of pSC101 which codes for tetracycline (Tc) resistance, and pMB8, carrying the ampicillin (Amp) transposon Tn3. Recombinants were selected in a recA^- Escherichia coli strain at 42°C in the presence of Tc. When the repressor gene tnpR of Tn3 was inactive, all cells examined contained cointegrate recombinants, even in the absence of selective conditions for the recombinant. Tn3's were present at both junctions between the two parental plasmids, in a direct orientation, and a 5-bp pHS1 sequence was directly duplicated at the plasmid junctions. Complementation of the cointegrate with active repressor (tnpR⁺) in a recA^- background at 30°C, resolved the recombinant into two plasmids, yielding the pMB8:: Tn3 parent and pHS1, which has acquired a copy of the Tn3 mutant. These results indicate that the repressor is responsible for this resolving recombination event. Recombinants were also formed between pHS1 and pMB8 carrying wild-type Tn3 or its mutant No. 25, which is tnpR⁺ and tnpA⁺ but has an EcoRI recognition site inserted at a position corresponding to the amino-terminus of the tnpA-coded transposase. Various sized recombinants were generated at 42°C (+Tc), all smaller than the cointegrates described above, but larger than the parental plasmids. The frequencies of recombination between wild-type Tn3 and pHS1, and mutant No. 25 and pHS1 were 10^-6-10^-7, i.e., drastically lower than the frequency of formation of cointegrates mediated by tnpR^- mutants. Genetic and nucleotide-sequence analysis suggest that the formation of these recombinants is through intermediate cointegrate structures, which subsequently undergo deletions removing all or part of a copy of Tn3, leaving one intact Tn3 in the plasmid. Interstingly, analysis of one recombinant showed that such a deletion occurred by recombination at a region of homology between the left inverted Tn3 repeat and pMB8, in a recA^- strain. Tn3 mutants that are defective in either of their inverted end repeats or in the carboxy-terminus of the transposase cannot mediate recombination between two plasmids, or do so at a very low frequency. Based on these results we discuss the role of genes and sites encoded by Tn3 in cointegration and transposition.