Abstract:
The transposition properties of Tn7 were studied using three different approaches: (i) Transposition structures formed while transposing into pHH507 and FΔ(0-15); (ii) Insertional specificity as given by sequence data at four different sites; (iii) Epidemiology of Tn7 among clinical isolates in Auckland (New Zealand). First, Tn7 transposition from two donor plasmids (ColEl::Tn7 and pCPL6::Tn7) was followed to observe the formation of cointegrates in an attempt to establish whether Tn7 transposes via a stable cointegrate pathway. With ColEl::Tn7 as a donor, a cointegrate that was stable in a polrecipient but unstable in a Rec- recipient was detected. However the cointegrate was resolved when transferred into a Rec- background. The resolution products followed classical cointegrate resolution pathway but in addition, recombinant plasmids encoding the colicin gene, tetracycline, and DHFR resistance markers were recovered as well as pHH507. The presence of tetracycline resistance amongst the recombinant products suggests that a transposon carrying a -tet gene may be present on pHH507, and this may have interfered with the Tn7 transposition assay system. The putative -tet transposon does not resemble Tn10 as judged by restriction analysis.
Transposition of Tn7 from pCPL6::Tn7 to FΔ(0-15) also occurred in the absence of stable cointegrate formation. A very low level of cointegrates (< 10-8/ F transconjugant) was recovered but these were probably the result of mobilisation of pBR322 by F.
These results suggest that Tn7 does not maintain a stable cointegrate pathway analogous to Tn5 and Tn9.
Secondly, the insertional specificity of Tn7 was studied by comparing the target insertion sequences from four different replicons. Sequence data from two replicons, RP4 and R483 were generated from this study. The base composition and the degree of homology between the insertion sites and the ends of Tn7 did not appear to play a significant part in determining specificity. A tetrameric sequence 'GAAG' was found to be present in close proximity to the Tn7 insertion sites and may represent part of the recognition sequence for the Tn7 transposase.
Finally, epidemiological studies in Europe and North America have shown that the incidence of trimethoprim resistance is increasing. This observation was linked to the fact that transposon Tn7 encodes a dihydrofolate reductase resistant to trimethoprim. Therefore sixteen local clinical bacterial isolates resistant to trimethoprim were examined for the nature of the resistance. All isolates carried several plasmids ranging in size from ~1-60 Mdal. However only eight of the isolates had the trimethoprim resistance borne on plasmids. These resistance plasmids were characterised further by the use of three radioactive probes in hybridization experiments. One of the probes came from an internal HindIII fragment of Tn7. It did not hybridize to any of the clinical plasmids. The remaining probes which specified Type II DHFR hybridized to plasmids in five of the clinical isolates. From these results I concluded that Tn7 was not present on the plasmids of these Tp resistant clinical isolates. Therefore Tn7 was unlikely to be implicated in the dissemination of Tp resistance among TpR isolates from the Auckland area at the time this investigation was carried out.