Characterisation of trimethoprim resistance transposons and their gene products

Abstract

A faecal enterobacterial strain, P-20, isolated from pigs, was shown to owe its trimethoprim resistance to two different plasmid/transposon mediated resistance genes. One gene, mobilised by RP4, was located on a 4 - 6 kb transposon designated Tn4135, and resulted in the mediation of transferable high level trimethoprim resistance of greater than 1000 ug/ml. Biochemical investigation of the transposon gene product revealed a close resemblence between the trimethoprim resistant dihydrofolate reductase (DHFR) of Escherichia coli J62(RP4::Tn4135) and that of the type I plasmid enzyme, encoded by RP4::Tn7_. Despite the differences in transposon size, this marked similarity between the two DHFRs suggests a similar evolutionary origin for the two transposons and reiterates the potential of trimethoprim resistance transfer between animal and human resevoirs

Detailed biochemical and genetic studies indicated that the second trimethoprim resistance gene of the pig isolate, P-20, mobilised by Sa-1 (Sa-1::Tn4135ORI), bore very little similarity with any previously isolated DHFR. The specific activity of the enzyme was 10 fold lower than that of the type I-like enzyme encoded by RP4::Tn4135, and this, coupled with differences in enzymic properties and the failure to hybridize with type I or type II gene probes, suggests the presence of a new enzyme - a type VI - of distinct evolutionary origin.

The incompatibility group W plasmid, Sa, was investigated for its role as a stable recipient for amplifiication studies of Tn4135, but molecular weight determination, resistance testing and restriction enzyme analysis revealed that this plasmid was not as stable as expected. This plasmid appeared in two forms, Sa-1 and Sa-2; the former being 15 kb smaller, which resulted from a deletion of DNA encoding the chloramphenicol resistance gene. This instability was further reflected in the size variations of Sa plasmid DNA harbouring trimethoprim resistance transposons. Examination of transconjugants from the transfer of Tn4135 from RP4 to Sa-1, in contrast with transfer to Sa-2, indicated that this transposon could transfer in an aberrant fashion, resulting in the formation of an enlarged plasmid species (Sa-1::Tn4135a). The mechanism(s) behind the generation of such a large species were obscure, but appear to involve a combination of multiple transposition, gene amplification, replicon fusion and transposon-mediated transfer of plasmid DNA. Examination of Tn7 transposition to Sa-2 indicated that this transposon was also capable of generating aberrant forms, and reiterated the similarity between the RP4::Tn7_ and RP4::Tn4135 encoded genes.