Summary
Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli β-galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage ϕ M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac+ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.
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Abbreviations
- bp:
-
base pairs
- Nm:
-
neomycin
- Km:
-
kanamycin
- Sm:
-
streptomycin
- Sp:
-
spectinomycin
- Gm:
-
gentamycin
- Tc:
-
tetracycline
- Tp:
-
trimethoprim
- Ot:
-
oxytetracycline
- Rf:
-
rifampicin
- Xgal:
-
5-bromo-4-chloro-3-indolyl-β-d-galactoside
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Communicated by J. Schell
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De Vos, G.F., Walker, G.C. & Signer, E.R. Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives. Molec Gen Genet 204, 485–491 (1986). https://doi.org/10.1007/BF00331029
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DOI: https://doi.org/10.1007/BF00331029