Molec. gen. Genet. 180, 123-127 (1980) © by Springer-Verlag 1980 Isolation of an IS/Flanked Kanamycin Resistance Transposon from Rldrdl9 Michel Clerget, Michael Chandler, and Lucien Caro D6partement de Biologic moRculaire, Universit6 de Gen~ve, CH-1211 Gen~ve 4, Switzerland Summary. We have isolated and identified an IS/- flanked transposon from the plasmid Rldrdl9. This transposon specifies resistance to kanamycin and is 10.4 kb long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, ISl-flanked transposon Tn9. We have named it Tn2350. Introduction The multiple antibiotic resistance plasmid Rldrdl9 specifies resistance to kanamycin/neomycin(Km/Nm), ampicillin (Ap), streptomycin (Sin), sulphonamides (Su), fusidic acid (Fu), and chloramphenicol (Cm). These genes are clustered in a single region of the plasmid, the r-determinant (r-det), and are flanked by two directly repeated copies of the insertion ele- ment IS1 (Ptashne and Cohen 1975; Hu et al. 1975). Rldrdl9 gives rise, fairly frequently, to variants which have lost either Km r or all of the other r-det resistance genes (Meynell and Cooke 1969; Kopecko and Cohen 1975; Molin et al. 1979). We have recently determined that, while the closely related plasmid R100.1 carries two copies of IS/ flanking its r-det, Rldrdl9 carries a third copy located within the r-det (Clerget, Chandler and Caro, in preparation). The presence of this third copy of IS/, separating the Km r determinant from the remaining r-det resistance genes, provides an explanation for the appearance of Rldrdl9 deletion derivatives: they could arise by recombination between two of the three directly re- peated copies of IS/, in a manner similar to that of r-det excision from R100.1 (Chandler et al. 1977). The r-det of R100.1 can behave as a transposon and insert itself into the bacteriophage P1 at very low frequencies (Arber et al. 1978). The Pl : :r-det de- rivatives undergo frequent deletions to generate smaller elements, flanked by directly repeated copies of IS1, which transpose at higher frequencies. The transposon Tn9 is a representative of this class (Got- tesman and Rosner 1975). Since the Km r determinant of Rldrdl9 is flanked by two copies of IS/, it seemed probable that it too might undergo transposition. In this communication, we present evidence that a 10.4 kb region of Rldrdl9 carrying the Km r deter- minant can be transposed to a 2 bacteriophage and thence to the RTF unit of R100.1. The results suggest that transposition of the Km r transposon, which we call Tn2350, is mediated by two directly repeated flanking copies of IS/. Materials and Methods Bacteria and Bacteriophage The bacterial strains employed in this study are described in Ta- ble i. Bacteriophage 2b515b519nin5xis6cI857S7 (2bbnin) was from the collection of G. Kellenberger-Gujer. ?kle dia Bacterial cultures were grown in L broth (Lennox 1955) supple- mented with 10 gg/ml thymine. Lambda Km ~ transductants were isolated on L agar plates supplemented with 20 gg/ml kanamycin. Selection of LC799 exconjugants carring RTF and RTF::Tn2350 was made on L agar, supplemented with 35 ~tg/ml nalidixic acid 25 gg/ml tetracycline, and 20 gg/ml kanamycin. Table 1. Escherichia coli K12 strains Strain Description C600 HB101 LC799 LC607 thr leu thi lacy pro leu thi lacy st/rKm ~ endo(-recA - (Bolivar et al. 1977) leu thr lac thi NaV PIr 2 r purE trp lys thi metE proC leu lac xyl ara T1r T5 r T6 r st/ 0026-8925/80/0180/0123/$01.00