Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K

Abstract

Transformation experiments showed that spontaneous deletions which result in loss of streptomvcin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10 4 in plasnid molecules of R6K. Similar deletions were thus readily selected by conjuLgal transfer of R6K, and their appearance was dependent upon lrecAactivity in either donor or recipient host. The deoxvribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA' strains.In four plasmids examined after transfer to a lrecA host, an inverted repeat of the preexisting TnA element was shown to have been inseited at a similar location and was in two instances associated with deletions which extended from the same TnA terminus and in the same direction as those described above. The deletions are ascribed to the result of recA--dependent recombination between direct repeats of TnA.R6K is a 26-megadalton plasmid that determines resistance to anmpicillin (Ap) and streptomvcin (Sm). Although it is a conjugative plasnmid, it has the unusual property foisuch plasmids that it is relaxed, or multicopv in its replication, under normal conditions, being present in approximately 10 copies per chromosonmal equivalent (20).The tiansfer efficiency of R6K is low compared with derepressed fertility plasmids such as F, so that only about 10 transconjugant (R6K+) cells are produced after overnight mating of a 1:1 mixture of 10' donor and recipient cells (20). Fertility repression may be inferred from the findings of Pinnev and Smith (33), who showed that when R6K and the N-group plasmid R46 coexist in a cell, R6K inhibits both R46 transfer and plaque formation by the bacteriophage IKe, which specifically infects cells carrying N-group plasmids.Ap riesistance of H6K is mediated by the TEM-2 /l-lactamase enzyme (2 7), and in fact R6K was isolated from Escherichia coli TEM, in which this enzyme was first characterized, the plasmid at that time being termed RTf:N (10). The transposable element TnA, which determines Ap resistance through TEM, was later shown to be located on R6K (16). (TnA has been termed alternativelv Tn], Tn2, or Tn3 [3] depending on the plasmid of its oi-igin. However, since the TnA sequence on R6K was not derived by transposition from any of the above, and since this sequence is not transposed to other DNA molecules in these experiments, we will retain the generic term TnA.) The Ap ti-ansposon, Tn3, has been shown to transpose to plasmids such as pSC101 and RSF1010 (21,29,38) at a number of clustered, nonrandom sites, and it is reported that insertion occurs in either orientation with about equal frequency and without influence of the lrecA gene of E. (c-l (21, 29, 38).Deletions resulting from the activity of insert...

Intramolecular transposition and inversion in plasmid R6K

“…Chemicals and enzymes. In addition to those previously cited (21), the biochemicals used included: ATP (Sigma Chemical Co.); ampicillin trihydrate (Beecham Laboratories); bromophenol blue (Sigma); chloramphenicol (B grade, Calbiochem); dithiothreitol (Sigma); disodium EDTA and hydroxylamine sulfate (Eastman Kodak); 2-hydroxyethyl mercaptan (2-mercaptoethanol) and kanamycin (Sigma); methylene blue (Merck & Co., Inc.); mitomycin C (from Strepto-TABLE 1. Bacterial strainsa Genotype thr leu thi lac tonA supE44 (A-) (F-) his argG metB leu rpsL thy xyl lacYpolA (Ts) met bfe (A-) (F-) (R6K) thr leu thi lac thy tonA deoC rpsL (A-) (ColEl) C600 A trpE5 recA RC656 (R6K) RC62(pSJC102) RC656(pSJC102) RC656 cys(ColE2) ColE2 derivative of LT2 a Gene symbols used in this table and throughout the text are those recommended by Bachmann et al (3) and Novick et al (36).…”

“…R6K and the Tn3-carrying plasmid rSF1030 show an approximately 4-kb homology, inferred to be the ampicillin transposon (19). Previous experiments in this laboratory have shown that insertions of a duplicate Tn2660 element into R6K are found after selection of a recA + R6K host strain for increased resistance to ampicillin (21). These insertions appeared to be unstable in recA+ host strains and were frequently accompanied by deletions, but could be stabilized after transfer to a recA host.…”

“…These insertions appeared to be unstable in recA+ host strains and were frequently accompanied by deletions, but could be stabilized after transfer to a recA host. A model was proposed in which in a recA host, transposition of the duplicate Tn2660 would occur into R6K with either orientation, leading to a direct or an inverse repeat with the resident Tn2660 (21). This paper reports the results of experiments to test this model, which led to the finding that all insertions ofa duplicate Tn2660 occurred as inverse repeats to the resident Tn2660, and were concluded to be due to transposition by a predominantly intramolecular event, accompanied in some instances, but not others, by an inversion of the DNA between the two transposons.…”

“…Media. Media used included those previously cited (21) and, in addition, Hi medium [100 mM KH2PO4-K2HPO4, 15 mM (NH4)2504, 1.8,uM FeSO4, 0.5% glucose; pH 7] (25) and L-broth (32).…”

“…Buffers. Buffers used were as cited previously (21) and also included: TEN (20 mM Tris-hydrochloride, 1 mM disodium EDTA, 20 mM NaCl; pH 8.0) (8); cleared lysate buffer (50 mM Tris-hydrochloride, 62.5 mM disodium EDTA, 0.2% Tnton X-100) (31), and ligation buffer (66 mM Tris, 100 uM disodium EDTA; pH 7.6) (44). Genetic techniques.…”