Plasmids containing insertion elements are potential transposons ( insertion sequence I / recombination in vivo / nucleotide sequence determination / direct duplications )

We studied in vivo recombination between the plasmid pHSl, a temperature-sensitive replication mutant carrying tetracycline resistance, and pSMl, a small plasmid carrying one copy of the insertion element IS1. Recombinant plasmids were found by selection for tetracycline resistance at 420C. Their formation was independent of recA function. Analysis of the physical structures of various recombinant DNA molecules with electron microscopy and restriction endonucleases revealed that pSMl was integrated at its ISI into numerous sites on pHSl, giving rise to a duplication of IS1 in the same orientation at both junctions. Nucleotide sequence analysis of recombinant plasmids and their parental p asmid DNA revealed that nine nucleotides at a target site were duplicated at the junction of each IS1. This phenomenon implies that plasmids containing a translocatable DNA element can be potential transposons. Recombination between two different genomes independent of recA function is an event frequently seen in vivo in the Escherichia coli system. The integration of phages such as X and 080 into a host chromosome is a typical example of such recombination, which generally occurs at specific sites on a phage and host chromosome and is catalyzed by an integration enzyme of the phages (1-3). Several lines of evidence indicate that there is another type of recombination, which is mediated by translocatable DNA elements responsible for insertion and transposition of particular DNA segments. The integration of the plasmid F into a host chromosome may be one example. Heteroduplex studies demonstrated that translocatable DNA elements such as insertion sequence 2 (IS2), insertion sequence 3 (IS3), or -y3 sequences appear at the recombination junctions between F and chromosomal DNA sequences (4-7). Gill et al. (8) reported that in a recombination between two plasmids, one of which carries a mutant Tn3, a transposable DNA element responsible for ampicillin resistance, the recombinant had Tn3 sequences at the junctions of the parental plasmids. Genetic analysis of fusion between a bacterial chromosome lysogenized by phage Mu and a defective X-gal demonstrated that Mu, itself a translocatable element, mediated the formation of a cointegrated genome in which Mu genomes sandwiched the X-gal genome (9). In this paper, we report studies on recombination between two different plasmid genomes, one of which carries a copy of an insertion DNA element IS1 (10-12). We developed a system to score cells having recombinant plasmids. Analysis of the recombinant molecules with electron microscopy and restriction enzyme cleavage demonstrated that they were formed by the transposition of the plasmid containing the IS element into the second plasmid such that the IS element appears as a direct repeat at both junctions of the two plasmid sequences. Based on the results obtained by riucleotide sequence analysis, models for the formation of these recombinants will be presented. MATERIALS AND METHODS Bacterial and Plasmid Strains. The bacterial strains used were JE5507 and JE5519, which is a recombination-deficient (recA-) mutant of JE5507; both were provided by K. Nakamura. pHS1 and pSM1, the plasmids (13-15) used in recombination experiments, as well as the recombinants between them, named pMZ, are described in the Results section. Covalently closed circular DNA was isolated-according to the method described by Ohtsubo et al. (16). Bacterial strains carrying both pHSl and pSMl were constructed by cotransformation of pSMl DNA, having no resistance genes, with pHSl DNA, having the tetracycline resistance (Tcr) gene as described (17). Analysis of Frequency of Recombination. Cells containing both plasmids were grown in 5 ml of penassay broth (Difco) at 30°C overnight. Samples (0.1 ml) of a 1:106 dilution were used to inoculate 20 tubes, each containing 5 ml (for recA+) or 10 ml (for recA-) of broth and the tubes were incubated overnight at 30°C. From each tube, 0.1 ml was plated on a 10 Ag/ml tetracycline plate and incubated at 42°C for 24 hr. The frequency of recombination was calculated in units of Tcr bacteria per division cycle as described by Luria and Delbruck (18). Crude Lysis. A simple way to visualize and compare the plasmid DNA in bacterial cells is the crude lysis method. We followed the procedure described by Mickel et al. (14). Molecular Lengths. Lengths of duplex DNA were determined from electron microscope measurements of molecular spreads by the aqueous basic protein-film technique (19). The length of our internal standard, ColEl DNA, was determined from aqueous spreading with phage OX174, which is known to be 5.38 kilobases (kb) long (20). Enzymes. EcoRI was purchased from Miles Laboratories and Pst I, Hha I, Alu I, Hinfl, and Hae II were obtained from New England BioLabs. These enzymes were assayed as recommended by the laboratory from which they were obtained. Hae III was isolated and assayed according to Roberts et al. (21). Gel Electrophoresis and Determination of Nucleotide Sequences. Native duplex DNA fragments were separated by gel electrophoresis on a 13 X 15 X 0.2 cm 0.7% agarose gel or on 4% polyacrylamide slab gels (acrylamide/bisacrylamide = 20:1). DNA bands were visualized by staining with ethidium bromide (0.4 ,g/ml) under short-wavelength UV light. The nucleotide sequence was determined as described by Maxam and Gilbert (22).