IS50-mediated inverse transposition: specificity and precision

The IS50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn5, can move in unison carrying with them any interstitial DNA segment. In consequence, DNA molecules such as a lambda::Tn5 phage genome are composed of two overlapping transposons - the kan segment bracketed by IS50 elements (Tn5), and lambda bracketed by IS50 elements. During direct transposition, mediated by IS50 "O" (outside) ends, the kan gene is moved and the lambda vector is left behind. During inverse transposition, mediated by the "I" (inside) ends of the IS50 elements, the lambda vector segment is moved and the kan gene is left behind. Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982). We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a lambda::Tn5 phage into the amp and tet genes of plasmid pBR322. Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively. Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn5 (by direct transposition). The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS50 junctions, and in eleven others by sequencing one junction. The sequence of the "I" end of IS50 was preserved and 9-bp target sequence duplications were present in every case analyzed. GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites. Our data indicate that transposition mediated by pairs of "I" ends is similar in its specificity and precision to the more frequent transposition mediated by IS50 "O" ends.     

Characterisation of Tn1721, a new transposon containing tetracycline resistance genes capable of amplification.

R plasmid pRSD1 contains tetracycline resistance (tet) genes in a 3.55 Mdal-region capable of amplification by forming tandem repeats (Mattes, Burkardt and Schmitt, Molec. gen. Genet., 1979). The repetitious tet element is itself part of a 7.2 Mdal-transposon, named Tn1721, as demonstrated by the following criteria; (i) Tn1721 has been translocated to phage lambda. The resulting hybrid phage lambda tet contains the 7.2 Mdal-insertion to the right of the attachment site, but not continguous with it indicating translocation of the element by non-homologous recombination. In addition, lambda tet has sustained a 3.4 Mdal-deletion adjacent to the insertion. (ii) Further transposition of Tn1721 to the 21.5 Mdal-plasmid R388 resulted in R388::Tn1721 derivatives, two of which were characterised. They contain Tn1721 inserted into different sites but in the same orientation as shown by restriction and heteroduplex analyses. These translocation of Tn1721 were not accompanied by deletions of DNA. (iii) The insertion plasmid pRSD102(R388::Tn1721) has conserved the capacity of the original plasmid pRSD1 to amplify the 3.55 Mdal-tet region. It has been concluded that Tn1721 constitutes a novel transposon encompassing a tet region capable of selective amplification. The model proposed for Tn1721 contains three short repeats. Two direct repeats, flanking the 3.55 Mdal tet region, provide sequence homology for amplification. The third repeat (located distally to tet) is inverted and provides the basis for transposition of the 7.2 Mdal-element.     

Polarity of Tn5 insertion mutations in Escherichia coli.

We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary.     

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.     

Regional preference of insertion of Tn501 and Tn802 into RP1 and its derivatives

Summary The sites of insertion of Tn501 into RP1 and into derivatives of this plasmid that either lack the Tn801 (TnA) element or contain it in a different location have been determined. Similarly, the sites of insertion of Tn802 into a derivative of RP1 that lacks the Tn801 element and into recombinants of this plasmid with Tn501 were determined. Hot spots for insertion were observed with both transposons; but it is clear that a particular DNA sequence is not sufficient to define a hot spot, since a particular region does contain many insertions when present in one plasmid but does not do so when part of another.     

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000.

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the HindIII site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of Tn1000 into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of Tn1000 insertions in the "plus" or gamma delta orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the Tn1000 insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter. The hutIG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of Tn1000 insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.     

Isolation of Escherichia coli K12 mutants with deficient in precise excision of transposons

Plasmid pNM1, the derivative of R100.1, has been constructed by insertion of transposon Tn5 into structural tet genet (Tn10) of the parental plasmid. The frequency of precise excision of Tn5 from plasmidic genome is 10(-5). The high frequency of precise excision obtained in this system permits one, to use it for isolation of mutants having low frequencies of precise excision. Two mutants were isolated in which the frequencies of precise excision of Tn5 were decreased for two orders. The pex1 and pex2 mutations responsible for the effect decrease the precise excision of Tn5 from R100.1 as well as from RP4 genomes.