Rüdiger Schmitt

TnMax — a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA) useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.

Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis

New derivatives of the tetracycline-resistance transposon Tn1721 that carry resistances to chloramphenicol, tetracycline, kanamycin and streptomycin are described. These elements are provided on various plasmid vehicles and as chromosomal insertions to extend the range of targets for Tn mutagenesis. Single EcoRI sites at the ends of these transposons proved most useful for physical mapping, for the generation of new EcoRI sites in cloning experiments, for end-labelling and for sequencing of DNA adjacent to an insertion.

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

SummaryDNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72–73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two clements. A TnpR− derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.

The tetracycline resistance transposons Tn1721 and Tn1771 have three 38-base-pair repeats and generate five-base-pair direct repeats

SummaryThe 10.7 kilobase (kb) tetracycline resistance transposons Tn1721 and Tn1771, isolated from disparate sources, are completely homologous on the basis of heteroduplex analyses. Both transposable elements are capable of forming multiple duplications of a 5.3 kb portion encompassing the resistance genes (tet region). A model accounting for both, recA-independent translocation and recA-dependent amplification, postulates two direct and one inverted repeat as essential constituents of the transposons. DNA sequence analyses of Tn1721 and Tn1771 have substantiated this model. They demonstrated three identical 38 base pair repeats identically in both transposons dividing them into a “minor transposon” and a tet region. Identical sequences of at least 87 base pairs providing recombination “hot spots” for gene duplication have been found at the ends of the repetitious tet region. Translocation of Tn1721 and Tn1771 generates five base pair direct repeats at the respective sites of insertion. On the basis of the heteroduplex molecules and sequences analyzed the two transposons are identical.

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

SummaryR 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 λ. The resulting hybrid phage λ tet contains the 7.2 Mdal-insertion to the right of the attachment site, but not contiguous with it indicating translocation of the element by non-homologous recombination. In addition, λ 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 orginal plasmid pRSD1 to amplify the 3.55 Mdal-tet region.nIt 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.

Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721

Abstract The transposons Tn21, Tn501, and Tn1721 are related to Tn3. Transposition-deficient mutants (tnpA) of these elements were used to test for complementation of transpostion. Transposition of tnpA mutants of Tn501 and Tn1721 was restored by the presence in trans of Tn21, Tn501, and Tn1721, but transposition of a tnpA mutant of Tn21 was restored in trans only by Tn21 itself. Tn3 did not complement transposition of Tn21, Tn501, or Tn1721, and these elements did not complement transposition of Tn3.

A transposable promoter and transposable promoter probes derived from Tn1721

We describe two types of new Tn1721-derivatives capable of random insertion and of generating transcriptional fusions at the site of insertion: transposable promoters (Tn1735) carrying a strong, inducible ptac promoter that turns on adjacent (cryptic) genes; and transposable promoter probes (Tn1736, Tn1737) carrying promoterless genes coding for chloramphenicol acetyl transferase or beta-galactosidase, and used to accurately determine the expression of external promoters. These elements are available with four different selectable resistance markers and on conjugative, temperature-sensitive and multicopy plasmid vehicles. Experiments are described that demonstrate the advantage of random insertions for expressing various genes and for studying gene regulation.

Replicon fusion mediated by a single-ended derivative of transposon Tn1721

SummaryTn1722Δ1K, a derivative of transposon Tn1721 lacking one terminal inverted repeat (IR) and conferring kanamycin resistance, promotes transposition of the resistance marker to a target replicon at about 100-fold lower frequency than the wild-type element. A study involving restriction analysis of 16 independent Tn1722‡1K-mediated events led to the following results: (i) Tn1722‡1K mediates fusions of the donor (pRU506) and target (RSF1010) replicons; the fused entities are non-permuted. (ii) Tn1722Δ1K promotes insertions of donor DNA at many different sites in the target replicon. (iii) The analyzed fusion plasmids contain the entire target and various lengths of donor DNA. Eleven products contain the entire donor plasmid plus a duplication of the IR (class A), whereas five products contain only portions adjacent to the single IR (class B). (iv) In each case the two replicons are joined at (or very close to) the single IR. The second junction is located shortly beyond the duplicated IR in class A and at different sites within the donor plasmid in class B. These results are interpreted in terms of asymmetric replicative transposition.

Transposon Tn1721: site-specific recombination generates deletions and inversions.

SummaryTransposon Tn1721 contains genes for transposase (tnpA), resolvase (tnpR) and a resolution site (res). The closely linked loci were localized within a 3.8 kb region their order being res-tnpR-tnpA with res at the translational start of tnpR. Genes tnpR and tnpA have identical transcriptional polarity but independent promoters. The tnpR promoter had 40% of lac promoter efficiency its activity being autoregulated by binding of resolvase to res, as shown by fusion to the galactokinase gene. The weak tnpA promoter was only detectable in the transposase-mediated transposition reaction. Resolvase-catalyzed, site-specific recombination was analyzed in hybrid plasmids with either direct or inverted repeats of res. The rate of the reaction was dependent on the relative orientation of the two sites and on the provision of tnpR in cis or in trans. Direct repeats were rapidly resolved leading to deletions of intervening DNA, if the tnpR gene was provided in cis, but required approximately 60 generations for completion of the reaction, if tnpR was located on a second plasmid (in trans). The reaction involving inverted repeats of res (leading to inversion of intervening DNA) was only detectable if tnpR was furnished in cis. After 50 generations about 10% of plasmid DNA showed the inversion. The reaction was reversible.

Resolution of a hybrid cointegrate between transposons Tn501 and Tn1721 defines the recombination site

SummaryThe related transposons Tn501 and Tn1721 have a 3.8 kb region in common that contains two genes (tnpA and tnpR) and a resolution site (res) required for transposition. Resolvase, the product of tnpR, catalyses site-specific recombination at res, a 186 base pair (bp) sequence located adjacent to tnpR at one end of the homology region. We describe here identification of the crossover site within res. It involved the construction of a plasmid containing copies of res (Tn501) and res (Tn1721) in direct orientation and tnpR-mediated intramolecular recombination between the two homologous (but non-identical) sites. The resulting hybrid contained Tn501 and Tn1721 fused at the crossover point. DNA sequence analysis of the recombinant indicates that recombination occurs in an 11 bp region of exact homology between Tn501 and Tn1721. The recombination site lies 161–172 bp upstream of tnpR at the transition from homology to non-homology between Tn501 and Tn1721 suggesting that site-specific recombination may have played a role in the evolution of these elements.