Transposon mutagenesis in Desulfovibrio desulfuricans: development of a random mutagenesis tool from Tn7.

The transposons Tn5, Tn7, Tn9, and Tn10 or their derivatives have been examined for transposition in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20. Tn7 inserted with a frequency of 10(-4) to 10(-3) into a unique attachment site that shows strong homology with those sites identified in other gram-negative bacteria. Inactivation of the tnsD gene in Tn7, encoding the function directing insertion into the unique site, yielded a derivative that transposed essentially randomly with a frequency of ca. 10(-6) per donor. Derivatives of Tn5, but not wild-type Tn5, were also found to undergo random transposition at a similar frequency. No evidence was obtained for transposition of Tn9 or Tn10.     

DNA sequence analysis of Tn10 insertions: origin and role of 9 bp flanking repetitions during Tn10 translocation.

The sequences of insertions of the translocatable tetracycline-resistance element Tn10 into the repressor (cl) gene of bacteriophage lambda have been analyzed. Each insertion contains the same discrete set of Tn10 sequences flanked by a direct repetition of a 9 bp cl-gene sequence. The flanking repititions are generated by duplication of information present only in the target DNA molecule rather than by a Campbell-type recombination event between one 9 bp sequence on the target DNA and a second one provided on the incoming element. The repetitions do not contain genetic or structural information important for translocation. A genetically constructed Tn10 insertion which lacks flanking repetitions is fully functional in translocation to a new position. Tn10 insertions cluster at preferred positions along a target DNA (Kleckner et al., 1979). Sequence analysis shows that four independently isolated cl::Tn10 insertions occur at identical positions in the cl gene. We speculate that homology between Tn10 and its target, at some distance from the site of the actual recombination event, could be relevant to the preference of Tn10 for particular insertion sites.     

Construction of a correlated physical and genetic map of the Klebsiella pneumoniae hisDGO region using transposon Tn5 mutagenesis.

Multicopy plasmids containing the hisDG region of Klebsiella pneumoniae were mutagenized with transposon Tn5. The resulting plasmids were examined for their ability to complement hisD and hisG mutations in Escherichia coli. The physical location of Tn5 on each of the hisD::Tn5 and hisG::Tn5 plasmids was determined by restriction endonuclease analysis. By combining the two types of data, a precise correlated physical and genetic map of the K. pneumoniae hisDG region was constructed. Based on this analysis, the minimum sizes of the hisD and the hisG genes were calculated to be 1100 bp and 900 bp, respectively. The hisO(P) region was also identified. The insertional specificity of transposon Tn5 was shown to be very low. One unanticipated result was obtained: Tn5 insertions in the plasmid-borne hisG gene were not polar on hisD.     

Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element.

A procedure is described that selects for the insertion of transposable antibiotic resistance elements in a variety of recipient replicons. The selected translocation procedure, which employs a plasmid having a temperature-sensitive defect in replication as a donor of transposable genetic elements, was used to investigate certain characteristics of the translocation process. Our results indicate that translocation of the Tn3 element from plasmid to plasmid occurs at a 10(3)- to 10(4)-times-higher frequency than from plasmid to chromosome. In both cases, continued accumulation of Tn3 on recipient genomes is prevented by development of an apparent equilibrium when only a small fraction of molecules in the recipient population contain Tn3. An alternative method for estimation of translocation frequency has shown that the translocation process is temperature sensitive and that its frequency is unaffected by the presence of host recA mutation. Insertions of Tn3 onto the 65 X 10(6)-dalton R6-5 plasmid in Escherichia coli are clustered on EcoRI fragments 3 (8 of 23 insertions) and 9 (7 of 23 insertions), which contain 12 and 5%, respectively, of the R6-5 genome. The occurrence of multiple insertions of Tn3 within EcoRI fragment 9, which contains the IS1 element and a terminus of the Tn4 element, is consistent with earlier evidence indicating that terminal deoxyribonucleic acid sequences of already present transposable elements may provide recognition sequences for subsequent illegitimate recombinational events.     

A system for in vivo selection of genomic rearrangements with predetermined endpoints in Escherichia coli using modified Tn10 transposons

Using recombinant DNA techniques, the Tn10-specific tetA gene (coding for tetracycline resistance) has been mutagenized by insertion of a streptomycin-resistance or a kanamycin-resistance gene. The insertions occurred at loci separated by 920 bp. The mutated tetA fragments, respectively designated as Tes (for tetracycline-streptomycin) and Tek (for tetracycline-kanamycin), were subsequently cloned into a phage lambda cIII+cIts857cII+ in replacement of the att lambda region. The two recombinant phages are convenient delivery vehicles which permit the in vivo substitution of the tetA locus of any Tn10 insertion with the Tes or the Tek fragment. The procedure involves two selectable steps: (i) integration of a lambda-Tes (or lambda-Tek) prophage into the Tn10 of interest; (ii) excision of the prophage by a second exchange which leaves the extra resistance gene installed within the Tn10. A major interest of the system is that, once a bacterium carries both Tn10-Tes and Tn10-Tek insertions, a recombination event between the two Tn10 sequences can reconstitute an active tetA gene. This selectable event may be associated with the rearrangement of the sequences surrounding the transposons. This unique property of the "Tes and Tek" system makes it very useful for selection of genomic rearrangements using the Tn10-Tes and Tn10-Tek as predetermined endpoints. The successful isolation of a chromosomal inversion is reported.     

Tn10-derived transposons active in Bacillus subtilis.

Small derivatives of the Escherichia coli transposon Tn10, comprising IS10 ends and a chloramphenicol resistance gene, were introduced in Bacillus subtilis on a thermosensitive plasmid, pE194. In the presence of the Tn10 transposase gene fused to signals functional in B. subtilis, these derivatives transposed with a frequency of 10(-6) per element per generation. They had no highly preferred insertion site or region, as judged by restriction analysis of the chromosomal DNA, and generated auxotrophic and sporulation-deficient mutants with a frequency of about 1%. These results suggest that Tn10 derivatives might be a useful genetic tool in B. subtilis and possibly other gram-positive microorganisms.     

Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

Genetic analysis of attTn7, the transposon Tn7 attachment site in Escherichia coli, using a novel M13-based transduction assay

The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Genetic mapping with Tn5-derived auxotrophs of Caulobacter crescentus.

Chromosomal insertions of Tn5 in Caulobacter crescentus displayed complete stability upon transduction and proved useful in strain building on complex media. RP4-primes constructed in vitro containing C. crescentus genomic sequences in the HindIII site of the kanamycin resistance gene failed to show enhanced or directed chromosome mobilization abilities. One of these kanamycin-sensitive RP4 derivatives, pVS1, was used as a mobilization vector in conjugation experiments on complex media where chromosomal Tn5 transfer to the recipient was selected. pVS1-mediated transfer of Tn5-induced auxotrophic mutations occurred at frequencies of 10(-6) to 10(-8) per donor cell. During conjugation with Tn5-encoded kanamycin resistance as the selected marker, Tn5 remained in its donor-associated locus in 85 to 100% of the transconjugants. A collection of eight temperature-sensitive donor strains bearing Tn5 insertion mutations from various regions of the C. crescentus genetic map were used to provide a rapid means for the determination of the map location of a new mutation. Use of the techniques described in this paper allowed an expansion of the C. crescentus genetic map to include the relative locations of 32 genes.     

Mutational analysis of att554, the target of the site-specific transposon Tn554.

Tn554 is a high-frequency, site-specific transposable element of Staphylococcus aureus which has integrative properties resembling those of temperate bacteriophages. Tn554 inserts at a unique chromosomal location, designated att554. att554 contains a core hexanucleotide sequence, 5'-GATGTA-3' (nucleotides numbered -3 to +3). Most of the time (greater than 99%) insertion occurs immediately 3' to this sequence; the resulting orientation of Tn554 to att554 is designated as the (+) orientation. Infrequent insertions immediately 5' to the core sequence result in the opposite, or (-) orientation. Mutational analysis of a cloned att554 site indicates that deletions extending from the left and ending at -15 or from the right ending between +8 and +12 reduced attachment site efficiency. Plasmids with deletions extending closer to the insertion site, although still retaining the core sequence from -3 to +3, were totally inactive. Tn554 insertions into partially active att554 sites retained normal site- and orientation-specificity with respect to att554, but they frequently contained abnormal sequences at the junction of att554 and the 3' end of Tn554. These data indicate that att554 contains a short nucleotide sequence essential for transposition and flanking sequences that greatly increase the frequency of recombination.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.     

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.