Tn5099, a xylE promoter probe transposon for Streptomyces spp.

Tn5099, a promoter probe transposon for Streptomyces spp., was constructed by inserting a promoterless xylE gene and a hygromycin resistance gene into IS493. Tn5099 transposed into different sites in the Streptomyces griseofuscus genome, and the xylE reporter gene was expressed in some of the transposition mutants. Strains containing Tn5099 insertions that gave regulated expression of the xylE gene were identified.     

Establishment of Tn5096-based transposon mutagenesis in Gordonia polyisoprenivorans

The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an alpha-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.     

Site-specific Tn7 transposition into the human genome

The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy.     

Structure and stability of transposon 5-mediated cointegrates

We have determined the structure of a set of independently derived, Tn5-mediated cointegrates and examined the stability of several examples. A variety of cointegrate structures was found, including those mediated by the entire compound transposon, and those mediated by a single flanking IS50 element, which was always IS50-R, and never IS50-L. IS50-R but not IS50-L is reported to code for a protein(s) required for transposition. This finding confirms that IS50-L is relatively inactive and suggests that the active transposition protein(s) acts largely in cis on IS50-R. Another class of cointegrate was created by inverse transposition of Tn5 (using the inside ends of the flanking elements). In addition, we found an unexpectedly large set of cointegrates, in which the joint between the two plasmids was not adjacent to the transposon. All cointegrates analysed were found to be stable. This suggests that Tn5, unlike the transposon Tn3, does not transpose via an obligate cointegrate intermediate. This finding is compared to previous results with Tn5 and Tn9, and is discussed in terms of current models of transposition.     

Transposition of Tn5096 and other IS493 derivatives in Streptomyces griseofuscus.

Tn5096 was constructed by inserting an apramycin resistance gene, aac(3)IV, into IS493 from Streptomyces lividans. By using conventional and pulsed-field gel electrophoresis, Tn5096 and related transposons were shown to insert into many different locations in the Streptomyces griseofuscus chromosome and in two linear plasmids. On insertion into the target site CANTg, 3 bp appeared to be duplicated. Independent transpositions were obtained by delivery of the transposon from a temperature-sensitive plasmid. The frequency of auxotrophy among cultures containing transpositions was about 0.2%.     

Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium

A transposon Tn10 vehicle was developed using a self transmissible (Tra⁺) plasmid pRK2013 having narrow host range ori of replication (ColEl). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli into various rhizobia. The ColEl replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause independent insertion mutations in rhizobia by Tn10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 10⁵ folds higher than the spontaneous Tet^R mutants. Reversion frequency of these mutants was less than 10^?8 indicating adequate stability of Tn10 mutations.     

Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans

The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.     

Structure and stability of Tn9-mediated cointegrates. Evidence for two pathways of transposition

We have measured the frequency of Tn9 transposition and cointegrate formation in several different ways and have examined the stability of the cointegrates. We have also physically analyzed the structure of 40 independently derived cointegrate molecules. We present evidence here that Tn9, unlike the transposable element Tn3, does not transpose via an obligate cointegrate intermediate. We suggest that transposition of Tn9 leads to two, mutually exclusive, end-products: either direct insertion of the element into a recipient replicon (transposition), or fusion between donor and recipient replicons (cointegrate formation). This conclusion is based on our observations that, while Tn9-mediated cointegrates are very stable, they are formed at a rate lower than the transposition frequency. This finding is discussed in terms of current models for transposition.We also present evidence that clearly demonstrates the compound nature of Tn9. We find that the individual flanking IS1 elements are more active than the entire Tn9 transposon in cointegrate formation. In addition, we find that one IS1 element that is proximal to the cam gene promoter, is more active than the other, and suggest that the difference in activity might be due to differences in nucleotide sequence at their extremities.     

Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.     

Transposition and transduction of plasmid DNA inStreptomyces spp.

Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.     

Site-specificity of the chromosomal insertion of Staphylococcus aureus transposon Tn554

Analysis of 15 strains of Staphylococcus aureus containing the transposon Tn554 by a technique (Southern blotting) involving the hybridization of radioactively labeled Tn554 DNA to restriction endonuclease-digested, gel-electrophoresed, high molecular weight S. aureus DNA, revealed that this transposon was present in the same site in all 15 strains examined. One of these strains contained additional Tn554-specific sequences. Tn554, therefore, seems to be highly site-specific, showing the same blotting pattern among natural isolates and a series of strains constructed by transposition under a variety of conditions. The possible role of phage φ11 in Tn554 intercell transfer is discussed, as are possible mechanisms that could account for this element's site-specificity.     

Cloning of a lac+, bamHI fragment into transposon Tn3 and transposition of the Tn3[lac] element

By use of recombinant DNA techniques, we have inserted the lac⁺ operon into a transposon (Tn3). We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac⁺ phenotype. Our results showed that transposition of the Tn3[lac⁺] element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.     

R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element.A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the Rprime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates our mutagenesis scheme. Mutants arising from Tn7 insertions outside of the genetic map have been isolated. Light harvesting II mutations have been isolated; one mutant lacks only the 14,000 M_r, polypeptide.     

Transposon Tagging Using Ty Elements in Yeast

We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.     

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.     

Architecture of the Tn7 Posttransposition Complex: An Elaborate Nucleoprotein Structure

Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein-protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7∷Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3′ end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.