Characterization of in vitro constructed IS30-flanked transposons

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.     

Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae.

The numbers of chromosomal copies of the insertion sequence IS1 in strains of Salmonella typhimurium (0 to 8 copies), Shigella sonnei (56 copies), and Shigella flexneri (41 copies) isolated in Mexico City, Mexico, were similar to those reported for these genera isolated in other countries. Of the 11 Shigella strains studied, all carried several small plasmids; however, in only one of these strains did a small plasmid contain IS1, IS1 recombination, cointegrate formation mediated by IS1 or by the IS1-flanked transposon Tn9, and transposition of Tn9 occurred at a higher frequency in S. typhimurium than in either Escherichia coli or S. sonnei strains. The frequencies of IS1 recombination in S. typhimurium strains containing either zero or eight copies of IS1 were similar.     

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.     

Molecular genetic analysis of the Tn5041 transposition system

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).     

Multiple copies of IS10 in the Enterobacter cloacae MD36 chromosome.

Repetitive sequences were isolated and characterized as double-stranded DNA fragments by treatment with S1 nuclease after denaturation and renaturation of the total DNA of Enterobacter cloacae MD36. One repetitive sequence was identical to the nucleotide sequence of IS10-right (IS10R), which is the active element in the plasmid-associated transposon Tn10. Unexpectedly, 15 copies of IS10R were found in the chromosomal DNA of E. cloacae MD36. One copy of the central region of Tn10 was found in the total DNA of E. cloacae MD36. IS10Rs in restriction fragments isolated from the E. cloacae MD36 total DNA showed 9-bp duplications adjacent to the terminal sequences that are characteristic of Tn10 transposition. This result suggests that many copies of IS10R in E. cloacae MD36 are due to transposition of IS10R alone, not due to transposition of Tn10 or to DNA rearrangement. I also found nine copies of IS10 in Shigella sonnei HH109, two and four copies in two different natural isolates of Escherichia coli, and two copies in E. coli K-12 strain JM109 from the 60 bacterial strains that were examined. All dam sites in the IS10s in E. cloacae MD36 and S. sonnei HH109 were methylated. Tn10 and IS10 transpose by a mechanism in which the element is excised from the donor site and inserted into the new target site without significant replication of the transposing segment; thus, the copy numbers of the elements in the cell are thought to be unchanged in most circumstances. Accumulation of IS10 copies in E. cloacae MD36 has interesting evolutionary implications.     

The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum

IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.     

Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.     

The effect of host-encoded nucleoid proteins on transposition: H-NS influences targeting of both IS903 and Tn10

Nucleoid proteins are small, abundant, DNA-binding proteins that profoundly affect the local and global structure of the chromosome, and play a major role in gene regulation. Although several of these proteins have been shown to enhance assembly of transpososomes before initiating transposition, no systematic survey has been carried out examining the in vivo role(s) of these proteins in transposition. We have examined the requirement of the six most abundant nucleoid proteins in transposition for three different transposons, IS903, Tn10 and Tn552. Most notably, H-NS was required for efficient transposition of all three elements in a papillation assay, suggesting a general role for H-NS in bacterial transposition. Further studies indicated that H-NS was exerting its effect on target capture. Targeting preferences for IS903 into the Escherichia coli chromosome were dramatically altered in the absence of H-NS. In addition, the alterations observed in the IS903 target profile emphasized the important role that H-NS plays in chromosome organization. A defect in target capture was also inferred for Tn10, as an excised transposon fragment, a precursor to target capture, accumulated in in vivo induction assays. Furthermore, a transposase mutant that is known to increase target DNA bending and to relax target specificity eliminated this block to target capture. Together, these results imply a role for H-NS in target capture, either by providing regions of DNA more accessible to transposition or by stabilizing transpososome binding to captured targets immediately before strand transfer.     

Involvement of E. coli dcm methylase in Tn3 transposition

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.     

Importance of illegitimate recombination and transposition in IS30-associated excision events.

In the present study we report on the excision of IS30 elements and IS30-derived composite transposons. Frequent loss of IS30 was observed during dissolution of dimeric IS30 structures, containing IR-IR junctions, leading to resealed donor molecules. In contrast, unambiguous transpositional excision resulting in resealed remainder products could not be identified in the case of a monomeric element. The bias in the excision of monomeric and dimeric IS30 structures indicates a difference in the molecular mechanism of transposition of IS30 monomers and dimers. Sequence data on the rarely detected plasmids missing full IS or Tn copies rather suggest that all products were derived from illegitimate recombination. The reaction occurred between short homologies and was independent of the transposase activity. Similar IS30 excision events accompanied by multiple plasmid or genome rearrangements were detected in Pseudomonas putida and Rhizobium meliloti, yielding stable replicons that retained the selective marker gene of the transposon. We provide evidence that both transposition and illegitimate recombination can contribute to the stabilization of replicons through the elimination of IS elements, which emphasizes the evolutionary significance of these events.     

Tn10 transposition and circle formation in vitro

We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.     

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.     

Low target site specificity of an IS6100-based mini-transposon, Tn1792, developed for transposon mutagenesis of antibiotic-producing Streptomyces

To improve transposon mutagenesis of antibiotic-producing Streptomyces, a mini-transposon, Tn1792, was constructed, based on IS6100, originally isolated from Mycobacterium fortuitum. Easily manageable transposition assays were developed to demonstrate inducible transposition of Tn1792 into the Streptomyces genome from a temperature-sensitive delivery plasmid. Introduction of the selectable aac1 gene between the inverted repeats in Tn1792 allowed for both reliable identification of transposition events in Streptomyces, and also subsequent cloning of transposon-tagged sequences in Escherichia coli. This enabled the target site specificity of Tn1792 to be determined at nucleotide resolution, revealing no significant shared homology between different target sites. Consequently, Tn1792 is well suited for random mutagenesis of Streptomyces.     

Target specificity of insertion element IS30

The Escherichia coli resident mobile element IS 30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS 30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS 30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS 30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS 30 -like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS 3 , IS 6 and IS 21 , too.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture.

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in ''divalent metal ion-dependent'' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.