Tn5 Transposition Research Articles

Use of the plasmid pRK 2013 as a vehicle for transposition inAzotobacter vinelandii

Plasmid stability inAzotobacter vinelandii has been determined and a way to introduce transposon into these cells using the plasmid pRK 2013 has been devised. Transposition of both Tn3 and Tn10 has been attained.

Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcus faecalis to Staphylococcus aureus and identification of some insertion sites in the staphylococcal chromosome.

The Streptococcus faecalis pheromone-dependent conjugative plasmid pAD1::Tn916 and the membrane filter-dependent conjugative plasmid pPD5::Tn916 were used to introduce Tn916 into Staphylococcus aureus by intergeneric protoplast fusions and intergeneric membrane-filter matings. In recombinants obtained by protoplast fusion where no plasmid DNA could be detected, tetracycline resistance resulted from transposition of Tn916 from pAD1 to the S. aureus chromosome. Transformation analyses showed that S. aureus Tn916 chromosomal insertions occurred near pig, ilv, uraA, tyrB, fus, ala, and the trp operon. DNA hybridization analyses of EcoRI- and HindIII-digested chromosomal DNAs confirmed the diversity of chromosomal sites involved and demonstrated that the inserts were Tn916 insertions rather than integrations of all or part of pAD1::Tn916. Both pAD1::Tn916 and pPD5::Tn916 were transferred to S. aureus by membrane-filter matings. These plasmids remained intact and expressed tetracycline resistance in S. aureus. S. aureus strains carrying pAD1::Tn916, but not a chromosomal insert of Tn916, and any one of several conjugal gentamicin-resistance plasmids lost their ability to serve as conjugal donors of the gentamicin-resistance plasmids.

Transposition of gram-positive transposon Tn916 in Acholeplasma laidlawii and Mycoplasma pulmonis.

Mycoplasma genetics has been limited by a lack of genetic tools such as selectable markers, methods to transfer DNA, and suitable vectors for cloning. Studies were undertaken to examine the potential of using the streptococcal transposon Tn916 as a mycoplasma genetic tool. The Escherichia coli plasmid pAM120, which contains Tn916, was transformed into Acholeplasma laidlawii and Mycoplasma pulmonis. Transposition of Tn916 into the mycoplasma chromosome apparently occurred by an excision-insertion mechanism. This example shows that newly introduced DNA from other bacteria can be successfully expressed in mycoplasma and that Tn916 should serve as a powerful genetic tool for the study of mycoplasmas.

Marking of a Sym plasmid of Rhizobium with Tn7

Transposon Tn7 was inserted into wide host range plasmid pSUP202 and used as a suicide plasmid vehicle for transposon mutagenesis in Rhizobium leguminosarum. Tn7 is transposed with high frequency into the self-transmissible plasmid pJB5JI without affecting the transfer, nodulation and nitrogen fixation functions. Tn7 transposition provides a useful tool for marking symbiotic plasmids.

Analysis of Tn7 transposition

Five gene products required for Tn7 transposition were identified using genetic complementation tests. Four of these (tnsA, tnsB, tnsC and tnsD) are essential for insertion into the attachment site, whereas the fifth (tnsE) is required for transposition to plasmids which lack this site. tnsD is not required for transposition to plasmids lacking the attachment site. This analysis used a chloramphenicol-resistant 'mini' transposon containing Tn7 termini but no complete Tn7 gene product and several compatible expression vectors containing cloned Tn7 fragments. The use of transcriptional and translational fusions allowed the identification of two promoters (P1 and P2) at the righthand end of the transposon and indicated that at least tnsA, tnsB and tnsC are translated in the same direction. Expression from P1 appears to be repressed by tnsB.

Replicative and conservative transposition in bacteria

When a bacterial transposon jumps to a new genomic location, does it replicate itself in the process thus generating two transposon copies, or does it excise itself from the old site and integrate into the new site without duplication? eplicative or conservative: this has been a central question concerning the mechanism of transposition. The lack of any well-characterized in vitro systems for studying transposition (with the exception of the bacteriophage Mu system) has restricted progress. However, thanks to some elegant genetic experiments, the answer now seems much clearer. Replicative Transposition Most mechanistic studies have been performed with members of three common groups of bacterial transposons: (i) the insertion sequences (IS) and their composite transposons; (ii) the Tn3 family of transposons; and (iii) the transposing bacteriophage Mu. The case for a replicative mechanism of transposition was established early and was a strong one. It came on two fronts-from studies of Tn3 and bacteriophage Mu. Transposition of Tn3 and related elements generally occurs by a two-step process (see Grindley, Cell 32, 3-5, 1983; Heffron, in Mobile Genetic Elements, ed. J. Shapiro, Academic Press, 1983, pp. 223-260). First, the donor and target replicons are fused to form a cointegrate molecule, which has a copy of the transposon at each junction of target and donor DNA (Figure 1). A site-specific recombination between the two transposon copies then reduces the cointegrate into its two constituent replicons, regenerating the donor and forming the target with a simple insertion. The two steps involve the consecutive action of two proteins, transposase and resolvase, both encoded by the transposon. The replicative nature of the transposasedependent step is clear from the nature of the cointegrate intermediate; although formed from a target and a donor with a single transposon copy, it contains two copies of the transposon. The replicative nature of Mu transposition was evident from the fact that replication of the phage during a lytic infection was absolutely dependent upon its transposition to many sites in the genome of a single cell. The short segments of random bacterial DNA joined to the ends of Mu in viral particles are a signature of this orgy of transposition. Elegant biochemical studies have firmly established that Mu transposition is generally replicative and, in addition, have elucidated the structure of an intermediate (see below) formed through the action of the Mu transposase (A protein), the B protein, and the E. coli histone-like protein HU (Mizuuchi, Cell 35, 785-794, 1983; Craigie and ireview

Tn10 transposition does not respond to environmental stress

Tn10 transposition does not respond to environmental stress

Evidence for replicative transposition of Tn5 and Tn9

Two basic types of models, conservative and replicative, have been proposed to account for the mechanism of transposition in bacteria. A method was developed to test these models by positive selection of various transposon-promoted events as galactose-resistant colonies from plasmid-containing cells. The results show that recA plays an important role in the transposition of Tn5 and Tn9 in Escherichia coli. All Tn5-promoted events (cointegrates, deletions and transpositions) are suppressed in recA − , and restored in recA + . In the case of Tn9, however, only transpositions (but not cointegrates or deletions) are diminished in recA − . Therefore, the recA function is required for cointegrate formation by Tn5, and for cointegrate resolution by Tn9. Both Tn5 and Tn9 cointegrates segregate transpositions (which can be seen as sectors on indicator plates) in recA + hosts. In recA − , the unresolved Tn9 cointegrates undergo a second round of cointegrate formation to excise plasmids bearing galactose-resistant deletions. In growing cultures, the proportion of cointegrates declines steadily while transpositions increase so that, in late stages, cultures are rich in transpositions and contain few cointegrates. This explains the failure of previous workers to identify cointegrates as essential intermediates in transposition. Hence, with the exception of the recA requirement, the mechanism of transposition of these composite transposons is not very different from simple transposons like Tn3. It is concluded that transposition of Tn5 and Tn9 is normally a replicative process.

Distribution and genetic location of Tn7 in trimethoprim-resistant Escherichia coli.

A series of 178 strains of Escherichia coli, highly resistant to trimethoprim, isolated from hospital patients and patients in the community between 1979 and 1983, was examined for the presence of Tn7 on a plasmid or on the chromosome only, by transposition to RP4 and restriction endonuclease digestion with Hind III. Of the isolates, 57% carried Tn7. Comparison of hospital isolates from 1979 to 1980 and 1982 showed that although the proportions that carried Tn7 were similar (63% and 57%) there had been a significant change in the genetic location of the transposon. The proportion of plasmid-mediated Tn7 had fallen from 62% to 30% with a corresponding rise in Tn7 located exclusively on the chromosome from 38% to 70%. This change may be the result of continuing transposition of Tn7 from plasmids to the bacterial chromosome followed by plasmid loss. The consequent reduction in the mobility of trimethoprim-resistance genes may in turn lead to changes in the incidence of resistance.

Insertion of transposon Tn7 into the Escherichia coli glmS transcriptional terminator.

The transposon Tn7 is unusual as it transposes at high frequencies from episomal elements to a unique site in the Escherichia coli chromosome. This unique site is within a region of dyad symmetry that we have demonstrated to be the transcriptional terminator of the glmS gene which encodes the glutamine amidotransferase, glucosamine synthetase. Transposition of Tn7 abolishes termination of glmS transcription at this site; the transcripts now extend into the left end of Tn7 and terminate at a new site, tm, 127 base pairs from the left end of Tn7. This region of the transposon contains a long open reading frame which encodes a protein sequence that is significantly related to the transposase proteins of the transposable elements IS1 and Tn3. A weak transcript has been identified that emanates from a promoter on the 5' side of this reading frame. This promoter is over-run by glmS transcripts and so it appears that expression of the Tn7 transposase may be regulated by promoter occlusion.

Tn7 transposition: a multigene process. Identification of a regulatory gene product.

Tn7 transposition is abolished by the deletion of a 2.2kb HindIII fragment from the central region of the transposon. Transposition is restored when the fragment is present in trans. When this fragment is present in trans with wild-type Tn7, transposition frequencies are stimulated 10-100-fold. The DNA sequence of this fragment has been determined and found to contain one long open reading frame coding for a protein of molecular weight 61 187. We have visualised this protein using a DNA-directed prokaryotic transcription-translation system. This gene may fill a regulatory role in the mechanism of Tn7 transposition. When present in trans the 2.2kb HindIII fragment alleviates a transcriptional block of promoter activity detected in Tn7.

Transposition of plasmid-borne Tn10 elements does not exhibit simple length-dependence.

The transposition frequencies of Tn10 elements from the bacterial chromosome to an F epitome decrease 40% for every kilobase increase in transposon length. The basis for this relationship is not known. We have now examined complemented transposition of defective Tn10 elements off small multicopy plasmids. We find that length dependence in this situation is either reduced or absent, depending on the specific class of transposition events involved. These observations can be interpreted as evidence against the model that chromosomal length dependence occurs because of decay of a transposition-associated replicative complex. This interpretation is consistent with unrelated experiments suggesting that Tn10 transposition is normally nonreplicative. Alternative explanations of length dependence phenomena are discussed.

Tn7-encoded proteins.

Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1::Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85-a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the left-hand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85-b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85-b and p40 products.

IS10 transposition is regulated by DNA adenine methylation.

We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.

Characterization of the in vivo RNA product of the pOUT promoter of IS10R

We characterized a single RNA species (RNAout1) which was the major in vivo RNA made from pOUT of IS10R. RNAout1 was 70 nucleotides long; its 5' end corresponded exactly to the in vitro start of pOUT transcription. The concentration of RNAout1 was estimated at 5 to 10 molecules per cell containing the single-copy plasmid NR1. RNA sequences from pOUT of IS10L were detected at a much lower (less than one molecule per cell) steady-state concentration and may be preferentially degraded in vivo. We suggest that the low level of the IS10L transcript led to the inability of IS10L sequences to translationally inhibit Tn10 transposition.

Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: Use of plasmid RP4::Tn76 as a suicide vector

Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. Beijerinckii::Tn76 isolates at a high frequency. Nitrogenfixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.

Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob::Tn5 from Escherichia coli

A Tn5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.

Genetic basis of non-transposition of Tn5inPseudomonas aeruginosafollowing mobilisation of RP4 Mob::Tn5fromEscherichia coli

A Tn5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.

Vector insertion mutagenesis of Rhizobium sp. strain ORS571: direct cloning of mutagenized DNA sequences.

When the limited-host-range plasmid pVP2021 carrying Tn5 was mobilized into Rhizobium sp. strain ORS571 and stable acquisition of Tn5 was selected, ORS571 plasmid-genome cointegrates were exclusively obtained; direct Tn5 transposition was never observed. In every case, genomic cointegrates exhibited an additional (third) IS50 element that bordered VP2021 DNA sequences but maintained a single Tn5 element. Genomic cointegrates containing IS50 triplications were stable; neither phenotypic reversion nor resolution was detectable. Auxotrophic mutant strains (vector insertion mutants) were identified at expected frequencies among derivatives carrying ostensibly random genomic pVP2021 insertions; N2 fixation (Nif)-defective vector insertion mutants were observed among these derivatives at a frequency of 10(-3). The presence of integrated pVP2021 in ORS571 nif::VP2021 mutant genomes enabled VP2021 to constitute an endogenous cloning vector. After EcoRI or KpnI digestions, genomic nif::pVP2021 DNA sequences contiguous with integrated pVP2021 were directly cloned as new replicons without addition of an exogenous vector. Recombinant plasmids derived from two such nif::pVP2021 mutants hybridized to previously analyzed ORS571 Nif DNA sequences. Recombinant plasmid DNA and ORS571 Nif region DNA were found to be colinear; pVP2021 insertions could be accurately mapped. pVP2021 insertion-mutagenesis thus allows the direct cloning of ORS571 gene sequences for which mutant phenotypes can be selected or screened.

Genetic analysis of Tn7 transposition.

The purpose of this work was to localize the DNA regions necessary for the transposition of Tn7. Several deletions of Tn7 were constructed by the excision of DNA fragments between restriction sites. The ability of these deleted Tn7s to transpose onto the recipient plasmid RP4 was examined. All the deleted Tn7s isolated in this work had lost their transposing capability. The possibility of complementing them was studied using plasmids containing all or part of Tn7. Two deleted Tn7s could not be complemented by an entire Tn7 indicating that a DNA sequence greater than the 42 bp terminal sequence is needed for recognition of the transposon by a transposition function. Four other deleted Tn7s could be complemented by Tn7. One of these was studied intensively in complementation experiments using different parts of Tn7 to obtain transposition. The results obtained allow us to propose that all genes needed for transposition of Tn7 onto plasmids are contained in a DNA segment of between 6.0 and 7.4 kb. Furthermore, one essential function must be contained in a DNA fragment longer than 2.5 kb on the right-hand end of Tn7. The classification of Tn7 with regard to the other transposable elements is discussed.