Cointegrate Formation Research Articles

The insE open reading frame of IS1 is not required for formation of cointegrates

The role of the insE open reading frame in transposition of IS1 was reexamined by using an insE nonsense mutation that does not alter the amino acid sequence of InsA inhibitor or InsAB transposase. The mutant was active in all strains tested, showing that insE is not essential for formation of cointegrates.

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn 1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn 1721 and the new derivative, Tn 5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites ( res) of Tn 1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn 5491 into the target sequence is selected by cointegrate formation of Tn 5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method. A 1304-bp sequence was found on this fragment with the features of insertion elements. The element called IS 1372 had 27-bp IR and two potential open reading frames. The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS 3 family. Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS 3 family was identified. Five to seven copies of IS 1372 were found in different strains of S. lividans but none in other Streptomyces species tested

Inducible transposition in Streptomyces lividans of insertion sequence IS6100 from Mycobacterium fortuitum.

Transposition of IS6100, originally isolated as part of the compound transposon Tn610 from Mycobacterium fortuitum, was tested in the related actinomycete Streptomyces lividans. Cointegrate formation was observed, as expected for this IS6-related element, and involved apparent random integration of the temperature-sensitive vector carrying IS6100 and concomitant duplication of the insertion sequence. This establishes that a single copy of the insertion sequence can promote transposition and is a precedent for the functioning of a heterologous transposable element in Streptomyces. Transposition could be induced 100-fold by external transcription emanating from a copy of the thiostrepton-inducible promoter ptipA located outwith the insertion sequence and resulting in overexpression of the transposase gene. Thus, in contrast to other prokaryotic transposable elements, IS6100 appears to have no effective means of protecting itself from external activation.

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn5053) has been determined. Tn5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD, and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn21 and Tn501. The transposition module of Tn5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ, and tniR. Transposition of Tn5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR. The same pathway of transposition is used by Tn402 (Tn5090) which carries the integron of R751. Transposition genes of Tn5053 and Tn402 are interchangeable. Sequence analysis suggests that Tn5053 and Tn402 are representatives of a new family of transposable elements, which fall into a recently recognized super-family of transposons including retroviruses, insertion sequences of the IS3 family, and transposons Tn552 and Tn7. We suggest that the tni genes were involved in the dissemination of integrons.

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event

All members of the IS1248 family residing in the genome of Paracoccus denitrificans have been isolated by using a set of insertion sequence entrapment vectors. The family consists of five closely related members that integrate the entrapment vectors at distinct sites. One of these, IS1248b, was sequenced and, except for a single base change, shown to be identical to the previously isolated IS1248a. Southern analysis of genomic DNA with labeled IS1248 revealed different hybridization patterns for different isolates of P. denitrificans and Thiosphaera pantotropha. No hybridization was observed with DNA from Thiobacillus versutus and more distantly related species. From a comparison of the fingerprints it was shown that one of the members of the IS1248 family found in P. denitrificans DSM413 is absent in strain NCIB8944, although they are catalogued in international strain catalogues as identical strains. Furthermore, strains Pd1222 and Pd1235, both derivatives of P. denitrificans DSM413, were shown to have different patterns of IS1248 hybridizing restriction fragments. In 14 of 18 strains, the entrapment vectors used in this study were incorporated into the genome via IS1248-mediated cointegrate formation. In the other four strains, the entrapment vectors were shown to be integrated through a different mechanism not involving IS1248.

Intermolecular transposition of IS10 causes coupled homologous recombination at the transposition site.

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 PR promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in delta recA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.

On the clustered exchanges of the RecBCD pathway operating on phage lambda.

Lytic cycle crosses of Red- Gam- phage lambda were conducted in rec+ Escherichia coli carrying one or another plasmid with homology to lambda. Lambda x lambda recombinants and lambda x plasmid recombinants were formed by RecBCD-mediated recombination. We showed previously that the act of recombining with a plasmid alters the disposition of selected lambda x lambda exchanges. This work reports that the relationships between the lambda x plasmid and the lambda x lambda exchanges is unaltered by the removal from one lambda parent of the homology shared with the plasmid. This result supports our view that a reciprocal exchange, allowing for cointegrate formation, is associated with but mechanistically separable from a (presumably) nonreciprocal lambda x lambda exchange. The nature of this relationship is independent of lambda's Rap function, which is shown to alter the ratio of cointegrate formation (splices) to marker pick-up (patches) in lambda x plasmid recombination mediated by the RecBCD pathway.

In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces.

Non-transmissible derivatives of the Streptomyces multi-copy plasmid plJ101 were mobilized, by cointegrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coelicolor A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

An Unusual Type of Cointegrate Formation between a Bacteroides Plasmid and the Excised Circular Form of an Integrated Element (NBU1)

Evidence for an unusual type of cointegrate formation was found as the result of analyzing three integration events that fused a mobilization-deficient Bacteroides plasmid (pEG920) with the excised circular form of a nonreplicating Bacteroides element (NBU1), NBU1 is capable of inserting itself into DNA segments, but the cointegrates were the result of invasion of NBU1 by pEG920, not vice versa. The same site on pEG920 was involved in all cases. Sequence analysis of the cointegrates suggested that the integration events may have been the result of a multistep process in which a conjugative transposon was involved.

Transposon mutagenesis of coryneform bacteria.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Structure and putative origin of a plasmid from Staphylococcus hyicus that mediates chloramphenicol and streptomycin resistance

The structure and functional organization of Staphylococcus hyicus plasmid pSCGp3EB that mediates chloramphenicol and streptomycin resistance (CmrSmr) is described and compared with another CmrSmr plasmid, pSCS12, from Staphylococcus sciuri. Both plasmids appeared to be formed by co-integrate formation between plasmids that very closely resemble the chloramphenicol resistance (Cmr) plasmid pC221 and the streptomycin resistance (Smr) plasmid pS194. In addition to the established recombination site B (RSB) in pC221 and pS194, another area suitable for recombination immediately downstream of the cat gene in pC221 and upstream of the str gene in pS194 has been identified. Co-integration at these sites would lead to the structures we have observed in the wild-type CmrSmr plasmids pSCGp3EB and pSCS12.

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac+) to a plasmid-free Lac−L. lactis strain. In each case, some Lac+ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra+), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.

Differential roles of the transposon termini in IS91 transposition.

Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target. After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC). We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli. IRR is absolutely required for transposition. Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient. Furthermore, the GTTC/CTTG target site is also required. The left inverted repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element. In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site. These results are easily accommodated by a rolling-circle replicative transposition mechanism. This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.

Transpositional activity of IS986 in Mycobacterium smegmatis.

The insertion sequence IS986 (also known as IS6110) has been widely used for typing Mycobacterium tuberculosis isolates, due to the extensive multiple polymorphism shown using this probe. Although this polymorphism is presumed to be due to the mobility of IS986, transposition of this element has not previously been demonstrated in the laboratory. Using artificial composite transposons constructed in a vector unable to replicate in mycobacteria, we have succeeded in demonstrating the mobility of IS986 in M. smegmatis, apparently through cointegrate formation and transposition. The apparently random nature of IS986 insertions in M. tuberculosis is advantageous for transposon mutagenesis, although its efficient use will require more effective delivery systems.

Identification of an insertion sequence-like element in a subspecies of Mycobacterium kansasii

Analysis of a genomic DNA clone library of a strain from the genetic subspecies of Mycobacterium kansasii determined the existence of a repetitive insertion sequence-like element. The element is 947 bp long and is present in a minimum of 1 to 11 copies per genome. Similar to insertion sequences, it contains a 3-bp (TAG) direct repeat at its extremities and a transcription promoter-like sequence. In addition, for one of the clones sequenced, a potential cointegrate formation, a characteristic frequently observed with insertion sequences, was revealed. This insertion sequence does not contain short inverted repeats near the ends or a large open reading frame to code for a transposase enzyme. Its host range is restricted to a previously described genetic subspecies of M. kansasii and is not present in typical M. kansasii or other mycobacterial species. When used as a probe for Southern blot hybridization, significant heterogeneity between different isolates of the M. kansasii subspecies was observed. This repeated element will be useful in further studies on the characterization, diagnosis, and epidemiology of M. kansasii.

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.

Macroevolution of plasmids: A model for plasmid speciation

A new evolutionary model for diversification in plasmid incompatibility groups (plasmid speciation) is suggested. The model is based on the formation of plasmid cointegrates from two compatible plasmids. The existence of plasmid cointegrates is well known, however, their potential key role in plasmid macroevolution has not yet been recognized. In a hypothesis presented here, one of the rep genes is supposed to be relaxed from selection in plasmid cointegrates and thus becomes free to accumulate mutations. These mutations can lead to a change in incompatibility specificity. Evidence supporting this hypothesis comes from the common occurrence of multi-replicon plasmids in nature as well as from experimental studies on plasmid cointegrate formation. A more speculative extension of this model hypothesizes an evolutionary scenario for origin of the eubacterial single-replicon genome and the eukaryotic multi-replicon genome, as well as the place of plasmids and viruses in this picture.

Excision of Tn10 from the donor site during transposition occurs by flush double-strand cleavages at the transposon termini.

Tn10 transposition is accomplished without extensive replication of the transposon sequences. Replicative cointegrate formation is precluded by efficient separation of transposon sequences from flanking donor DNA at an early stage in the transposition reaction. We report here that excision of Tn10 from its donor site occurs by a pair of flush double-strand breaks. Breaks occur at each end of the element precisely between the terminal base pair of the element and the first base pair of flanking DNA. This observation provides definitive evidence that cleavage of both strands of the element occurs under the direct control of Tn10 transposase protein. It is highly likely that transposase itself is directly responsible for these cleavages. The implications of this possibility are discussed.