Cointegrate Formation Research Articles

Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.

Pseudomonas aeruginosa plasmids as suicide vectors in Escherichia coli: resolution of genomic cointegrates through short regions of homology

Pseudomonas aeruginosa plasmids which cannot replicate in Escherichia coli have been used to introduce specific modifications into the E. coli chromosome by homologous recombination (‘gene targeting’). The E. coli gene ( gpt) encoding guanine-xanthine phosphoribosyltransferase (Gpt) was used for initial targeting studies owing to the availability of a powerful positive selection for loss of the Gpt + phenotype (6-thioguanine resistance or 6TG R or Gpt −). P. aeruginosa plasmids containing selectable markers flanked by gpt sequences were introduced as supercoiled DNA into an E. coli strain which contained a normal gpt locus. Primary cointegration of such plasmids into the E. coli genome results in a gene duplication event which maintains Gpt function; a secondary recombinational event which resolves the cointegrate either reverses the primary event or results in replacement of the original gpt copy with the modified version. A 316-bp region of homology was sufficient for cointegrate formation, and resolution of the cointegrates through a shorter (92 bp) homologous flank was selectable through loss of Gpt function. The frequency of cointegrate resolution under these conditions was significantly above the spontaneous gpt mutational loss rate.

Chlorobenzoate catabolic transposon Tn5271 is a composite class I element with flanking class II insertion sequences.

The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described. Tn5271 is a 17-kilobase (kb) transposon that resides in the plasmid or chromosome of Alcaligenes sp. strain BR60 and allows this organism to grow on 3- and 4-chlorobenzoate. The transposon is flanked by a directly repeated sequence of 3201 base pairs (bp), which in turn is flanked by 110-bp inverted repeats. The 3.2-kb repeated sequence, designated IS1071, exists in multiple copies in the genome of Alcaligenes sp. strain BR60 and is involved in recombination of the catabolic genes into the chromosome of this strain. Sequence analysis revealed that the inverted repeat of IS1071 and the derived amino acid sequence of the single open reading frame within IS1071 are related to the inverted repeats and transposase (TnpA) proteins of the class II (Tn3 family) transposable elements. The absence of a resolvase gene within IS1071 suggests that this element is capable of determining the first step in class II transposition only. This was confirmed by observations on the IS1071-dependent formation of stable cointegrates in a recombination-deficient Escherichia coli. These results support an evolutionary scheme in which the class II transposable elements descended from simple insertion sequences.

IS900-promoted stable integration of a foreign gene into mycobacteria.

An artificial mycobacterial transposon was constructed by placing two copies of the insertion sequence IS900 flanking a kanamycin resistance gene into a non-(mycobacterial) replicating vector. Constructs were introduced into mycobacteria by electroporation and transposition events conferring kanamycin resistance were selected. Integration of IS900 into several genomic sites was analysed by Southern blotting and shown to involve both simple insertions and cointegrate formation, suggesting that IS900 can transpose by a replicative mechanism. Kanamycin resistance of IS900-integrated transformants was shown to be stable in the absence of selection.

Molecular cloning in Lactobacillus helveticus by plasmid pSA3::pVA797 co-integrate formation and conjugal transfer.

A gene encoding beta-glucanase activity from Bacillus amyloliquefaciens was subcloned in both orientations into plasmid shuttle vector pSA3. In only one orientation could a co-integrate be generated with the conjugative plasmid pVA797. The plasmid co-integrate was conjugated into Lactobacillus helveticus strain CNRZ450, where it was stably maintained without antibiotic selection and exhibited beta-glucanase activity. This method of introducing cloned DNA into thermophilic lactobacilli will facilitate the study of heterologous gene expression in non-transformable species.

Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli.

Transposition events mediated by plasmidborne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene: however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 bp long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation condons of ORF I and II in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

IS10 promotes adjacent deletions at low frequency.

Some transposable elements move by a replicative mechanism involving cointegrate formation. Intramolecular cointegration can generate a product called an "adjacent deletion" in which a contiguous chromosomal segment adjacent to the transposon is deleted while the element responsible remains intact. Insertion sequence IS10 is thought to transpose by a nonreplicative mechanism. In the simplest models, nonreplicative transposition cannot give rise to an adjacent deletion because an intrinsic feature of such transposition is excision of the IS element from the donor location. We report here that IS10 can generate adjacent deletions, but at a frequency which is approximately 1/30th the frequency of transposition for the same element. We suggest that these deletions might arise either by nonreplicative transposition events that involve two IS10 elements located on sister chromosomes or by aberrant nonreplicative events involving cleavage and ligation at only one end of the element.

A comparison of intramolecular rearrangements promoted by transposons Tn 5 and Tn 10

The bacterial transposon Tn10 has previously been shown to move to other genomic sites by a conservative mechanism, whereby the transposon is excised by double-strand breaks and inserted between a pair of staggered nicks at the target. Other transposons, like Tn3, have been shown to transpose by a replicative mechanism that involves symmetrical nicking of the element and formation of the 'Shapiro intermediate', which can mature into either a cointegrate or a simple insert. The situation with respect to Tn5 is unclear; it was originally reported to use a conservative mechanism, but other evidence suggests that the mechanism might be replicative. In this paper, rearrangements of adjacent DNA promoted by Tn10 and Tn5 have been compared using positive selection for galactose-resistance to detect such rearrangements. Tn10 promoted the formation of adjacent deletions (that started from an inside end of Tn10), deletion/inversions and simple IS10 insertions, but no cointegrates. This behaviour is fully consistent with a conservative mechanism. In contrast, Tn5 was found to promote formation of adjacent deletions (that started mainly from an outside end of Tn5), IS50 insertions (that were frequently accompanied by inversions of adjacent DNA) and cointegrates. These characteristics seem compatible with a replicative, rather than a conservative, mode of transposition. Clearly, Tn5 and Tn10 exhibit some significant differences in their transposition. These results, and results of some previous experiments, have been interpreted to mean that Tn5 could use a replicative mechanism for its transposition.

The istA gene of insertion sequence IS21 is essential for cleavage at the inner 3′ ends of tandemly repeated IS21 elements in vitro.

The bacterial 2.1 kb insertion sequence IS21 occurs as a tandem repeat [=(IS21)2] on the broad host range plasmid R68.45. In (IS21)2, the two IS21 elements are separated by 3 bp termed junction sequence. Plasmids carrying (IS21)2 form cointegrates with other replicons at high frequencies. The two IS21 genes, istA and istB, were found to be necessary for cointegrate formation in vivo. Since the outer ends of (IS21)2 are dispensable for cointegrate formation, we favor a transposition model according to which a plasmid carrying (IS21)2 is cleaved at the junction sequence; the opened plasmid is then inserted into a target replicon. Here we show that Escherichia coli cell extracts, which contained over-produced IstA protein, nicked a supercoiled (IS21)2 plasmid precisely at the inner 3' termini of IS21; the resulting staggered cut generated 5' protrusions. The istA gene, but not the istB gene, was required for in vitro cleavage of an IS21-IS21 junction. Because of this cleavage and our previous findings (generation of 4 bp target duplications and loss of the junction sequence after cointegrate formation in vivo) we propose that plasmids with (IS21)2 produce cointegrates by a mechanism which involves joining of the inner 3' ends of IS21 to the 5' ends of the target.

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.

The regulatory role of the IS 1‐encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS1 transposition activity. We demonstrate that it inhibits both IS1-mediated cointegrate formation and transposition of a synthetic IS1-based transposon ('omegon'; omega-on). These results also indicate that the omega-on which does not itself encode IS1 transposition functions can be complemented in trans, presumably by the copies of IS1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS1-encoded genes both in cis or in trans. The experiments involving omega-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS1.

Construction of an R‐prime plasmid carrying the hup genes of Azorhizobium caulinodans ORS571 and its transfer to and stability in Rhizobium meliloti

AbstractAn R‐prime plasmid (pJB3JI‐18) carrying a 10 kb region of Azorhizobium caulinodans ORS571 DNA which codes for uptake hydrogenase activity was isolated from a Km8 derivative of R68.45. R‐prime plasmids were obtained by transferring R68.45 to ORS571, retransferring it to E. coli, and selecting transconjugants for hydrogenase activity by means of a colour test based on methylene blue. The R‐prime plasmid DNA was hybridized with a DNA region which contained the hup genes of Bradyrhiobium japonicum, indicating homology between the heterologous hup genes. Transfer of the R‐prime plasmid to different Hup− mutants restored the Hup+ phenotype. When it was introduced to R. meliloti strains, it maintained its molecular weight and did not affect indigenous plasmids in two of three recipients. In the third recipient cointegrate formation and incompatibility with indigenous plasmids were found.

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.

Characterization of the staphylococcal beta-lactamase transposon Tn552.

The staphylococcal beta-lactamase transposon Tn552 is a member of a novel group of transposable elements. The organization of genes in Tn552 resembles that of members of the Tn21 sub-group of Tn3 family transposons, which transpose replicatively by cointegrate formation and resolution. Thus, a possible resolution site ('resL') and a resolvase gene (tnpR or 'binL') have been identified. However, consistent with the fact that Tn552 generates 6 bp (rather than 5 bp) flanking direct repeats of target DNA, neither the putative transposase protein, nor the terminal inverted repeats of Tn552 are homologous to those of Tn3 elements. Tn552, like phage Mu and retroelements, is defined by the terminal dinucleotides 5' TG .. CA 3'. A naturally occurring staphylococcal plasmid, pI9789, contains a Tn552-derived resolution system ('resR-binR') that acts as a 'hotspot' for Tn552 transposition; insertion creates a segment of DNA flanked by inversely repeated resolution sites, one (resR) on pI9789 and the other (resL) on Tn552. The putative Tn552 resolvase, the most closely related of known resolvases to the homologous DNA invertases, initially was identified as a DNA invertase ('Bin') as a result of its ability to mediate efficient inversion of this segment in vivo.

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007 was examined. A plasmid of 88 MDa (pJS88) was identified in addition to the previously reported conjugally transferred plasmids of 32 (pKB32) and 4.8 MDa. Proteinase activity, reduced bacteriophage sensitivity, bacteriocin resistance, and conjugal transfer ability were encoded by pJS88. The ability to metabolize lactose (Lac +) was encoded by pKB32, and the 4.8-MDa plasmid was cryptic. When a strain containing both pKB32 and pJS88 was mated with a recipient deficient in host-mediated homologous recombination (Rec −), a plasmid of 40 MDa (pJS40) was observed in approximately 50% of the Lac + transconjugants. DNA-DNA hybridization results indicated that pJS40 contained homology with both pKB32 and pJS88. These results indicated that pKB32 was conjugally transferred via conduction and suggested that pJS40 is a deletion derivative of a pKB32::pJS88 cointegrate. A Rec − strain containing pKB32 and pJS88 mediated Lac + conjugal transfer, suggesting that the pKB32::pJS88 cointegrate could form via a rec-independent event. Resolution of the pKB32::pJS88 cointegrate was observed in both Rec − and Rec + hosts. Cointegrate formation and resolution via rec-independent mechanisms suggest the involvement of a transposable element in the Tn3 family.

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.

Mutational Analysis of the Open Reading Frames in the Transposable Element IS1

IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.

Mutational analysis of the open reading frames in the transposable element IS1.

IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.

IS1-mediated mobility of the aerobactin system of pColV-K30 in Escherichia coli.

Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system.