Cointegrate Resolution Research Articles

Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance.

The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.

Tn502 and Tn512 are res site hunters that provide evidence of resolvase-independent transposition to random sites.

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.

Cointegrate resolution following transposition of Tn 1792 in Streptomyces avermitilis facilitates analysis of transposon-tagged genes

The insertion sequence IS 6100, belonging to the IS 6 family, normally forms a cointegrate as an end product of transposition. The IS 6100-based minitransposon, Tn 1792, has been developed as a genetic tool to mutagenise antibiotic-producing Streptomyces. Here, we describe resolution of Tn 1792 cointegrates in Streptomyces avermitilis that can facilitate both the initial isolation of Tn 1792 insertion mutants and also the subsequent rescue of Tn 1792-tagged sequences. This is the first reported example of cointegrate resolution for an IS 6-type transposable element. As a result of mutagenesis, several putative genes involved in morphological development and antibiotic production have been isolated.

The shuffling function of resolvases

Redistribution (shuffling) of genetic material between replicons requires consecutive recombination at four points, two of which (X, X′) are involved in the replicon fusion and the other two (Y, Y′), in the cointegrate resolution. The fusion of replicons by bacterial resolvases makes the second recombination round at sites Y and Y′ problematic because of the high probability of the reverse reaction. Structural differences of the res sites recognized by resolvase could delay the reverse reaction, thus enhancing the probability of recombination at sites Y, Y′, but the direct reaction ensuring it (i.e. the fusion of replicons via different res sites) has not been described yet. Here, a genetic system to test intermolecular recombination at heterogeneous res sites has been developed. The system was based on the res site (RS2) of the novel resolution system, cinH-RS2, encoded by pKLH2, pKLH204 and pKLH205. As its partner, the res site of RP4 located in the par locus, the res site of transposon gammadelta or Tn1721, the incomplete site RS1 consisting only of the (crossover) subsite resI also found in pKLH2/204/205 and others were used. Except for the pairing of RS2 × gammadelta res, recombination was observed in each case even when the homology shared by partners did not exceed 35% (as in RS2 × par). In the latter case, the presence in cis of an additional, enhancer-like-acting element was required. Pairing of crossover subsites during site-specific recombination occurred in either orientation, depending on the structure of res partners and the kind of resolvase acting on the sites. With the complete res sites, the antiparallel alignment resulted in the production of an unusual res having the accessory subsites II and III at both sides of I, and a res lacking II and III. The wide range of frequencies was observed not only in the fusion formation but also in the dissociation of the resulting cointegrates. Hence, the resolvase-mediated interreplicon exchange of the DNA segments by fusion via an inefficient reaction (at sites X, X′) and dissociation via an efficient one (at sites Y, Y′) become possible.

Stability by multimer resolution of pJHCMW1 is due to the Tn1331 resolvase and not to the Escherichia coli Xer system.

The plasmid pJHCMW1 encodes resistance to several aminoglycosides and beta-lactams and consists of a copy of the transposon Tn1331, a region including the replication functions, and a sequence with homology to ColE1 cer, designated mwr. In this work, the role of this cer-like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied. The Escherichia coli Xer site-specific recombination system acts at sites such as ColE1 cer to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance. Despite its high similarity to ColE1 cer, the pJHCMW1 mwr was a poor substrate for Xer recombination in E. coli and did not contribute significantly to plasmid stability. Instead, the Tn1331 co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1. Although Xer recombination at pJHCMW1 mwr was inefficient in E. coli, the recombination that did occur was dependent on ArgR, PepA, XerC and XerD. A supercoiled circular DNA molecule containing two pJHCMW1 mwr sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD in vitro, confirming that pJHCMW1 mwr is a functional recombination site. However, unlike cer, some Holliday-junction-containing product could be detected for mwr in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions. Binding experiments demonstrated that XerD bound to pJHCMW1 mwr core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD.

The uvp1 gene on the R46 plasmid encodes a resolvase that catalyzes site-specific resolution involving the 5′-conserved segment of the adjacent integron In1

The product of the uvp1 gene of the R46 plasmid, a member of the DNA invertase-resolvase family, was studied to characterize its recombination activity on the R46 plasmid. The purified Uvp1 protein specifically binds to a 256-bp DNA fragment located immediately upstream of the uvp1 gene itself, and overlapping the 5'-conserved segment (5'-CS) of the R46 integron In1. We identified on this fragment a putative resolution (res) site. Using an in vitro assay, we demonstrated the ability of the protein to resolve a synthetic cointegrate containing a direct repeat of the res site. In vivo, we obtained cointegrate resolution in Uvp1-expressing recA- cells. Sites I and II, subsites of the putative res site, lie within the outer boundary of the integron 5'-CS which is common to all the known integrons. Furthermore, a 69-bp DNA element (containing site I) is required for cointegrate resolution. We propose that this recombination mechanism protects R46 plasmid against unequal distribution following fusion with either identical or different integron-bearing plasmids. Moreover, Uvp1 might have a role in generating gene cassette diversity between the two conserved segments of the integron.

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

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.

Tn5401, a new class II transposable element from Bacillus thuringiensis.

A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp. morrisoni EG2158 following its insertion into a recombinant plasmid. Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats. The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B. thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases. The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430. Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified. Southern blot analysis indicated that Tn5401, in contrast to Tn4430, is not commonly found among different subspecies of B. thuringiensis and is not typically associated with known insecticidal crystal protein genes. Transposition was studied with B. thuringiensis by using plasmid pEG922, a temperature-sensitive shuttle vector containing Tn5401. Tn5401 transposed to both chromosomal and plasmid target sites but displayed an apparent preference for plasmid sites. Transposition was replicative and resulted in the generation of a 5-bp duplication at the target site. Transcriptional start sites within Tn5401 were mapped by primer extension analysis. Two promoters, designated PL and PR, direct the transcription of orf1-orf2 and tnpI-tnpA, respectively, and are negatively regulated by TnpI. Sequence comparison of the promoter regions of Tn5401 and Tn4430 suggests that the conserved sequence element ATGTCCRCTAAY mediates TnpI binding and cointegrate resolution. The same element is contained within the 53-bp terminal inverted repeats, thus accounting for their unusual lengths and suggesting an additional role for TnpI in regulating Tn5401 transposition.

Analysis of the genes and gene products of Xanthomonas transposable elements IS Xc5 and IS Xc4

A series of deletion mutants have been constructed for the gene analyses of transposable elements, IS Xc5 and IS Xc4, derived from Xanthomonas. At least two element-encoded polypeptides of 48 kDa and 40 kDa have been identified in the minicell-producing Escherichia coli strain, TC410. A study of the element transposition and cointegrate resolution revealed that the 48-kDa and 40-kDa polypeptides are both involved in translocation of the elements, the 48-kDa product being involved in transposition of the elements and the 40-kDa product being involved in cointegrate resolution.

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.

Tn552, a novel transposable element from Staphylococcus aureus

Tn552, one of several closely related beta-lactamase-encoding transposons from Staphylococcus aureus, has a novel set of putative transposition functions. Each is homologous with a well-characterized function from a different type of mobile genetic element. Thus, Tn552 encodes: (i) resL-binL, a co-integrate resolution system homologous with those of Tn3 family elements; (ii) p480, a potential transposase significantly homologous with the DNA integrases of eukaryotic retroviruses and retrotransposons; and (iii) p271, a potential ATP-binding protein that shows homology with the B protein of phage Mu. The 3' terminal nucleotides of Tn552 (CA), adjacent to which p480 might cleave, are the same as those of retroviruses, retrotransposons and phage Mu. The presumptive resolvase (BinL) is very closely related to BinR, which was identified as a DNA invertase and is now shown to resolve an artificial co-integrate in vivo. Furthermore, the structure of the derivative of Tn552 found in the staphylococcal plasmid pI258 can be explained by a BinL (or BinR)-mediated site-specific deletion ('resolution') event. Thus, pI258 contains only the right-hand half of Tn552, which encodes the beta-lactamase and two regulatory proteins. The latter are homologous with the beta-lactamase gene repressor and co-inducer of Bacillus licheniformis. Interestingly, the order of the regulatory genes is reversed in S. aureus compared with Bacillus licheniformis.

Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.

The 4149-bp transposon Tn4430 from Bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (TnpA) of Tn3, Tn21 and Tn501. Through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of Tn4430 molecules. These features are characteristic of transposons of the Tn3 family (class II elements). The second step of the transposition process, the co-integrate resolution, is mediated by a 32-kd protein. This protein (TnpI) displays regional similarities with site-specific recombinases of the integrase family, such as Int of bacteriophage lambda, Cre of bacteriophage P1 or TnpA and TnpB of the Tn554 transposon. Moreover, the 250-bp sequence upstream to the tnpI gene contains several structural features that are reminiscent of the attP attachment site of phage lambda. This unique association between the integrase-like TnpI recombinase and the TnpA transposase qualifies Tn4430 as a member of a new group within the class II mobile genetic elements.

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWW0.

Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWW0 are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in Escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointegrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.

The R46 site-specific recombination system is a homologue of the Tn3 and gamma delta (Tn1000) cointegrate resolution system.

The nucleotide sequence of the R46 site-specific recombination system has been determined. The organization of the recombination gene (perR46) and the site at which it acts (per site), together with the extensive sequence homology displayed with the tnpR genes and res sites of the transposons Tn3 and gamma delta (Tn1000), suggests that they have been derived from a Tn3-like element. These site-specific recombination functions of R46 play a role in plasmid maintenance.

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.

TnpM: A novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution

We have identified a new gene, tnpM, in Tn21 that encodes the 12.6 kilodalton modulator protein. The Tn21 modulator enhances Tn21 transposition and suppresses resolution of cointegrate replicons in vivo. A putative binding site may be located in the N-terminal portion of the TnpR (resolvase) structural gene sequences. Tn501 transposition and cointegrate resolution can be regulated by the subcloned tnpM gene of Tn21 in trans-complementation experiments. Examination of the Tn501 DNA sequence also reveals a potential tnpM coding sequence upstream of the Tn501 resolvase gene. We conclude that Tn21 and Tn501 are different from Tn3 and Tn1000 both in genome organization and in regulation of transposition functions.

Tn5 transposes independently of cointegrate resolution: Evidence for an alternative model for transposition

The replicon fusion model for transposition predicts that each transposable element encodes a site-specific recombination activity (resolvase). A series of experiments were performed to test for the presence of such a resolvase in Tn5. None could be found when assays were devised to detect inter- or intramolecular recombination. An experiment was performed to determine if expression of resolvase occurs simultaneously with the transposition process, thus explaining the undetectable levels of site-specific recombination. In this case, no resolvase activity could be detected in cells that had undergone transposition. Instead, rearrangements were found that could be explained best by an alternative model of transposition. The rearrangements were consistent with a pathway that invokes excising the transposon from its initial site of insertion, followed by circularization of the element before its eventual transposition. The excision event may occur either with or without DNA synthesis.

Does Tn10 transpose via the cointegrate molecule?

It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods. In the first method, lambda 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from lambda 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec+ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.(ABSTRACT TRUNCATED AT 250 WORDS)

R46 encodes a site-specific recombination system interchangeable with the resolution function of Tn A

Transposition of Tn A onto the IncN plasmid R46 generates unstable DNA molecules. The R46::Tn A recombinant plasmids undergo further DNA rearrangements which depend on the orientation in which the Tn A element is inserted into the plasmid, and deletions and inversions of R46 and Tn A sequences have been observed. Both types of rearrangement have the same specific endpoints, one within Tn A and one located between the R46 coordinates, 36.0 and 37.0. The results are consistent with the operation of a recA-independent, site-specific recombination system utilizing, at least in part, the transposon cointegrate resolution system of Tn A, together with R46-encoded functions. Data are presented that indicate that R46 encodes analogs of both the res site of Tn A and its tnpR gene, although little homology between this element and the plasmid is apparent. Models for the Tn A-induced generation of site-specific deletions and inversions upon transposition of Tn A to R46 are presented.