Xis protein binding to the left arm stimulates excision of conjugative transposon Tn916

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.     

Conjugative transposition of Tn916: the transposon int gene is required only in the donor.

Conjugative transposition of transposon Tn916 has been shown to proceed by excision of the transposon in the donor strain and insertion of this element in the recipient. This process requires the product of the transposon int gene. We report here the surprising finding that the int gene is required only in the donor during conjugative transposition. We find that Tn916 int-1, whose int gene has been inactivated by an insertion mutation, transposes when a complementing wild-type int gene is present only in the donor during mating. When the int+ gene is present in a plasmid and is expressed from the spac promoter, conjugative transposition is very inefficient. However, when the Int+ function is supplied from a coresident distantly linked Tn916 tra-641 mutant, which is defective in a function required for conjugation, efficient conjugative transposition of Tn916 int-1 occurs. This suggests either that Int is not required for integration of Tn916 in gram-positive bacteria or that the protein is transferred from the donor to the transconjugant during the mating event. When the nonconjugative plasmid pAT145 was present in the donor, it was rarely cotransferred with Tn916. This suggests that complete fusion of mating cells is not common during conjugative transposition.     

The structure of the excisionase (Xis) protein from conjugative transposon Tn916 provides insights into the regulation of heterobivalent tyrosine recombinases

Heterobivalent tyrosine recombinases play a prominent role in numerous bacteriophage and transposon recombination systems. Their enzymatic activities are frequently regulated at a structural level by excisionase factors, which alter the ability of the recombinase to assemble into higher-order recombinogenic nucleoprotein structures. The Tn916 conjugative transposon spreads antibiotic resistance in pathogenic bacteria and is mobilized by a heterobivalent recombinase (Tn916Int), whose activity is regulated by an excisionase factor (Tn916Xis). Unlike the well-characterized (lambda)Xis excisionase from bacteriophage lambda, Tn916Xis stimulates excision in vitro and in Escherichia coli only modestly. To gain insights into this functional difference, we have performed in vitro DNA-binding studies of Tn916Xis and Tn916Int, and we have solved the solution structure of Tn916Xis. We show that the heterobivalent Tn916Int protein is capable of bridging the DR2-type and core-type sites on the left arm of the tranpsoson. Consistent with the notion that Tn916Int is regulated only loosely, we find that Tn916Xis binding does not alter the stability of DR2-Tn916Int-core bridges or the ability of Tn916Int to recognize the arms of the transposon in vitro. Despite a high degree of divergence at the primary sequence level, we show that Tn916Xis and (lambda)Xis adopt related prokaryotic winged-helix structures. However, they differ at their C termini, with Tn916Xis replacing the flexible integrase contacting tail found in (lambda)Xis with a positively charged alpha-helix. This difference provides a structural explanation for why Tn916Xis does not interact cooperatively with its cognate integrase in vitro, and reveals how subtle changes in the winged-helix fold can modulate the functional properties of excisionase factors.     

Excision of a conjugative transposon in vitro by the Int and Xis proteins of Tn916.

The roles of purified Int and Xis proteins of the conjugative transposon Tn 916 in excision of a deletion derivative of the closely related element Tn 1545 were investigated. At a low salt concentration (37.5 mM NaCl), Int alone was able to promote limited excision to produce a covalently closed circular form of the transposon, showing that Tn 916 Int can catalyze both DNA cleavage and strand exchange. This reaction was stimulated by Xis. At higher salt concentrations (150 mM NaCl), excision by Int alone was reduced to barely detectable levels and Xis was required for excision. The low salt, Xis-stimulated reaction was approximately 8-fold more efficient than the high salt, Xis-dependent reaction. These results reflect in vivo requirements for Int and Xis in excision.     

Excision of the conjugative transposon Tn916 in Lactococcus lactis

In Lactococcus lactis excision of Tn916 is limited by the concentration of integrase and is increased by providing more excisionase. However, even with increased excision of Tn916 in L. lactis, no conjugative transfer is detectable. This suggests that L. lactis is deficient in a host factor(s) required for conjugative transposition.     

DNA binding by the Xis protein of the conjugative transposon Tn916.

We purified the Xis protein of the conjugative transposon Tn916 and showed by nuclease protection experiments that Xis bound specifically to sites close to each end of Tn916. These specific binding sites are close to, and in the same relative orientation to, binding sites for the N-terminal domain of Tn916 integrase protein. These results suggest that Xis is involved in the formation of nucleoprotein structures at the ends of Tn916 that help to correctly align the ends so that excision can occur.     

Coupling sequences flanking Tn916 do not determine the affinity of binding of integrase to the transposon ends and adjacent bacterial DNA

Coupling sequences are the 6 bp flanking the conjugative transposon Tn916 and are thought to play a role in determining the frequency of conjugative transposition. The affinity of binding of a chimeric protein, which consisted of maltose binding protein fused to the carboxy-terminal DNA binding domain of Tn916 integrase (Int), to different double-stranded oligonucleotide substrates containing coupling sequences associated with high- and low-frequency conjugative transposition was measured using a competition binding assay. The relative affinity of the chimeric protein was unaffected by the nature of the coupling sequences tested. The same results were obtained when the coupling sequences were placed in a different surrounding sequence context. It therefore appears that the effects of different coupling sequences on the frequency of conjugative transposition are not due simply to differences in Int binding.     

Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision

The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.     

Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.     

A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition

In matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919 are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild-type form of the transposon or the mutant Tn916-int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.     

The presence of conjugative transposon Tn916 in the recipient strain does not impede transfer of a second copy of the element.

Transfer of the conjugative transposon Tn916 from the chromosome of Bacillus subtilis to a transposon-free Streptococcus pyogenes strain occurs at the same frequency as transfer to a Tn916-containing recipient. This rules out a model for conjugal transfer of Tn916 in which a copy of the element in the recipient represses transposition of a copy introduced by conjugation. Homology-directed integration of the incoming transposon into the resident one is less frequent than insertion elsewhere in the chromosome. This shows that after conjugation, transposition occurs more frequently than homologous recombination. However, because transconjugants arising from homologous recombination can be selected, it is possible to use Tn916 as a shuttle for gram-positive organisms for which there is no easy means of introducing DNA.     

Conjugative transposition of Tn916 requires the excisive and integrative activities of the transposon-encoded integrase.

Transposon Tn916 is a 16.4-kb broad-host-range conjugative transposon originally detected in the chromosome of Enterococcus faecalis DS16. Transposition of Tn916 and related transposons involves excision of a free, nonreplicative, covalently closed circular intermediate that is substrate for integration. Excisive recombination requires two transposon-encoded proteins, Xis-Tn and Int-Tn, whereas the latter protein alone is sufficient for integration. Here we report that conjugative transposition of Tn916 requires the presence of a functional integrase in both donor and recipient strains. We have constructed a mutant, designated Tn916-int1, by replacing the gene directing synthesis of Int-Tn by an allele inactivated in vitro. In mating experiments, transfer of Tn916-int1 from Bacillus subtilis to E. faecalis was detected only when the transposon-encoded integrase was supplied by trans-complementation in both the donor and the recipient. These results suggest that conjugative transposition of Tn916 requires circularization of the element in the donor followed by transfer and integration of the nonreplicative intermediate in the recipient.     

Specific binding of integrase to the origin of transfer (oriT) of the conjugative transposon Tn916

Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding to oriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.     

Control of SXT integration and excision

The Vibrio cholerae SXT element is a conjugative self-transmissible chromosomally integrating element that encodes resistance to multiple antibiotics. SXT integrates in a site-specific fashion at prfC and excises from the chromosome to form a circular but nonreplicative extrachromosomal form. Both chromosomal integration and excision depend on an SXT-encoded recombinase, Int. Here we found that Int is necessary and sufficient for SXT integration and that int expression in recipient cells requires the SXT activators SetC and SetD. Although no xis-like gene was annotated in the SXT genome, Int was not sufficient to mediate efficient SXT chromosomal excision. We identified a novel SXT Xis that seems to function as a recombination directionality factor (RDF), facilitating SXT excision and inhibiting SXT integration. Although unrelated to any previously characterized RDF, Xis is similar to five hypothetical proteins that together may constitute a new family of RDFs. Using real-time quantitative PCR assays to study SXT excision from the chromosome, we determined that while SXT excision is required for SXT transfer, the percentage of cells containing an excised circular SXT does not appear to be a major factor limiting SXT transfer; i.e., we found that most cells harboring an excised circular SXT molecule do not act as SXT donors. In the absence of prfC, SXT integrated into several secondary attachment sites but preferentially into the 5' end of pntB. SXT excision and transfer from a donor containing pntB::SXT were reduced, suggesting that the SXT integration site may also influence the element's transmissibility.     

Genetic organization of the bacterial conjugative transposon Tn916.

Tn916, which encodes resistance to tetracycline, is a 16.4-kilobase conjugative transposon originally identified on the chromosome of Streptococcus faecalis DS16. The transposon has been cloned in Escherichia coli on plasmid vectors, where it expresses tetracycline resistance; it can be reintroduced into S. faecalis via protoplast transformation. We have used a lambda::Tn5 bacteriophage delivery system to introduce Tn5 into numerous sites within Tn916. The Tn5 insertions had various effects on the behavior of Tn916. Some insertions eliminated conjugative transposition but not intracellular transposition, and others eliminated an excision step believed to be essential for both types of transposition. A few inserts had no effect on transposon behavior. Functions were mapped to specific regions on the transposon.     

Broad host range gene transfer: plasmids and conjugative transposons

Abstract Conjugation is the primary route of broad host range DNA transfer between different genera of bacteria. Plasmids are the most familiar conjugative elements, but there are also self-transmissible integrated elements called conjugative transposons. Conjugative transposons have been found in many genera of gram-positive bacteria, in mycoplasmas and in gram negative bacteria such as Bacteriodes spp. and Moraxella spp., and they have a very broad host range. The best-studied conjugative transposons are: the ones related to Tn 916 , a 16 kb conjugative transposon found originally in Gram-positive bacteria; Tn 5276 , a 70 kb conjugative transposon from Lactococcus lactis ; and a group of large (> 70 kb) conjugative transposons found in Bacteroides spp. Transfer of conjugative transposons takes place in three steps: excision to form a circular intermediate, transfer of one strand of the circular intermediate to a recipient, and integration into the recipient genome. Some conjugative transposons integrate almost randomly, whereas other integrate site-specifically. Conjugative transposons not only transfer themselves but also mobilize co-resident plasmids, either by providing transfer functions in trans or by inserting themselves into the plasmid. In addition, the conjugative transposons found in Bacteroides spp. can excise and mobilize unlinked integrated elements, called NBUs. Transfer of many of the Bacteroides conjugative transposons is regulated by tetracycline, whereas transfer of Tn 916 and other conjugative transposons appears to be constitutive. The conjugative transposons are clearly widespread in clinical isolates, but their distribution in environmental isolates remains to be determined.     

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.     

Roles of Exc protein and DNA homology in the CTnDOT excision reaction

Excision from the chromosome is the first step during the transfer of conjugative transposons (CTns) to a recipient. We previously showed that the excision of CTnDOT is more complex than the excision of lambdoid phages and CTns such as Tn916. The excision in vivo of CTnDOT utilizes four CTnDOT-encoded proteins, IntDOT, Xis2c, Xis2d, and Exc, and a host factor. We previously developed an in vitro excision reaction where the recombination sites attL and attR were located on different plasmids. The reaction was inefficient and did not require Exc, suggesting that the reaction conditions did not mimic in vivo conditions. Here, we report the development of an intramolecular excision reaction where the attL and attR sites are located on the same DNA molecule. We found that Exc stimulates the reaction 3- to 5-fold. The efficiency of the excision reaction was also dependent on the distance between the attL and attR sites and on the sequences of the overlap regions between the sites of the strand exchanges. Substrates with identical overlap sequences recombined more efficiently than ones with heterologous overlap sequences. This was surprising, because the integration reaction is not sensitive to heterology in the overlap regions of the attDOT and attB sites.