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At least 19 recordsLinked to original sources
PubMed1995

A transposon-like sequence adjacent to the AccI restriction-modification operon.

We have cloned and sequenced the accIRM genes from Weeksella zoohelcum (the original identification of this strain as Acinetobacter calcoaceticus was incorrect). Our sequence differs in the coding regions from a previously published sequence by the addition of three nucleotides near the 3' end of the DNA methyltransferase-encoding gene (accIM). We have sequenced approx. 3 kb beyond this operon. Two genes were found, convergently transcribed with the R-M operon. The first of these genes encodes a protein which shows significant similarity to the recombinases of the phage integrase family. The W. zoohelcum recombinase may function as a transposon resolvase, as in Tn4430. The recombinase-encoding gene is followed by a putative transposase (Tnp), which is in turn followed by a terminator which is predicted to be Rho-dependent for the recombinase-Tnp operon and Rho-independent for the convergent R-M operon. Since the G + C content of the two operons is notably different, it is possible that the terminator is at the extremity of the mobile element and serves to protect it from incoming transcription.

Amino Acid Sequence↗
PubMed2005

Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites.

Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.

Amino Acid Sequence↗
PubMed2003

The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB.

Integration of the mycobacteriophage Bxb1 genome into its host chromosome is catalyzed by a serine-integrase, a member of the transposon-resolvase family of site-specific recombinases. These enzymes use a concerted mechanism of strand exchange involving double-stranded cleavages with two-base extensions, and covalent protein-DNA linkages via phosphoserine bonds. In contrast to the resolvase/invertase recombination systems--where there are strict requirements for a specific synaptic complex within which the catalytic potential of the enzyme is activated--synapsis of attP and attB by Bxb1 integrase is completely promiscuous, aligning the sites with equal proclivity in parallel and antiparallel alignments. Moreover, the catalytic potential of Bxb1 integrase is fully active in either alignment. As a consequence, the nonpalindromic central dinucleotide (5'-GT) at the center of attP and attB is the sole determinant of Bxb1 prophage orientation, and a single base pair substitution in the two sites is sufficient to eliminate orientation control.

Attachment Sites, Microbiological↗
PubMed2001

Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B.

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.

Amino Acid Sequence↗
PubMed2000

Transposon targeting determined by resolvase.

The Mu-related transposon Tn5090, also called Tn402, was observed to be highly selective for targets clustered in or close to recombination sites of serine-type recombinases in plasmids R388 and RP1. Transposition to the par area of RP1 responded strongly to a deletion in the gene of resolvase ParA. A search in sequence databanks revealed further insertions of Tn5090/Tn402 close to different genes of resolvases. These results imply that the target selection of Tn5090 depends on a property that is shared among several serine recombinases.

Base Sequence↗
PubMed1985

The resolvase protein from the transposon Tn21.

The tac promoter was inserted into Tn21 upstream of the tnpR gene and the resultant plasmid was used to generate substantial amounts of resolvase. This protein was purified to homogeneity. The protein was characterized by amino acid sequence studies (which showed that an open-reading frame previously identified by DNA sequencing had been correctly assigned to the tnpR gene) and by molecular weight measurements (which demonstrated that the only active for of the protein in solution was dimeric). Pure Tn21 resolvase catalysed site-specific recombinations between directly repeated res sites from Tn21 or Tn1721 but not from Tn3 nor on inverted res sites from Tn21.

Amino Acid Sequence↗
PubMed2003

Production of unmarked mutations in mycobacteria using site-specific recombination.

Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon gammadelta site-specific recombination system. A res-OmegaKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon gammadelta resolvase gene, res-OmegaKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants.

Cloning, Molecular↗
PubMed1997

Topoisomerase IV, not gyrase, decatenates products of site-specific recombination in Escherichia coli.

DNA replication and recombination generate intertwined DNA intermediates that must be decatenated for chromosome segregation to occur. We showed recently that topoisomerase IV (topo IV) is the only important decatenase of DNA replication intermediates in bacteria. Earlier results, however, indicated that DNA gyrase has the primary role in unlinking the catenated products of site-specific recombination. To address this discordance, we constructed a set of isogenic strains that enabled us to inhibit selectively with the quinolone norfloxacin topo IV, gyrase, both enzymes, or neither enzyme in vivo. We obtained identical results for the decatenation of the products of two different site-specific recombination enzymes, phage lambda integrase and transposon Tn3 resolvase. Norfloxacin blocked decatenation in wild-type strains, but had no effect in strains with drug-resistance mutations in both gyrase and topo IV. When topo IV alone was inhibited, decatenation was almost completely blocked. If gyrase alone were inhibited, most of the catenanes were unlinked. We showed that topo IV is the primary decatenase in vivo and that this function is dependent on the level of DNA supercoiling. We conclude that the role of gyrase in decatenation is to introduce negative supercoils into DNA, which makes better substrates for topo IV. We also discovered that topo IV has an unexpectedly strong DNA relaxation activity that, together with gyrase and topo I, is able to set the supercoiling levels in Escherichia coli.

Bacteriophage lambda↗
PubMed2004

The large resolvase TnpX is the only transposon-encoded protein required for transposition of the Tn4451/3 family of integrative mobilizable elements.

Chloramphenicol resistance in Clostridium perfringens and Clostridium difficile is often encoded by catP genes located within the 6.3 kb integrative mobilizable elements Tn4451 and Tn4453 respectively. This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element. Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate. In this study, in vivo deletion analysis of the transposon-encoded tnpV and tnpY genes showed that they are not essential for excision or integration of this group of elements. Similar studies on tnpW suggested either that this gene is not essential for these functions or that TnpW does not function when provided in trans. Development and use of an in vivo insertion assay showed that TnpX is the only transposon-encoded protein required for the integration reaction. Subsequently, a TnpXLEH6 protein was purified and shown to catalyse excision in vitro in the absence of any other protein and preferentially to excise a supercoiled DNA substrate. In summary, these studies have shown that TnpX is the only transposon protein required in vivo and in vitro for the excision process and that, like excision, integration also occurs by a serine recombinase-mediated site-specific recombination mechanism.

Chloramphenicol Resistance↗
PubMed1998

Characterization of the Pac25I restriction-modification genes isolated from the endogenous pRA2 plasmid of Pseudomonas alcaligenes NCIB 9867.

Genes for the class II Pseudomonas alcaligenes NCIB 9867 restriction-modification (R-M) system, Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. The Pac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of the Pac25I methylase revealed significant similarity with the XcyI, XmaI, Cfr9I, and SmaI methylases. High sequence similarity was displayed between the Pac25I endonuclease and the XcyI, XmaI, and Cfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5'-C CCGGG-3') and are thus perfect isoschizomers. However, no sequence similarity was detected between the Pac25I endonuclease and the SmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5'-CCCGGG-3'). Both the Pac25I methylase and endonuclease were expressed in Escherichia coli. An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of the Pac25I R-M operon. In addition, a transposon designated Tn5563 was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.

Amino Acid Sequence↗
PubMed2000

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.

Base Sequence↗
PubMed1995

Nucleotide sequence within Tn3926 confirms this as a Tn21-like transposable element and provides evidence for the origin of the mer operon carried by plasmid pKLH2.

The DNA sequence of the resolvase gene, resolution sites, and the region between the transposition functions and the end of the mercury resistance operon of the bacterial transposon, Tn3926, is presented. The sequence of Tn3926 upstream of the resolution sites is homologous to that bordering the 11.2-kb insert of Tn21, supporting the idea that this insert transposed into a progenitor of Tn3926. This region of Tn3926 also shows 97.0% identity to the mercury-resistant determinant of the plasmid, pKLH2, suggesting that this plasmid once harbored a close relative of Tn3926. It is proposed that the mercury resistance operon of Tn3926 will have a structure very similar to that found on pKLH2.

Base Sequence↗
PubMed2004

DNA binding properties of TnpX indicate that different synapses are formed in the excision and integration of the Tn4451 family.

Site-specific recombination is an important mechanism for genetic exchange. Insertional recombination mediated by the recently delineated large resolvase or serine recombinase proteins is unique within the resolvase family as integration was thought to be a reaction catalysed only by members of the integrase or tyrosine recombinase family of site-specific recombinases. The large resolvase TnpX is a serine recombinase that is responsible for the movement of the Tn4451/3 family of chloramphenicol resistance elements, which are found within two genera of the medically important clostridia. Deletion analysis of TnpX showed that the last 110 amino acids (aa) of TnpX, which comprise a cysteine rich region, were not essential for its biological function and that a region required for DNA binding was located between aa 493-597. Purified TnpX was shown to bind to the ends of the element and to the joint of the circular intermediate with high affinity but, most unusually, to bind to its target sites with a considerably lower affinity. Therefore, it was concluded that the resolvase-like excision and insertion reactions mediated by TnpX were distinct processes even though the same serine recombinase mechanism was involved. TnpX is the first large serine recombinase in which differential binding to its transposon and target sites has been demonstrated.

Base Sequence↗
PubMed1992

Tn21-specific structures in gram-negative bacteria from clinical isolates.

A total of 807 gram-negative clinical isolates were treated with five different probes: intragenic segments for the transposase gene tnpA; the resolvase gene tnpR; the modulator of the resolvase, tnpM; the integraselike factor gene tnpI; and a 20-mer oligonucleotide for the recombinational site of action for the integrase. A total of 8% of the isolates hybridized with all five Tn21-related probes, and another 11% represented transposons in which one or more of the tested genes were missing. This 11% included groups whose descriptions have been published as well as groups that have not yet been described. The not-yet-described groups include various deletion products and some precursor structures, as is predicted for the evolution of Tn21-like transposons. The integration system appears to be coupled with Tn21-like structures and yet independent from these structures, implying an independent evolution of this system from Tn21-like transposons. The structures were found with similar incidence levels in all species tested except Pseudomonas aeruginosa, for which a novel separate family of class II transposons has been described before.

Base Sequence↗
PubMed1994

Stereoselectivity of DNA catenane fusion by resolvase.

Communications between distant sites on DNA often depend on the way in which the sites are connected. For example, site-specific recombination catalysed by Tn3 resolvase is most efficient when the 114-base-pair res recombination sites are directly repeated in the same DNA molecule. In vitro a supercoiled plasmid substrate containing two directly repeated res sites gives a resolution product in which the two recombinant circles are topologically linked as a simple (two-noded) catenane (Fig. 1a). Resolvase is highly selective in forming this product rather than unlinked circles or more complex catenanes. It does not catalyse recombination between sites on separate supercoiled molecules, or between inverted sites in the same supercoiled molecule. Tn3 resolution removes four negative supercoils from the substrate, an energetically favourable change which may drive the reaction: in relaxed or nicked circular substrates, resolution is incomplete and slower. Resolvase can catalyse fusion of the circles of a nicked or relaxed catenane, giving a single unknotted circular product. The fusion is the precise topological reversal of resolution, introducing four negative supercoils into a relaxed catenane substrate, and should therefore not proceed if the catenane is already negatively supercoiled. Here we study recombination between res sites in non-supercoiled DNA circles linked into simple catenanes. We used (+2) and (-2) catenanes, which differ only in the direction in which one circle is threaded through the other (Fig. 2a). Although stereoselectivity is a feature of enzyme catalysis, it is not obvious how resolvase can distinguish between these subtly different catenane diastereomers. A model for the intertwining of the res site DNA in the catalytically active complex predicts that only the (-2) catenane will recombine, giving unknotted and 4-noded knot circular products. We have confirmed this prediction for the Tn3 and Tn21 resolvases.

DNA, Circular↗