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PubMed2000

Primate foamy virus Pol proteins are imported into the nucleus.

Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127(Pol), p85(RT/RN) and p40(IN) served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.

3T3 Cells↗
PubMed1993

Functional and physiological characterization of the Tn21 cassette for resistance genes in Tn2426.

The Tn21 subgroup of class II transposons plays an important role in the dissemination of resistance genes and especially in the epidemic spread of multi-resistance. This ability reflects the variety of resistance genes that associate with the streptomycin/spectinomycin-resistance gene (aadA) of Tn21. Deletion experiments with Tn2426, a typical member of the Tn21 subgroup, and sequencing of the region that accommodates additional resistance genes revealed significant structural characteristics. Each resistance gene was flanked by short, directly repeated recombinationally active sequences with unexpected variability in their sequence and length. The consensus for a recombinationally active sequence appeared to be 13 bp in length (TAAAACAANGNNA), compared to previous estimates of 54 bp. This sequence, in combination with the product of the integrase gene, is responsible for the genetic variability of members of the Tn21 family of transposable elements and the dissemination of multi-resistance.

Amikacin↗
PubMed1997

Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element.

Dichelobacter nodosus is the causative agent of ovine footrot. The vap regions of the D. nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence. The virulent D. nodosus strain A198 has multiple copies of the vap regions. In the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain A198 were analysed by Southern blotting and DNa sequencing. The results suggest that vap regions 1 and 2 rose by independent integration events into different tRNA genes. The discovery of a second integrase gene (intB), a gene with similarity to bacteriophage repressor proteins (regA), and a gene similar to an ORF from a conjugative transposon (gepA), suggests that a second genetic element, either a bacteriophage or a conjugative transposon, is integrated next to vap region 3 in the D. nodosus genome. The arrangement of intB and the vap regions in three other virulent strains and one benign strain was determined using using Southern blotting and PCR. One strain, H1215, contained vapE' and not vapE, and thus resembles vap region 3, suggesting that vap region 3 also may have arisen by an independent integration event. In all strains, a copy of intB was found next to the vap regions. The vap regions contain two genes, vapA and toxA, with similarity to the hig genes of the killer plasmid Rts1. Evidence is presented that vapA and toxA have a similar function in D. nodosus.

Amino Acid Sequence↗
PubMed1997

Tn5041: a chimeric mercury resistance transposon closely related to the toluene degradative transposon Tn4651.

This paper reports the discovery and characterization of Tn5041, a novel-type transposon vehicle for dissemination of mercury resistance in natural bacterial populations. Tn5041 (14876 bp), identified in a Pseudomonas strain from a mercury mine, is a Tn3 family mercury resistance transposon far outside the Tn21 subgroup. As in other Tn3 family transposons, Tn5041 duplicates 5 bp of the target sequence following insertion. Tn5041 apparently acquired its mer operon as a single-ended relic of a transposon belonging to the classical mercury resistance transposons of the Tn21 subgroup. The putative transposase and the 47 bp terminal inverted repeats of Tn5041 are closely related to those of the toluene degradative transposon Tn4651 and fall into a distinct subgroup on the fringe of the Tn3 family. The amino acid sequence of the putative resolvase of Tn5041 resembles site-specific recombinases of the integrase family. Besides the mer operon and putative transposition genes, Tn5041 contains a 4 kb region that accommodates a number of apparently defective genes and mobile elements.

Amino Acid Sequence↗
PubMed2000

The Vibrio cholerae O1 chromosomal integron.

Until the discovery of the Vibrio cholerae repeat (VCR), the gene capture and expression systems termed integrons had been typically associated with antibiotic-resistance gene cassettes with usually less than five genes in an array. A method is described for the cloning of the ends of large cassette arrays. Conserved restriction sites within VCRs facilitated the mapping by Southern hybridization and cloning of the 5' end of the VCR array, and using appropriate fragments it was possible to develop a physical map of the region of the V. cholerae chromosome. Sequence determination of the predicted beginning of this region revealed intI4, a member of the integron family of integrases. Comparison of these sequences from El Tor, Classical and serotype O134 V. cholerae strains identified the 3' end of the attI site, thereby defining the class 4 integron in one of the V. cholerae chromosomes, and providing the first evidence for integron-like site-specific recombination within V. cholerae. Conduction assays demonstrated IntI1-mediated recombination between VCRs. Restriction mapping places the sequences of intI4 and 26 VCR gene cassettes in arrays within a 120 kb region of the V. cholerae O1 strain 569B genome. This region contains an estimated 150 VCR gene cassettes, dwarfing previously described arrays. Southern analysis of genomic DNA from strains of Vibrio anguillarum, Vibrio mimicus and a number of V. cholerae serotypes revealed fragments that hybridized with VCR-specific probes but showed a high degree of restriction fragment length polymorphism. These data facilitate the identification of part of a new class 5 integron from V. mimicus.

Blotting, Southern↗
PubMed2001

Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules.

Comparative analysis of the conjugative transposons Tn5397 from Clostridium difficile and Tn916 from Enterococcus faecalis, and the CW459tet(M) element from Clostridium perfringens, has revealed that these tetracycline-resistance elements are closely related. All three elements contain the tet(M) resistance gene and have sequence similarity throughout their central region. However, they have very different integration/excision modules. Instead of the int and xis genes that are found in Tn916, Tn5397 has a large resolvase gene, tndX. The C. perfringens element encodes the putative Int459 protein, which is a member of the integrase family of site-specific recombinases but is not closely related to Int from Tn916. Based on these studies it is concluded that the clostridial elements have a modular genetic organization and were derived independently from distinct mobile genetic elements.

Base Sequence↗
PubMed1994

Trimethoprim resistance in urinary pathogens in northern Scotland: epidemic spread of a resistance plasmid encoding the type Ib trimethoprim-resistant dihydrofolate reductase.

The prevalence of trimethoprim resistance in enterobacterial urinary pathogens from hospitalised patients in the Angus district of northern Scotland (22.8%) was twice that found in similar isolates from patients attending general practitioners (11.2%). Thirty-three of the 143 trimethoprim-resistant strains were shown to harbour transferable plasmids conferring high-level trimethoprim resistance. In total, 17 different plasmid types were distinguished. Two plasmids, pUK1184 and pUK1185, accounted for 36% of the trimethoprim resistance plasmids and were shown by restriction endonuclease digestion fingerprints to be closely related to plasmid pUK28, previously demonstrated to be endemic in urinary pathogens in the Edinburgh area. Only 21% of the plasmids were shown to encode the type Ia trimethoprim-resistant dihydrofolate reductase, whereas 70% of the trimethoprim resistance plasmids were found to encode the type Ib dihydrofolate reductase. Hybridisation of the trimethoprim resistance plasmids identified in this study with gene probes specific for the integrase genes of transposons Tn7 and Tn21 indicates that the dhfrIa is rarely present within Tn7 or related transposons in these plasmids and may be more prevalent within Tn21-like transposons. In contrast, with the exception of the two endemic plasmids that harboured the dhfrIb gene within a Tn7-like transposon, the majority of dhfrIb genes were not found to be associated with either Tn7- or Tn21-like structures.

Bacteriuria↗
PubMed2004

The complete sequences of plasmids pB2 and pB3 provide evidence for a recent ancestor of the IncP-1beta group without any accessory genes.

The nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1beta backbone, which diverged early from the sequenced IncP-1beta plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14.4 kb or 19.0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1beta plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)-tetR-tnpA(IS26) fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D beta-lactamase (bla(NPS-2)); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)-tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1beta plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1beta plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1beta plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.

Antiporters↗
PubMed2005

Two site-specific recombinases are implicated in phenotypic variation and competitive rhizosphere colonization in Pseudomonas fluorescens.

The biocontrol agent Pseudomonas fluorescens F113 undergoes phenotypic variation during rhizosphere colonization, and this variation has been related to the activity of a site-specific recombinase encoded by the sss gene. Here, it is shown that a second recombinase encoded by the xerD gene is also implicated in phenotypic variation. A putative xerD gene from this strain was cloned, and sequence analysis confirmed that it encoded a site-specific recombinase of the lambda integrase family. Mutants affected in the sss or xerD genes produced a very low quantity of phenotypic variants compared to the wild-type strain, both under prolonged cultivation in the laboratory and after rhizosphere colonization, and they were severely impaired in competitive root colonization. Overexpression of the genes encoding either recombinase resulted in a substantial increment in the production of phenotypic variants under both culture and rhizosphere colonization conditions, implying that both site-specific recombinases are involved in phenotypic variation. Overexpression of the sss gene suppressed the phenotype of a xerD mutant, but overexpression of the xerD gene had no effect on the phenotype of an sss mutant. Genetic analysis of the phenotypic variants obtained after overexpression of the genes encoding both the recombinases showed that they carried mutations in the gacA/S genes, which are necessary to produce a variety of secondary metabolites. These results indicate that the Gac system is affected by the activity of the site-specific recombinases. Transcriptional fusions of the sss and xerD genes with a promoterless lacZ gene showed that both genes have a similar expression pattern, with maximal expression during stationary phase. Although the expression of both genes was independent of diffusible compounds present in root exudates, it was induced by the plant, since bacteria attached to the root showed enhanced expression.

Amino Acid Sequence↗
PubMed2004

Natural simian foamy virus infection in wild-caught gorillas, mandrills and drills from Cameroon and Gabon.

A survey for the presence of simian foamy retroviruses (SFVs) was performed in 44 wild-caught apes and monkeys, including 27 gorillas, 11 mandrills and six drills, originating from south Cameroon or Gabon. Combined serological and/or nested-PCR assays indicated SFV infection among five Gorilla gorilla gorilla, seven Mandrillus sphinx and two Mandrillus leucophaeus. Sequences of a 425 bp fragment of the integrase gene were obtained for 11 animals. Phylogenetic studies indicated that strains from gorillas, mandrills and drills each formed a highly supported phylogenetic clade with, moreover, the existence of two different gorilla SFVs. This study demonstrates for the first time that these animals are naturally infected with specific SFVs. In the context of simian-to-human interspecies transmission, the results confirm that such viruses can also infect humans, as the SFVs identified in wild-caught animals were the same as those recently reported as infecting hunters living in the same geographical areas.

Animals↗
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↗
PubMed1994

Complete transposition requires four active monomers in the mu transposase tetramer.

A tetramer of Mu transposase (MuA) cleaves and joins multiple DNA strands to promote transposition. Derivatives of MuA altered at two acidic residues that are conserved among transposases and retroviral integrases form tetramers but are defective in both cleavage and joining. These mutant proteins were used to analyze the contribution of individual monomers to the activity of the tetramer. The performance of different protein combinations demonstrates that not all monomers need to be catalytically competent for the complex to promote an individual cleavage or joining reaction. Furthermore, the results indicate that each pair of essential residues is probably donated to the active complex by a single monomer. Although stable, tetramers composed of a mixture of mutant and wild-type MuA generate products cleaved at only one end and with only one end joined to the target DNA. The abundance of these abortive products and the ratios of the two proteins in complexes stalled at different steps indicate that the complete reaction requires the activity of all four monomers. Thus, each subunit of MuA appears to use the conserved acidic amino acids to promote one DNA cleavage or one DNA joining reaction.

Bacteriophage mu↗
PubMed2006

High efficiency site-specific genetic engineering of the mosquito genome.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

Aedes↗
PubMed1991

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

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.

Bacillus subtilis↗
PubMed1992

Gene cassettes from the insert region of integrons are excised as covalently closed circles.

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.

Base Sequence↗
PubMed1993

Sequence requirements for target activity in site-specific recombination mediated by the Int protein of transposon Tn 1545.

Excision and integration of Tn1545 occur by reciprocal site-specific recombination between 6 (or 7) bp variable sequences present in the recombining attachment (att) sites and designated overlap regions. We devised an assay for Tn1545 transposition in which derivatives containing the cis-acting transposition sequences (attTn 1545) integrate into a target replicon when complemented in trans by the transposon-encoded integrase Int-Tn. This assay was used to determine the characteristics of the DNA sequence that influence target site selection. Characterization of several integration sites indicated that a 20 bp segment, designated attB, contains the sequences required for target activity. It also appeared that (i) the target activity depends upon the extent of homology between the 7bp segments flanking the overlap regions in attB and attTn 1545, and (ii) the degree of homology between the two recombining overlap regions does not affect the level of target activity and has no influence on integration orientation.

Bacterial Proteins↗
PubMed1993

Site-specific insertion of gene cassettes into integrons.

Site-specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5' to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.

Base Sequence↗