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1.
The 34,734-bp element ICESt1 from Streptococcus thermophilus CNRZ368 is site-specifically integrated into the 3(') end of the gene fda. ICESt1 encodes integrative functions and putative transfer functions. Six proteins of the putative conjugative system of ICESt1 are related to those encoded by the conjugative transposon Tn916 from Enterococcus faecalis. A comparison of these proteins with those encoded by the complete or partial genome sequences of various low G+C bacteria including Bacillus subtilis, Clostridium difficile, E. faecalis, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans revealed the presence of numerous putative site-specific integrative conjugative elements and/or conjugative transposons within these genomes. Sequence comparisons revealed that these elements possess a modular structure and that exchanges of unrelated or distantly related modules and genes have occurred between these elements, and also plasmids and prophages. These exchanges have probably led to modifications in the site specificity of integration of these elements. Therefore, a distinction between low specificity integrative conjugative elements (i.e., conjugative transposons) and site-specific integrative conjugative elements does not appear to be relevant. We propose to call all the conjugative elements that excise by site-specific recombination and integrate by recombination between a specific site of a circular intermediate and another site, "Integrative and Conjugative Elements" (ICEs), irrespective of the integration specificity. 相似文献
2.
Characterization of a Novel Integrative Element, ICESt1, in the Lactic Acid Bacterium Streptococcus thermophilus
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Vincent Burrus Yvonne Roussel Bernard Decaris Grard Gudon 《Applied microbiology》2000,66(4):1749-1753
The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3′ end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element. 相似文献
3.
Bellanger X Morel C Decaris B Guédon G 《Journal of molecular microbiology and biotechnology》2008,14(1-3):16-21
The integrative and conjugative elements (ICEs) excise by site-specific recombination between attL and attR flanking sites, self-transfer the resulting circular form and integrate into the genome of the recipient cell. Two putative ICEs, ICESt1 and ICESt3, are integrated in the same locus in 2 strains of Streptococcusthermophilus. ICESt1 is a composite element harbouring an internal recombination site, attL'. The recombination between attL' and attR leads to the excision of a shorter putative ICE, ICESt2. ICESt1/ICESt2 and ICESt3 carry related regulation modules sharing the open reading frame arp1 that encodes a protein related to the cI repressor of the phage lambda. The repressors belonging to this family autoproteolyse in the presence of damaged DNA. Treatments with mitomycin C induce an increase in the excision of ICESt1, ICESt2 and ICESt3. Furthermore, the arp1 deletion leads to a 1,000-fold increase in the excision of ICESt1 and ICESt2 and to a decrease in the excision induction by mitomycin C. Thus, all together, these results suggest that the autocleavage of the arp1 repressor is involved in derepression of the S. thermophilus putative ICE excision by mitomycin C. 相似文献
4.
Derepression of excision of integrative and potentially conjugative elements from Streptococcus thermophilus by DNA damage response: implication of a cI-related repressor
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A DNA-damaging agent, mitomycin C, derepresses the site-specific excision of two integrative and potentially conjugative elements from Streptococcus thermophilus, ICESt1 and ICESt3. The regulation pathway involves a repressor related to phage lambda cI repressor. It could also involve a putative regulator related to another type of phage repressors, the "cI-like" repressors. 相似文献
5.
Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916. 总被引:1,自引:1,他引:0
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The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases. 相似文献
6.
Conjugative transposition: Tn916 integrase contains two independent DNA binding domains that recognize different DNA sequences. 总被引:10,自引:0,他引:10
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Transposition of the conjugative transposon Tn916 requires the activity of a protein, called Int, which is related to members of the integrase family of site-specific recombinases. This family includes phage lambda integrase as well as the Cre, FLP and XerC/XerD recombinases. Different proteins, consisting of fragments of Tn916 Int protein fused to the C-terminal end of maltose binding protein (MBP) were purified from Escherichia coli. DNase I protection experiments showed that MBP-INT proteins containing the C-terminal end of Int bound to the ends of the transposon and adjacent plasmid DNA. MBP-INT proteins containing the N-terminal end of Int bound to sequences within the transposon close to each end. Competition binding experiments showed that the sites recognized by the C- and N-terminal regions of Int did not compete with each other for binding to MBP-INT. We suggest that Tn916 and related conjugative transposons are unique among members of the integrase family of site-specific recombination systems because the presence of two DNA binding domains in the Int protein might allow Int to bridge recombining sites, and this bridging seems to be the sole mechanism ensuring that only correctly aligned molecules undergo recombination. 相似文献
7.
The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family. 总被引:8,自引:3,他引:5
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NBU1 is a 10.3-kbp integrated Bacteroides element that can be induced to excise from the chromosome and can be mobilized to a recipient by trans-acting functions provided by certain Bacteroides conjugative transposons. The NBU1 transfer intermediate is a covalently closed circle, which is presumed to be the form that integrates into the recipient genome. We report here that a 2.4-kbp segment of NBU1 was all that was required for site-specific integration into the chromosome of Bacteroides thetaiotaomicron 5482. This 2.4-kbp region included the joined ends of the NBU1 circular form (attN1) and a single open reading frame, intN1, which encoded the integrase. Previously, we had found that NBU1 integrates preferentially into a single site in B. thetaiotaomicron 5482. We have now shown that the NBU1 target site is located at the 3' end of a Leu-tRNA gene. The NBU1 integrase gene, intN1, was sequenced. The predicted protein had little overall amino acid sequence similarity to any proteins in the databases but had limited carboxy-terminal similarity to the integrases of lambdoid phages and to the integrases of the gram-positive conjugative transposons Tn916 and Tn1545. We also report that the intN1 gene is expressed constitutively. 相似文献
8.
Conjugative transposition of Tn916 requires the excisive and integrative activities of the transposon-encoded integrase. 总被引:2,自引:0,他引:2
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M J Storrs C Poyart-Salmeron P Trieu-Cuot P Courvalin 《Journal of bacteriology》1991,173(14):4347-4352
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. 相似文献
9.
Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E. 相似文献
10.
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. 相似文献
11.
The large resolvase TndX is required and sufficient for integration and excision of derivatives of the novel conjugative transposon Tn5397
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Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile. This element can transfer between C. difficile strains and to and from Bacillus subtilis. It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the role of tndX, the gene was cloned and the protein was expressed in Escherichia coli. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion in E. coli. 相似文献
12.
Tn552, a novel transposable element from Staphylococcus aureus 总被引:37,自引:5,他引:32
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. 相似文献
13.
Transposition genes of the Bacteroides mobilizable transposon Tn4555: role of a novel targeting gene
Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein. 相似文献
14.
Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX
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Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile. Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916. However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916. Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916. Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C. difficile. Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide. The Tn5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile. A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis. This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients. 相似文献
15.
The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn4451.
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Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostridium perfringens and is found on the conjugative plasmid pIP401. The element undergoes spontaneous excision from multicopy plasmids in Escherichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is probably the transposition intermediate. Excision of Tn4451 is dependent upon the site-specific recombinase TnpX, which contains potential motifs associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid residues within these domains was used to show that the resolvase/invertase domain was essential for TnpX-mediated excision of Tn4451 from multicopy plasmids in E. coli. An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity. The Tn4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanked by directly repeated GA dinucleotides, and there was also a GA at the junction of the circular form, where the left and right termini of Tn4451 were fused. We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp staggered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives with altered GA dinucleotides provided experimental evidence to support the model. 相似文献
16.
Identification and nucleotide sequence analysis of a transfer-related region in the streptococcal conjugative transposon Tn5252. 总被引:4,自引:1,他引:3
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To obtain a functional map of Tn5252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORF1 and ORF2, adjoining the att site. The organization of this region and the structure of the predicted integrase encoded by ORF1 were found to be similar to those of other site-specific recombination systems. 相似文献
17.
A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to phiST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5'-GATC-3'. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5'-GATC-3'. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems. 相似文献
18.
Nucleotide sequence analysis of the termini and chromosomal locus involved in site-specific integration of the streptococcal conjugative transposon Tn5252. 总被引:4,自引:1,他引:3
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The 47-kb, broad-host-range, streptococcal conjugative transposon Tn5252 is capable of site-specific integration into the pneumococcal chromosome. We present the nucleotide sequence of the terminal regions of the transposon and its target site in the pneumococcal genome. No inverted repeats were found at the termini of the transposon. A 72-bp region of the target was present on either side following the insertion of Tn5252 and appeared to serve as a signal for its integration and excision. The data suggest that the left copy of the 72-bp segment was a part of the conjugative element, the crossover point of integration was nonrandom within this region, and the mechanism of insertion could resemble that of the site-specific temperate phages. 相似文献
19.
The integrase protein catalyzes the excision and integration of the Tn916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel beta-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-Ppol-DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent approximately 35 degrees and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon. 相似文献
20.
Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases
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In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange. 相似文献