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Vincent Burrus - One of the best experts on this subject based on the ideXlab platform.
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Replication and Active Partition of Integrative and Conjugative Elements (ICEs) of the SXT/R391 Family: The Line between ICEs and Conjugative Plasmids Is Getting Thinner
PLoS genetics, 2015Co-Authors: Nicolas Carraro, Dominique Poulin, Vincent BurrusAbstract:Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-Transfer to other bacteria by conjugation. Conjugative Transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative Transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the Origin of Transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative Transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.
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Integrating conjugative elements of the SXT/R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands.
Molecular microbiology, 2010Co-Authors: Aurélie Daccord, Daniela Ceccarelli, Vincent BurrusAbstract:Summary In vibrios and enterobacteria lateral gene Transfer is often facilitated by integrating conjugative elements (ICEs) of the SXT/R391 family. SXT/R391 ICEs integrate by site-specific recombination into prfC and Transfer by conjugation, a process that is initiated at a specific locus called the Origin of Transfer (oriTSXT). We identified genomic islands (GIs) harbouring a sequence that shares > 63% identity with oriTSXT in three species of Vibrio. Unlike SXT/R391 ICEs, these GIs are integrated into a gene coding for a putative stress-induced protein and do not appear to carry any gene coding for a conjugative machinery or for mobilization proteins. Our results show that SXT/R391 ICEs trigger the excision and mediate the conjugative Transfer in trans of the three Vibrio GIs at high frequency. GIs' excision is independent of the ICE-encoded recombinase and is controlled by the ICE-encoded transcriptional activator SetCD, which is expressed during the host SOS response. Both mobI and traI, two ICE-borne genes involved in oriT recognition, are essential for GIs' Transfer. We also found that SXT/R391 ICEs mobilize in trans over 1 Mb of chromosomal DNA located 5′ of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.
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identification of the Origin of Transfer orit and a new gene required for mobilization of the sxt r391 family of integrating conjugative elements
Journal of Bacteriology, 2008Co-Authors: Daniela Ceccarelli, Aurélie Daccord, Mélissa René, Vincent BurrusAbstract:Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs Transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the Origin of Transfer (oriT). The SXT/R391 family of ICEs includes SXTMO10, R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXTMO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriTSXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB′. The oriTSXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriTSXT and oriTR391 by R391 and SXTMO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriTSXT, which proved to be essential for SXTMO10 Transfer and SXTMO10-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXTMO10-dependent Transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXTMO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriTSXT.
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Identification of the Origin of Transfer (oriT) and a New Gene Required for Mobilization of the SXT/R391 Family of Integrating Conjugative Elements
Journal of bacteriology, 2008Co-Authors: Daniela Ceccarelli, Aurélie Daccord, Mélissa René, Vincent BurrusAbstract:Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs Transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the Origin of Transfer (oriT). The SXT/R391 family of ICEs includes SXTMO10, R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXTMO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriTSXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB′. The oriTSXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriTSXT and oriTR391 by R391 and SXTMO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriTSXT, which proved to be essential for SXTMO10 Transfer and SXTMO10-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXTMO10-dependent Transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXTMO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriTSXT.
Joel F. Schildbach - One of the best experts on this subject based on the ideXlab platform.
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tracking f plasmid trai relaxase processing reactions provides insight into f plasmid Transfer
Nucleic Acids Research, 2011Co-Authors: Lubomir Dostal, Sichen Shao, Joel F. SchildbachAbstract:Early in F plasmid conjugative Transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the Origin of Transfer called nic. The reaction covalently links TraI Tyr16 to the 5'-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3'-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during Transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is Transferred to the recipient with a free 3'-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for Transfer. Therefore, two TraI molecules are required for F plasmid Transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3'-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization.
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examination of an inverted repeat within the f factor Origin of Transfer context dependence of f trai relaxase dna specificity
Nucleic Acids Research, 2006Co-Authors: Sarah L Williams, Joel F. SchildbachAbstract:Prior to conjugative Transfer of plasmids, one plasmid strand is cleaved in a site- and strand-specific manner by an enzyme called a relaxase or nickase. In F and related plasmids, an inverted repeat is located near the plasmid strand cleavage site, and others have proposed that the ability of this sequence to form a hairpin when in single-stranded form is important for Transfer. Substitutions were introduced into a cloned F oriT region and their effects on plasmid Transfer were assessed. For those substitutions that substantially reduced Transfer, the results generally correlated with effects on in vitro binding of oligonucleotides to the F TraI relaxase domain rather than with predicted effects on hairpin formation. One substitution shown previously to dramatically reduce both plasmid Transfer and in vitro binding to a 17-base oligonucleotide had little apparent effect on binding to a 30-base oligonucleotide that contained the hairpin region. Results from subsequent experiments strongly suggest that the relaxase domain can bind to hairpin oligonucleotides in two distinct manners with different sequence specificities, and that the protein binds the oligonucleotides at the same or overlapping sites.
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dna recognition by f factor trai36 highly sequence specific binding of single stranded dna
Biochemistry, 2001Co-Authors: Jennifer C Stern, Joel F. SchildbachAbstract:The TraI protein has two essential roles in Transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid Origin of Transfer (oriT), cutting the DNA strand that is Transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to Transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affiniti...
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Specific DNA Recognition by F Factor TraY Involves β-Sheet Residues
Journal of Biological Chemistry, 1999Co-Authors: Joel F. SchildbachAbstract:Abstract The F Factor TraY protein is a sequence-specific DNA-binding protein required for efficient conjugal Transfer. Genetic and biochemical studies indicate that TraY has two functional roles in conjugation. TraY binds to the PY promoter to up-regulate transcription of tra genes. TraY also binds to the plasmid Origin of Transfer (oriT), serving as an accessory protein in the nicking of F Factor in preparation for Transfer. TraY is thought to belong to the ribbon-helix-helix family of transcription factors. These proteins contact DNA using residues of an antiparallel β-sheet. We engineered and characterized six TraY mutants each having a single potential β-sheet DNA contact residue replaced with Ala. Most TraY mutants had significantly reduced affinity for the TraY oriT binding site while possessing near wild-type stability and nonspecific DNA recognition. These results indicate that TraY β-sheet residues participate in DNA recognition, and support inclusion of TraY in the ribbon-helix-helix family.
Daniela Ceccarelli - One of the best experts on this subject based on the ideXlab platform.
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Integrating conjugative elements of the SXT/R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands.
Molecular microbiology, 2010Co-Authors: Aurélie Daccord, Daniela Ceccarelli, Vincent BurrusAbstract:Summary In vibrios and enterobacteria lateral gene Transfer is often facilitated by integrating conjugative elements (ICEs) of the SXT/R391 family. SXT/R391 ICEs integrate by site-specific recombination into prfC and Transfer by conjugation, a process that is initiated at a specific locus called the Origin of Transfer (oriTSXT). We identified genomic islands (GIs) harbouring a sequence that shares > 63% identity with oriTSXT in three species of Vibrio. Unlike SXT/R391 ICEs, these GIs are integrated into a gene coding for a putative stress-induced protein and do not appear to carry any gene coding for a conjugative machinery or for mobilization proteins. Our results show that SXT/R391 ICEs trigger the excision and mediate the conjugative Transfer in trans of the three Vibrio GIs at high frequency. GIs' excision is independent of the ICE-encoded recombinase and is controlled by the ICE-encoded transcriptional activator SetCD, which is expressed during the host SOS response. Both mobI and traI, two ICE-borne genes involved in oriT recognition, are essential for GIs' Transfer. We also found that SXT/R391 ICEs mobilize in trans over 1 Mb of chromosomal DNA located 5′ of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.
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identification of the Origin of Transfer orit and a new gene required for mobilization of the sxt r391 family of integrating conjugative elements
Journal of Bacteriology, 2008Co-Authors: Daniela Ceccarelli, Aurélie Daccord, Mélissa René, Vincent BurrusAbstract:Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs Transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the Origin of Transfer (oriT). The SXT/R391 family of ICEs includes SXTMO10, R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXTMO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriTSXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB′. The oriTSXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriTSXT and oriTR391 by R391 and SXTMO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriTSXT, which proved to be essential for SXTMO10 Transfer and SXTMO10-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXTMO10-dependent Transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXTMO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriTSXT.
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Identification of the Origin of Transfer (oriT) and a New Gene Required for Mobilization of the SXT/R391 Family of Integrating Conjugative Elements
Journal of bacteriology, 2008Co-Authors: Daniela Ceccarelli, Aurélie Daccord, Mélissa René, Vincent BurrusAbstract:Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs Transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the Origin of Transfer (oriT). The SXT/R391 family of ICEs includes SXTMO10, R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXTMO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriTSXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB′. The oriTSXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriTSXT and oriTR391 by R391 and SXTMO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriTSXT, which proved to be essential for SXTMO10 Transfer and SXTMO10-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXTMO10-dependent Transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXTMO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriTSXT.
Aurélie Daccord - One of the best experts on this subject based on the ideXlab platform.
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Integrating conjugative elements of the SXT/R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands.
Molecular microbiology, 2010Co-Authors: Aurélie Daccord, Daniela Ceccarelli, Vincent BurrusAbstract:Summary In vibrios and enterobacteria lateral gene Transfer is often facilitated by integrating conjugative elements (ICEs) of the SXT/R391 family. SXT/R391 ICEs integrate by site-specific recombination into prfC and Transfer by conjugation, a process that is initiated at a specific locus called the Origin of Transfer (oriTSXT). We identified genomic islands (GIs) harbouring a sequence that shares > 63% identity with oriTSXT in three species of Vibrio. Unlike SXT/R391 ICEs, these GIs are integrated into a gene coding for a putative stress-induced protein and do not appear to carry any gene coding for a conjugative machinery or for mobilization proteins. Our results show that SXT/R391 ICEs trigger the excision and mediate the conjugative Transfer in trans of the three Vibrio GIs at high frequency. GIs' excision is independent of the ICE-encoded recombinase and is controlled by the ICE-encoded transcriptional activator SetCD, which is expressed during the host SOS response. Both mobI and traI, two ICE-borne genes involved in oriT recognition, are essential for GIs' Transfer. We also found that SXT/R391 ICEs mobilize in trans over 1 Mb of chromosomal DNA located 5′ of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.
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identification of the Origin of Transfer orit and a new gene required for mobilization of the sxt r391 family of integrating conjugative elements
Journal of Bacteriology, 2008Co-Authors: Daniela Ceccarelli, Aurélie Daccord, Mélissa René, Vincent BurrusAbstract:Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs Transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the Origin of Transfer (oriT). The SXT/R391 family of ICEs includes SXTMO10, R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXTMO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriTSXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB′. The oriTSXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriTSXT and oriTR391 by R391 and SXTMO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriTSXT, which proved to be essential for SXTMO10 Transfer and SXTMO10-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXTMO10-dependent Transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXTMO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriTSXT.
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Identification of the Origin of Transfer (oriT) and a New Gene Required for Mobilization of the SXT/R391 Family of Integrating Conjugative Elements
Journal of bacteriology, 2008Co-Authors: Daniela Ceccarelli, Aurélie Daccord, Mélissa René, Vincent BurrusAbstract:Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs Transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the Origin of Transfer (oriT). The SXT/R391 family of ICEs includes SXTMO10, R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXTMO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriTSXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB′. The oriTSXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriTSXT and oriTR391 by R391 and SXTMO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriTSXT, which proved to be essential for SXTMO10 Transfer and SXTMO10-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXTMO10-dependent Transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXTMO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriTSXT.
Fernando De La Cruz - One of the best experts on this subject based on the ideXlab platform.
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Cis-Acting Relaxases Guarantee Independent Mobilization of MOBQ4 Plasmids
Frontiers Media, 2020Co-Authors: Garcillán-barcia M. Pilar, Cuartas-lanza Raquel, Cuevas Ana, Fernando De La CruzAbstract:© 2019 Garcillán-Barcia, Cuartas-Lanza, Cuevas and de la Cruz.Plasmids are key vehicles of horizontal gene Transfer and contribute greatly to bacterial genome plasticity. In this work, we studied a group of plasmids from enterobacteria that encode phylogenetically related mobilization functions that populate the previously non-described MOBQ4 relaxase family. These plasmids encode two Transfer genes: mobA coding for the MOBQ4 relaxase; and mobC, which is non-essential but enhances the plasmid mobilization frequency. The Origin of Transfer is located between these two divergently transcribed mob genes. We found that MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative dissemination among clinically relevant enterobacteria. While highly similar in their mobilization module, two sub-groups with unrelated replicons (Rep_3 and ColE2) can be distinguished in this plasmid family. These subgroups can stably coexist (are compatible) and Transfer independently, despite Origin-of-Transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such a strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2017-86378-P, AEI/FEDER, UE, to FC) and Consejo Superior de Investigaciones Científicas (201820I143 to MG-B). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)
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relaxase dna binding and cleavage are two distinguishable steps in conjugative dna processing that involve different sequence elements of the nic site
Journal of Biological Chemistry, 2010Co-Authors: María Lucas, Gabriel Moncalián, Matilde Cabezas, Blanca Gonzalezperez, German Rivas, Fernando De La CruzAbstract:TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand Transfer reactions on a specific site (nic) of its Origin of Transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.
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conjugative dna metabolism in gram negative bacteria
Fems Microbiology Reviews, 2010Co-Authors: Fernando De La Cruz, Laura S Frost, Richard J. Meyer, Ellen L. ZechnerAbstract:Bacterial conjugation in Gram-negative bacteria is triggered by a signal that connects the relaxosome to the coupling protein (T4CP) and Transferosome, a type IV secretion system. The relaxosome, a nucleoprotein complex formed at the Origin of Transfer (oriT), consists of a relaxase, directed to the nic site by auxiliary DNA-binding proteins. The nic site undergoes cleavage and religation during vegetative growth, but this is converted to a cleavage and unwinding reaction when a competent mating pair has formed. Here, we review the biochemistry of relaxosomes and ponder some of the remaining questions about the nature of the signal that begins the process.
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Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Olga Draper, Fernando De La Cruz, Cristina Machón, Carolina Elvira César, Matxalen LlosaAbstract:Conjugative relaxases are the proteins that initiate bacterial conjugation by a site-specific cleavage of the Transferred DNA strand. In vitro, they show strand-Transferase activity on single-stranded DNA, which suggests they may also be responsible for recircularization of the Transferred DNA. In this work, we show that TrwC, the relaxase of plasmid R388, is fully functional in the recipient cell, as shown by complementation of an R388 trwC mutant in the recipient. TrwC transport to the recipient is also observed in the absence of DNA Transfer, although it still requires the conjugative coupling protein. In addition to its role in conjugation, TrwC is able to catalyze site-specific recombination between two Origin of Transfer (oriT) copies. Mutations that abolish TrwC DNA strand-Transferase activity also abolish oriT-specific recombination. A plasmid containing two oriT copies resident in the recipient cell undergoes recombination when a TrwC-piloted DNA is conjugatively Transferred into it. Finally, we show TrwC-dependent integration of the Transferred DNA into a resident oriT copy in the recipient cell. Our results indicate that a conjugative relaxase is active once in the recipient cell, where it performs the nicking and strand-Transfer reactions that would be required to recircularize the Transferred DNA. This TrwC site-specific integration activity in recipient cells may lead to future biotechnological applications.
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Recognition and processing of the Origin of Transfer DNA by conjugative relaxase TrwC
Nature Structural & Molecular Biology, 2003Co-Authors: Alicia Guasch, María Lucas, Gabriel Moncalián, Matilde Cabezas, Rosa Pérez-luque, Fernando De La Cruz, F. Xavier Gomis-rüth, Miquel CollAbstract:Relaxases are DNA strand Transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA Transfer. Upon binding to the Origin of Transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer � /� sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a � -ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.