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R. Devoret - One of the best experts on this subject based on the ideXlab platform.
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Introduction of a UV-damaged replicon into a Recipient Cell is not a sufficient condition to produce an SOS-inducing signal.
Mutation research, 1991Co-Authors: Suzanne Sommer, Alvaro C. Leitão, Alberto Bernardi, Adriana Bailone, R. DevoretAbstract:Abstract Three models have been proposed for the nature of the SOS-inducing signal in E. coli . One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein. We tested the models by measuring SOS induction produced by introducing into Recipient Cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the Recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a Recipient Cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.
Fernando De La Cruz - One of the best experts on this subject based on the ideXlab platform.
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CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids
RNA Biology, 2013Co-Authors: Edze R Westra, Søren Høgh, Sarah Neumann, Peter C Fineran, Raymond H.j. Staals, Gerrit Gort, Fernando De La Cruz, Stan J. J. BrounsAbstract:Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid’s mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the Recipient Cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the Recipient Cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against ...
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Conjugative transfer can be inhibited by blocking relaxase activity within Recipient Cells with intrabodies
Molecular Microbiology, 2007Co-Authors: M. Pilar Garcillán-barcia, Paola Jurado, Blanca González-pérez, Luis A Fernandez, Gabriel Moncalián, Fernando De La CruzAbstract:Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to Recipient Cells during bacterial conjugation, we investigated whether blocking relaxase activity in the Recipient Cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli Cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the Recipient Cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the Recipient Cell and open a new approach to identify specific inhibitors of bacterial conjugation.
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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.
Joel F. Schildbach - One of the best experts on this subject based on the ideXlab platform.
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assembly and mechanisms of bacterial type iv secretion machines
Philosophical Transactions of the Royal Society B, 2012Co-Authors: Ellen L Zechner, Silvia Lang, Joel F. SchildbachAbstract:Type IV secretion occurs across a wide range of prokaryotic Cell envelopes: Gram-negative, Gram-positive, Cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the Cell surface as a pilus structure important for direct contact and penetration of the Recipient Cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the Cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host Cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.
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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...
Matxalen Llosa - One of the best experts on this subject based on the ideXlab platform.
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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.
Suzanne Sommer - One of the best experts on this subject based on the ideXlab platform.
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Introduction of a UV-damaged replicon into a Recipient Cell is not a sufficient condition to produce an SOS-inducing signal.
Mutation research, 1991Co-Authors: Suzanne Sommer, Alvaro C. Leitão, Alberto Bernardi, Adriana Bailone, R. DevoretAbstract:Abstract Three models have been proposed for the nature of the SOS-inducing signal in E. coli . One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein. We tested the models by measuring SOS induction produced by introducing into Recipient Cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the Recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a Recipient Cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.
