Conjugative Relaxase TrwC Research Articles

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus.

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.

DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens.

We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery.IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

A Functional oriT in the Ptw Plasmid of Burkholderia cenocepacia Can Be Recognized by the R388 Relaxase TrwC.

Burkholderia cenocepacia is both a plant pathogen and the cause of serious opportunistic infections, particularly in cystic fibrosis patients. B. cenocepacia K56-2 harbors a native plasmid named Ptw for its involvement in the Plant Tissue Watersoaking phenotype. Ptw has also been reported to be important for survival in human cells. Interestingly, the presence of PtwC, a homolog of the conjugative relaxase TrwC of plasmid R388, suggests a possible function for Ptw in conjugative DNA transfer. The ptw region includes Type IV Secretion System genes related to those of the F plasmid. However, genes in the adjacent region shared stronger homology with the R388 genes involved in conjugative DNA metabolism. This region included the putative relaxase ptwC, a putative coupling protein and accessory nicking protein, and a DNA segment with high number of inverted repeats and elevated AT content, suggesting a possible oriT. Although we were unable to detect conjugative transfer of the Ptw resident plasmid, we detected conjugal mobilization of a co-resident plasmid containing the ptw region homologous to R388, demonstrating the cloned ptw region contains an oriT. A similar plasmid lacking ptwC could not be mobilized, suggesting that the putative relaxase PtwC must act in cis on its oriT. Remarkably, we also detected mobilization of a plasmid containing the Ptw oriT by the R388 relaxase TrwC, yet we could not detect PtwC-mediated mobilization of an R388 oriT-containing plasmid. Our data unambiguously show that the Ptw plasmid harbors DNA transfer functions, and suggests the Ptw plasmid may play a dual role in horizontal DNA transfer and eukaryotic infection.

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

BackgroundBacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.Methodology/Principal FindingsWe have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.Conclusions/SignificanceThe results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.

TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a "closed" conformation. In an IHF-deficient (IHF-) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF- background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an "open" form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

A new domain of conjugative relaxase TrwC responsible for efficient oriT‐specific recombination on minimal target sequences

We show that relaxase TrwC promotes recombination between two directly repeated oriTs while related relaxases TraI of F and pKM101 do not. Efficient recombination required also relaxosome accessory protein TrwA even after deletion of TrwA binding sites at oriT, suggesting that the effect of TrwA is mediated by protein-protein interactions. TrwC relaxase domain was necessary but not sufficient to catalyse recombination efficiently. Full recombinase activity was obtained with the N-terminal 600 residues of TrwC. The minimal target sequences required for recombination were different at each of the two involved oriTs: oriT1 could be reduced to the nic site and TrwC binding site, while oriT2 required an extended sequence including a set of iterons that are not required for conjugation. TrwC-mediated integration of a transferred DNA into a resident oriT copy required a complete oriT in the recipient. We observed dramatic changes in the efficiency of recombination between tandem oriTs linked to the direction of plasmid replication and transcription through oriT1. We propose that recombination is triggered by the generation of a single-stranded DNA at oriT1 that causes TrwC nicking. The resulting TrwC-DNA complex reacts with oriT2, excising the intervening DNA. This intermediate can be resolved by host-encoded replication functions.

Erratum: Corrigendum: Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Corrigendum: Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC.

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 alpha/beta 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 beta-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.