Noncanonical cell-to-cell DNA transfer in Thermus spp. is insensitive to argonaute-mediated interference.

Horizontal gene transfer drives the rapid evolution of bacterial populations. Classical processes that promote the lateral flow of genetic information are conserved throughout the prokaryotic world. However, some species have nonconserved transfer mechanisms that are not well known. This is the case for the ancient extreme thermophile Thermus thermophilus. In this work, we show that T. thermophilus strains are capable of exchanging large DNA fragments by a novel mechanism that requires cell-to-cell contacts and employs components of the natural transformation machinery. This process facilitates the bidirectional transfer of virtually any DNA locus but favors by 10-fold loci found in the megaplasmid over those in the chromosome. In contrast to naked DNA acquisition by transformation, the system does not activate the recently described DNA-DNA interference mechanism mediated by the prokaryotic Argonaute protein, thus allowing the organism to distinguish between DNA transferred from a mate and exogenous DNA acquired from unknown hosts. This Argonaute-mediated discrimination may be tentatively viewed as a strategy for safe sharing of potentially "useful" traits by the components of a given population of Thermus spp. without increasing the genome sizes of its individuals.

Nuclear targeting of a bacterial integrase that mediates site-specific recombination between bacterial and human target sequences.

TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian 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.

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells.

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.