MicroReviewBacterial conjugation: a two-step mechanism for DNA transport the recipient cytoplasm. This is the first step in conjugation. The second step is the active pumping of the DNA to the recipient, using the already available T4SS transport conduit. It is proposed that this second step is catalysed by the coupling proteins. Our 'shoot and pump' model solves the protein-DNA transport paradox of T4SS. Conjugation as the merging of two ancient bacterial systemsDNA replication and macromolecule transport across membranes are basic processes of life. If linked, they could form the basis for a new mechanism, DNA secretion, by simply driving the displaced replicating DNA strand to the macromolecular transporter in the membrane. It is tempting to imagine that bacterial conjugation once arose in this way. For this to happen, some DNAreplicating enzymes on one side and protein transporters on the other would have to adjust to new substrates. In addition, a new protein is needed to bring the displaced DNA in contact with the transporter: a coupling protein.With this one and only new protein (and a few adjustments), bacteria could have acquired the basics for conjugation, a mechanism that provides them with a unique means for genetic exchange and a powerful source of genetic variability.Indeed, bacterial conjugation systems (including the related Vir system that Agrobacterium tumefaciens uses to transfer DNA to plants) show striking similarities to both DNA replication and macromolecular transport systems. Conjugative DNA-processing enzymes and their substrate DNA sequence (components of the so-called relaxosome) show extended sequence similarity to rolling-circle replication (RCR) systems (Waters and Guiney, 1993). Moreover, the set of conjugative proteins that assembles the membrane transporter belong to the type IV secretion system (T4SS) family (Christie, 2001). In addition to proteins with similarity to RCR or T4SS, all conjugative systems include one protein that has no obvious counterpart in either system. This is considered to be the coupling protein that connects the relaxosome with the membrane transporter. The evidence for this role will be discussed below. SummaryBacterial conjugation is a promiscuous DNA transport mechanism. Conjugative plasmids transfer themselves between most bacteria, thus being one of the main causal agents of the spread of antibiotic resistance among pathogenic bacteria. Moreover, DNA can be transferred conjugatively into eukaryotic host cells. In this review, we aim to address several basic questions regarding the DNA transfer mechanism. Conjugation can be visualized as a DNA rolling-circle replication (RCR) system linked to a type IV secretion system (T4SS), the latter being macromolecular transporters widely involved in pathogenic mechanisms. The scheme 'replication + secretion' suggests how the mechanism would work on the DNA substrate and at the bacterial membrane. But, how do these two parts come into contact? Furthermore, how is the DNA transported? T4SS are known to be invo...

Conjugative coupling proteins (CPs) are proposed to play a role in connecting the relaxosome to a type IV secretion system (T4SS) during bacterial conjugation. Here we present biochemical and genetic evidence indicating that the prototype CP, TrwB, interacts with both relaxosome and type IV secretion components of plasmid R388. The cytoplasmic domain of TrwB immobilized in an affinity resin retained TrwC and TrwA proteins, the components of R388 relaxosome. By using the bacterial two-hybrid system, a strong interaction was detected between TrwB and TrwE, a core component of the conjugative T4SS. This interaction was lost when the transmembrane domains of either TrwB or TrwE were deleted, thus suggesting that it takes place within the membrane or periplasmic portions of both proteins. We have also analyzed the interactions with components of the related IncN plasmid pKM101. Its CP, TraJ, did not interact with TrwA, suggesting a highly specific interaction with the relaxosome. On the other side, CPs from three different conjugation systems were shown to interact with both their cognate TrwE-like component and the heterologous ones, suggesting that this interaction is less specific. Mating experiments among the three systems confirmed that relaxosome components need their cognate CP for transfer, whereas T4SSs are interchangeable. As a general rule, there is a correlation between the strength of the interaction seen by two-hybrid analysis and the efficiency of transfer.

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 strandtransferase 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.bacterial conjugation ͉ plasmid R388 ͉ site-specific integration ͉ strand transferase ͉ TrwC B acterial conjugation is a widespread mechanism for horizontal DNA transfer among prokaryotes. Under laboratory conditions, conjugation has also been reported between bacteria and eukaryotic cells (1-3). Any DNA molecule containing a short segment called the origin of transfer (oriT) can be conjugatively transferred to a recipient cell if the rest of the conjugation machinery is provided, either in cis or in trans. The transfer apparatus can be divided into three functional modules (4 -6). (i) A nucleoprotein complex known as relaxosome consists of oriT-binding proteins and their cognate DNA. These proteins include one relaxase plus one or more accessory nicking proteins; the relaxase introduces a site-specific nick at the oriT and remains covalently bound to the 5Ј end of the strand that is to be transferred. (ii) A type IV secretion system (T4SS) is a multiprotein complex spanning the inner and outer membranes through which the substrate is secreted. (iii) The conjugative coupling protein (T4CP) is responsible for connecting the two other functional modules. Current models for DNA transfer through T4SS, in both conjugation and the Agrobacterium tumefaciens T-DNA transfer system (7,8), postulate that a T4CP recruits the relaxosome to the T4SS via protein-protein interactions.For Ͼ20 years, models for conjugative DNA transport have postulated that the relaxase catalyzes the final recircularization step of the transferred DNA because of its strand-transfer activity (8, 9). Nevertheless, it was not until r...

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During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, crosslinking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.