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.

Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwB⌬N70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwB⌬N70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwB⌬N70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwB⌬N70 -DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F 1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor. molecular motor ͉ DNA transfer C onjugative plasmids provide a main route for acquisition of new genetic information in bacteria. Bacterial conjugation systems (including the related Vir system that Agrobacterium tumefaciens uses to transfer DNA to plants) show similarities to both DNA replication and protein transport systems (1, 2). The translocated substrate is a nucleoprotein particle that crosses the bacterial envelope into other bacterial or eukaryotic cells, crossing the kingdom boundaries. A two-step mechanism for DNA transport was proposed (2), in which conjugation is visualized as a DNA rolling-circle replication process linked to a type IV protein secretion system. Conjugation is encoded by two gene clusters in most conjugative plasmids. One cluster (dtr) encodes the DNA transfer replication proteins, and the other (mpf ) codes for the proteins that assemble the type IV protein secretion system channel. R388 is a 34-kb plasmid, which displays the simplest known dtr region, encoding only three proteins (TrwA, TrwB, and TrwC). The mpf region codes for 11 proteins (TrwD-N) involved in the formation of an extracellular pilus and a membrane-associated complex that translocates the nucleoprotein substrate across membranes.TrwB, an integral membrane and DNA-binding protein, is a key player in R388 conjugation (3-6). It has a counterpart in all conjugative systems, and it is thought to be responsible for recruiting the relaxosome DNA-pr...

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.