Dimerization of the quorum-sensing transcription factor TraR enhances resistance to cytoplasmic proteolysis

TraR is a LuxR-type quorum-sensing protein encoded by the tumour-inducing plasmid of Agrobacterium tumefaciens . TraR requires the pheromone N-3-oxooctanoyl- l -homoserine lactone (OOHL) for biological activity, and is dimeric both in solution and when bound to DNA. Dimerization is mediated primarily by two α-helices, one in the N-terminal OOHL binding domain, and the other in the C-terminal DNA binding domain. Each of these helices forms a parallel coiled coil with the identical helix of the opposite subunit. We have previously shown that OOHL is essential for resistance to proteolysis, and here we asked whether dimerization is also required for protease resistance. We constructed a series of site-directed mutations at the dimer interface, and tested these mutants for activity in vivo . Alteration of residues A149, A150, A153, A222 and I229 completely abolished activity, while alteration of three other residues also caused significant defects. All mutants were tested for dimerization as well as for specific DNA binding. The cellular abundance of these proteins in A. tumefaciens was measured using Western immunoblots and OOHL sequestration, while the half-life was measured by pulse-chase radiolabelling. We found a correlation between defects in in vivo activity, in vitro dimerization, DNA binding and protein half-life. We conclude that dimerization of TraR enhances resistance to cellular proteases.     

Molecular basis of transcriptional antiactivation. TraM disrupts the TraR-DNA complex through stepwise interactions

Conjugative transfer of Agrobacterium Ti plasmids is regulated by TraR, a quorum-sensing activator. Quorum dependence requires TraM, which binds to and inactivates TraR. In this study, we showed that TraR and TraM form a 151-kDa stable complex composed of two TraR and two TraM dimers both in vitro and in vivo. When interacted with TraR bound to tra box DNA, wild-type TraM formed a nucleoprotein complex of 77 kDa composed of one dimer of each protein and DNA. The complex converted to the 151-kDa species with concomitant release of DNA with a half-life of 1.6 h. TraR in the complex still retained tightly bound autoinducer. From these results, we conclude that TraM interacts in a two-step process with DNA-TraR to form a large, stable antiactivation complex. Mutagenesis identified residues of TraR important for interacting with TraM. These residues form two patches, possibly defining the binding interfaces. Consistent with this interpretation, comparison of the trypsin-digested polypeptides of TraR and of TraM with that of the TraR-TraM complex revealed that a tryptic site at position 177 of TraR around these patches is accessible on free TraR but is blocked by TraM in the complex. From these genetic and structural considerations, we constructed three-dimensional models of the complex that shed light on the mechanism of TraM-mediated inhibition of TraR and on TraM-mediated destabilization of the TraR-DNA complex.     

Quorum sensing in Rhizobium sp. strain NGR234 regulates conjugal transfer (tra) gene expression and influences growth rate

Rhizobium sp. strain NGR234 forms symbiotic, nitrogen-fixing nodules on a wide range of legumes via functions largely encoded by the plasmid pNGR234a. The pNGR234a sequence revealed a region encoding plasmid replication (rep) and conjugal transfer (tra) functions similar to those encoded by the rep and tra genes from the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens, including homologues of the Ti plasmid quorum-sensing regulators TraI, TraR, and TraM. In A. tumefaciens, TraI, a LuxI-type protein, catalyzes synthesis of the acylated homoserine lactone (acyl-HSL) N-3-oxo-octanoyl-L-homoserine lactone (3-oxo-C8-HSL). TraR binds 3-oxo-C8-HSL and activates expression of Ti plasmid tra and rep genes, increasing conjugation and copy number at high population densities. TraM prevents this activation under noninducing conditions. Although the pNGR234a TraR, TraI, and TraM appear to function similarly to their A. tumefaciens counterparts, the TraR and TraM orthologues are not cross-functional, and the quorum-sensing systems have differences. NGR234 TraI synthesizes an acyl-HSL likely to be 3-oxo-C8-HSL, but traI mutants and a pNGR234a-cured derivative produce low levels of a similar acyl-HSL and another, more hydrophobic signal molecule. TraR activates expression of several pNGR234a tra operons in response to 3-oxo-C8-HSL and is inhibited by TraM. However, one of the pNGR234a tra operons is not activated by TraR, and conjugal efficiency is not affected by TraR and 3-oxo-C8-HSL. The growth rate of NGR234 is significantly decreased by TraR and 3-oxo-C8-HSL through functions encoded elsewhere in the NGR234 genome.     

The F plasmid-encoded TraM protein stimulates relaxosome-mediated cleavage at oriT through an interaction with TraI

Conjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer - they introduce a site- and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5'-end of the cleaved DNA strand. The action of the initiator protein at the origin of transfer (oriT) is governed by auxiliary proteins that alter the architecture of the DNA molecule, allowing binding of the initiator protein. In the F plasmid system, two auxiliary proteins have roles in establishing the relaxosome: the host-encoded IHF and the plasmid-encoded TraY. Together, these proteins direct the loading of TraI which contains the catalytic centre for the transesterification. The F-oriT sequence includes a binding site for another plasmid-encoded protein, TraM, which is required for DNA transfer. Here the impact of TraM protein on the formation and activity of the F plasmid relaxosome has been examined. Purified TraM stimulates the formation of relaxed DNA in a reaction that requires the minimal components of the relaxosome, TraI, TraY and IHF. Unlike TraY and IHF, TraM is not essential for the formation of the relaxosome in vitro and TraM cannot substitute for either TraY or IHF in this process. The TraM binding site sbmC, along with both IHF binding sites, is essential for stimulation of the relaxase reaction. In addition, stimulation of transesterification appears to require the C-terminal domain of TraI suggesting that TraM and TraI may interact through this domain on TraI. Taken together, these results provide additional evidence of a role for TraM as a component of the relaxosome, suggest a previously unknown interaction between TraI and TraM, and allow us to propose a molecular role for the C-terminal domain of TraI.     

Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM

F factor TraM is essential for efficient bacterial conjugation, but its molecular function is not clear. Because the physical properties of TraM may provide clues to its role in conjugation, we have characterized the TraM oligomerization equilibrium. We show that the reversible unfolding transition is non-two-state, indicating the presence of at least one intermediate. Analytical ultracentrifugation experiments indicate that the first phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state in the second phase. Furthermore, we show that a C-terminal domain isolated by limited proteolysis is tetrameric in solution, like the full-length protein, and that its loss of structure correlates with dissociation of the TraM tetramer. Unfolding of the individual domains indicates that the N- and C-terminal regions act cooperatively to stabilize the full-length protein. Together, these experiments suggest structural overlap of regions important for oligomerization and DNA binding. We propose that modulating the oligomerization equilibrium of TraM may regulate its essential activity in bacterial conjugation.     

Mutations in the C-terminal region of TraM provide evidence for in vivo TraM-TraD interactions during F-plasmid conjugation

Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.