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.     

Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM-sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM's N-terminal ribbon-helix-helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore.     

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.     

The TraM protein of plasmid R1 is a DNA-binding protein

The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA-binding studies. Both gel retardation- and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist. The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.     

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.     

The essential transfer protein TraM binds to DNA as a tetramer

The TraM proteins encoded by F-like plasmids are sequence specific DNA binding proteins that are essential for conjugative DNA transfer. We investigated the quarternary structure and the DNA binding properties of the TraM wild-type protein of the resistance plasmid R1 and two mutant forms thereof. Size-exclusion chromatography and differential scanning calorimetry showed that purified TraM protein (amino acids 2-127) forms stable tetramers in solution. A truncated version of the protein termed TraMM26 (amino acids 2-56) forms dimers. Thus, the dimerization and tetramerization domains can be assigned to the N-terminal and C-terminal domains of TraM, respectively. Further analyses using chemical cross-linking and light scattering corroborated the preferentially tetrameric nature of the protein but also suggest that TraM has a tendency to form higher aggregates. Band-shift and fluorescence spectroscopy investigations of TraM-DNA complexes revealed that the TraM protein is also tetrameric when bound to its minimal DNA binding site. The deduced binding constant in the range of 10(8) M(-1) demonstrated a very strong binding of TraM to its preferred DNA sequence. Secondary structure analysis based on CD measurements showed that TraM is mainly alpha-helical with a significant increase in alpha-helicity (48 to 58%) upon DNA-binding, indicating an induced fit mechanism.     

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.