One of the genes that OccR regulates is a regulatory gene called traR. TraR and a protein called TraI are members of the "quorum-sensing" family of regulatory proteins. They regulate conjugal transfer genes and allows conjugation to occur only when a "quorum" of conjugal donors is present. We are studying a number of questions about these proteins: (i) we are identifying cis-acting sites required for TraR activity; (ii) we are also attempting to reconstitute the activities of the TraR protein in vitro; (iii) we are studying an antagonist of TraR denoted TraM, whose synthesis is induced by TraR; (iv) we are studying the regulation of the traR gene by OccR, as additional environmental signals appear to affect transcription; (v) we are attempting to find additional genes induced by TraR (see J. Bacteriol. 176: 2796; J. Bacteriol. 178: 435).
We recently purified the TraI protein and reconstituted its activities in a purified system. We identified the two substrates for this reaction. The fatty acid moiety of AAI is derived from acyl ACP, while the homoserine lactone moiety is derived from S-adenosyl-methionine (see Science, 272:1655).
Enzymatic synthesis of Agrobacterium autoinducer using purified substrates.
Recently, a gene over 90% identical to traR was found on the Ti plasmid (S. K. Farrand, IS-MPMI Meeting, 1996). We have found that this gene, provisionally designated traS, is expressed in response to another opine called mannopine. However, this gene contains a frame shift mutation, and is predicted to encode a protein that is not only inactive, but which blocks the activity of TraR. This predicts first, that artificial overexpression of TraS should prevent conjugation, second, that mannopine should inhibit conjugation in wild type strains, and, third, that this inhibition should be dependent on TraS. In preliminary experiments, all three predictions have been confirmed. Directly downstream from traS lies an open reading frame that is strongly similar to the family of MCPs. This ORF is truncated at its 5' end, and could encode a dominant negative MCP that could interfere with chemotaxis toward opines or other chemoattractants (Mol. Microbiol. 27:289-297).
We tested 32 AAI analogs for their ability to induce tra gene expression and to act as antagonists of AAI. In near wild type strains, only AAI was a powerful agonists, and only a small number of closely related compounds showed any detectable activity. However, several compounds were strong antagonists of AAI. In strains that overexpress TraR, almost all compounds were agonists, and none showed significant activity as antagonists (J. Bacteriol. 180:5398-5405).
We have purified TraR to homogeneity and used it to demonstrate specific binding to predicted DNA binding sites and activation of transcription of two divergent TraR-regulated promoters. TraR binds AAI with a ratio of 1:1. AAI increased the affinity of TraR for its DNA binding sites. AAI also strongly stabilized TraR against cytoplasmic proteases, increasing the half life of this protein about 20 fold (P.N.A.S. 96:4832-4837). TraR-AAI complexes are dimeric in vitro, while apo-TraR is monomeric. TraR is also dimeric when bound to DNA.