Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes

Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum-sensing system involving the transcriptional activator TraR and the acyl homoserin e lactone autoinducer N-(3-oxo-octanoyl)-L-homoserine lactone (AAI). Activa tion of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid. Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activat or protein. Using the yeast two-hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmi d pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 p roduced beta-galactosidase and grew on medium lacking leucine, both phenoty pes indicative of an interaction between the two proteins. Early terminatio n mutants and substitution mutants mapping to the C-terminus of TraM were i solated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens. These mutants all failed to interact with the Tr aR fusion in the two-hybrid system. An N-terminal deletion mutant of TraM l acking the first 27 residues weakly interacted with TraR in the two-hybrid system whereas deletions of 48 amino acids or more abolished the interactio n. As assessed by Western blot analysis, the mutant fusion proteins were pr oduced at levels indistinguishable from that of the wild-type TraM in the y east tester strain. Mutants of TraR that were not inhibited by TraM in A. t umefaciens were isolated and fell into two classes. In the first, the mutat ion resulted in increased expression of wild-type TraR. In the second, a pr oline residue at position 176 was changed to serine (P176 --> S) or to leuc ine (P176 --> L). The P176 --> S mutant interacted with wild-type TraM, but at a detectably lower level, in the two-hybrid assay. Mutants of TraR with N-terminal deletions as large as 105 amino acids interfered with the abili ty of TraM to inhibit wild-type TraR in A, tumefaciens, Two-hybrid assays i ndicated that these mutants, as well as a C-terminal 49 residue fragment of TraR, can interact with TraM, We conclude that TraM and TraR interact in v ivo and that this interaction is responsible for inhibition of TraR-mediate d activation. We also conclude that the two proteins interact with each oth er through domains located at their respective C-termini.