1.5.6
THE ROLE OF PROTEIN-PROTEIN INTERACTIONS IN GENE REGULATION BY QUORUM SENSING

AJ SMYTH1, I HWANG1,3 and SK FARRAND1,2

Departments of 1Crop Sciences and 2Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
3Present address: Korea Research Institute of Bioscience and Biotechnology, Yusung, Taejeon, South Korea

Background and objectives
Transcription of the genes mediating conjugal transfer of the Agrobacterium tumefaciens Ti plasmid pTiC58 is regulated by the transcriptional activator TraR and its co-inducer, Agrobacterium auto-inducer [AAI; N-(3-oxo-octanoyl)-L-homoserine lactone]. An additional regulatory element, TraM, modulates TraR-mediated auto-induction of the tra regulon [1]. TraM does not directly affect expression of traR or the tra and trb operons. Rather, it apparently functions by suppressing the action of TraR; TraM functions only when over-expressed with respect to TraR, and this suppression can be overcome by over-expressing TraR. However, suppression by TraM cannot be overcome by adding excess AAI. Our analysis using the yeast two-hybrid system indicates that TraM can interact directly with TraR at the protein level. We thus propose that TraM modulates the activity of TraR by interacting directly with the activator protein. In this work we examine further the mechanisms involved in interaction of these two proteins.

Results and conclusions
To study further the interaction of TraM and TraR, we have used the yeast two-hybrid system which examines protein-protein interactions, and the lamda cI repression assay, which examines protein oligomerization. Using the latter system, we show that TraM is capable of oligomerization. We previously identified point mutants of TraM that were unable to suppress TraR activity in Agrobacterium. Nonsense or mis-sense mutations that resulted in removal or alteration of the hydrophobic C-terminus of TraM rendered the protein unable to interact with TraR in the yeast two-hybrid system. To examine whether these mutants are altered in their oligomerization domain, or in the domain at which interaction with TraR occurs, we examined their ability to oligomerize in the lamda cI repression assay system. The mutants were found to be impaired in their ability to oligomerize in the lamda cI repression assay, suggesting that TraM interacts with TraR as an oligomer, and that the hydrophobic C-terminus of TraM plays an important structural role in this process.

TraR also has been proposed to function as a dimer. When tested in the lamda cI repression system, TraR did indeed oligomerize, but only in the presence of AAI. Moreover, oligomerization was dependent upon AAI concentration. In contrast, oligomerization of TraM was not affected by AAI. Oligomerization of TraR was optimal in the presence of N-(3-oxo-octanoyl)-L-homoserine lactone, the cognate Agrobacterium AI; N-(3-oxo-hexanoyl)-L-homoserine lactone, the cognate AI of LuxR, the transcriptional activator of the bioluminescence genes in the marine bacterium Vibrio fischeri, could only promote a similar level of oligomerization of TraR at approximately 100-fold higher concentrations. These results strongly support the hypothesis that AIs function to promote oligomerization of the quorum-sensing gene activators.

References
1. Hwang I, Cook DM, Farrand SK, 1996. Journal of Bacteriology 177, 449-458.