Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity

TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmi d conjugal transfer. The protein requires as coinducer an acyl-homoserine l actone signal molecule called AAI (Agrobacterium autoinducer) that is produ ced by the bacteria themselves. TraR only activates its target genes, makin g it difficult to determine whether interaction with AAI is required for bi nding DNA or for initiating transcription. To assess this, we converted Tra R into a repressor by placing a copy of the tra box, an 18-bp inverted repe at believed to be the recognition site for this protein, over the -10 regio n of a promoter driving expression of lacZ, Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of Vibrio fischeri, C-terminal deletions as short as 2 aa and N-ter minal deletions as short as 4 aa in TraR abolished both repressor and activ ator functions. The C-terminal mutants were strongly dominant over TraR, su ggesting that they can form heteromultimers with the wild-type activator. M utants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric prom oter in an AAI-dependent manner. We conclude that TraR recognizes the tra b ox as its binding site, that binding of TraR to this site depends on AAI, a nd that the N-terminal half of the protein contains one or more domains tha t are required for activation but not for multimerization, for interaction with the acyl-homoserine lactone, or for DNA binding.