Hierarchical gene regulatory systems arising from fortuitous gene associations: Controlling quorum sensing by the opine regulon in Agrobacterium

Abstract

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum-sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5' to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR-regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine-responsive transcriptional regulator.