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

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierar chy involving induction by the opines agrocinopines A and B and a quorum-se nsing system. Regulation by the opines is mediated by the repressor AccR, w hile quorum sensing is effected by the transcriptional activator TraR and i ts ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoin ducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that t raR is the fourth gene of an operon, which we named are, that is transcribe d divergently from accR. Complementation analysis of mutations in the genes 5' to traR showed that the other members of the are operon are not require d for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that Ac cR-regulated expression of traR initiates from a promoter located in the in tergenic region between accR and orfA, the first gene of the are operon. Re verse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the are transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a mo del in which quorum sensing is subordinate to the opine regulon because tra R has become associated with an operon controlled by the opine-responsive t ranscriptional regulator.