Co-evolution of the agrocinopine opines and the agrocinopine-mediated control of TraR, the quorum-sensing activator of the Ti plasmid conjugation system

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is controlled by a hierarchical system in which opines, substrates produced by crown gall t umours, induce a quorum-sensing system. The cascade results from the contro l of expression of traR, the quorum-sensing activator, by a regulator respo nsive to the opine. In the two cases studied to date, the gene arrangements responsible for the cascade differ remarkably, suggesting that considerabl e diversity exists among the many Ti-like plasmids in the agrobacteria. In this study, we demonstrated that the novel Ti plasmid pTiChry5 is induced t o transfer at high frequency by extracts from tumours initiated by strain C hry5. The purified inducer had the chemical and biological properties of ag rocinopines C and D, a set of sugar phosphodiester opines known to induce t ransfer of another Ti plasmid, pTiBo542. The T-region of pTiChry5 contained a gene whose product, called Acs(Chry5), is virtually identical to the agr ocinopine C+D synthase from the T-region of pTiBo542. The two genes are les s closely related to acs of pTiC58, which is responsible for the production of agrocinopines A+B, a similar but not identical set of phosphodiester op ines by tumours induced by strain C58. Agrocinopines A+B induce transfer of pTiC58 but did not induce transfer of pTiChry5. A single copy of traR was identified at the 11 o'clock region of pTiChry5, where it is part of a two- gene operon called arc(Chry5). Although altered by deletions, arcChry5 is r elated to the five-gene arc operon that controls the expression of traR on pTIC58. Expression of traR(Chry5) was induced by agrocinopines C+D and the opines isolated from Chry5 tumours but not by agrocinopines A+B. A mutation in traR(Chry5) abolished transfer, and transfer was restored by complement ation in trans. We conclude that the agrocinopine opines and the correspond ing opine-meditated conjugal regulatory regions of pTiChry5 and pTiC58 shar e a common origin, but that the opine signals for the two Ti plasmids have evolved divergently through changes in the opine synthase enzymes. The alte rations in the opines, in turn, necessitated a coevolutionary change in the opine recognition systems responsible for controlling expression of the tr aR genes on these two types of Ti plasmids.