Quorum sensing but not autoinduction of Ti plasmid conjugal transfer requires control by the opine regulon and the antiactivator TraM

Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is cont rolled by autoinduction via the transcriptional activator TraR and the acyl -homoserine lactone ligand, Agrobacterium autoinducer (AAI). This control p rocess is itself regulated by opines, which are small carbon compounds prod uced by the crown gall tumors that are induced by the bacteria, Opines cont rol autoinduction by regulating the expression of traR. Transfer of pTiC58 from donors grown with agrocinopines A and B, the conjugal opines for this Ti plasmid, was detected only after the donors had reached a population lev el of 10(7) cells per cm(2). Donors incubated with the opines and AAI trans ferred their Ti plasmids at population levels about 10-fold lower than thos e incubated with opines only, Transcription of the tra regulon, as assessed by monitoring a traA::lacZ reporter, showed a similar dependence on the de nsity of the donor population. However, even in cultures at low population densities that were induced with opines and AAI, there was a temporal lag o f between 15 and 20 h in the development of conjugal competence. Moreover, even after this latent period, maximal transfer frequencies required severa l hours to develop. This lag period was independent of the population densi ty of the donors but could be reduced somewhat by addition of exogenous AAI . Quorum dependent development of conjugal competence required control by t he opine regulon; donors harboring a mutant of pTiC58 deleted for the maste r opine responsive repressor accR transferred the Ti plasmid at maximum fre quencies at very low population densities. Similarly, an otherwise wild-typ e derivative of pTiC58 lacking traM, which codes for an antiactivator that inhibits TraR activity, transferred at high frequency in a population-indep endent manner in the absence of the conjugal opines, Thus, while quorum sen sing is dependent upon autoinduction, the two phenomena are not synonymous. We conclude that conjugal transfer of pTiC58 is regulated in a quorum-depe ndent fashion but that supercontrol of the TraR-AAI system by opines and by TraM results in a complex control process that requires not only the accum ulation of AAI but also the expression of TraR and the synthesis of this pr otein at levels that overcome the inhibitory activity of TraM.