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 regulated directly by the transcriptional activator TraR and its acyl-homoserine lactone (acyl-HSL) ligand, Agrobacterium autoinducer [AAI; N-(3-oxo-octanoyl)-l-homoserine lactone] (16, 34, 40; reviewed in reference 12). TraR, in its interaction with AAI, controls conjugation by autoinduction, a process by which the bacteria induce gene sets in response to signals they themselves produce. This regulatory strategy is believed to tie plasmid transfer to the population density of the donor in what has come to be called the quorum-sensing effect (reviewed in reference 18). The acyl-HSL autoinducers, which are produced by the bacteria themselves, are released into the environment, where they accumulate to ever higher concentrations. Moreover, because these molecules apparently can exchange between the intracellular and extracellular compartments, they transduce the signal among the individual members of the population. The quorum-sensing phenomenon results from the need for the accumulation of the autoinducer to some threshold concentration within the habitat. Not until it reaches this critical level does the autoinducer productively interact with the transcriptional activator, thereby initiating expression of the target genes. Thus, the bacteria gauge their population size by sensing the amount of the autoinducer present in the environment. Expression of bioluminescence in Vibrio fischeri, the paradigm quorum-sensing system, is controlled by the transcriptional activator LuxR and the acyl-HSL signal molecule, Vibrio autoinducer [VAI; N-(3-oxo-hexanoyl)-l-homoserine lactone] (reviewed in reference 7). At low population densities, the lux operon is not expressed. However, luminescence is strongly induced when, due to population growth, VAI accumulates to its threshold level. Experimentally, the quorum dependence of lux gene activation can be circumvented by adding the acyl-HSL signal to cultures of cells at low population density (8, 10). LuxR is produced at a relatively high basal level during growth (reviewed in reference 36); under such conditions, the activator is not limiting and the lux operon is almost immediately induced. Autoinduction of Ti plasmid transfer is somewhat more complex than that of lux-mediated bioluminescence. Expression of the tra regulon also is controlled by a second set of exogenous signals, highly specific compounds called the conjugal opines that are produced by the crown gall tumors induced by the phytopathogen (reviewed in reference 12). These compounds, the production of which is coded for by genes inherited from the bacterium by the transformed plant cells, control conjugation by regulating the expression of traR (15, 35). Thus, unlike lux, autoinduction of tra is tightly controlled at the transcriptional level by regulating the expression of traR. Furthermore, the activity of TraR itself is modulated by the antiactivator, TraM (17, 21). This small protein interacts with TraR to form a complex that no longer can bind to promoters of the tra regulon (22, 26a). Like the lux system, the expression of traI, the gene responsible for the production of AAI, requires activated TraR (23). However, given that the expression of traR requires the opine signal, only very small amounts of AAI are produced when donor populations are growing in the absence of crown gall tumors. Thus, autoinduction of the tra regulon first requires the induction of the expression of traR. Then, the activator must accumulate to levels sufficient to overcome the inhibitory activity of TraM. In the meantime, autoinduction and its attendant quorum dependence require that AAI accumulate to the levels necessary to activate the newly synthesized TraR. The requirements for TraR and AAI indicate that conjugal transfer is controlled by autoinduction and predict that transfer is regulated in a quorum-dependent manner. This appears to be the case; transfer of the octopine-type Ti plasmid pTiR10 occurs only when the donor population has reached a critical size (14). Moreover, density dependence could be circumvented by addition of exogenous AAI to the culture. However, how the hierarchical control exerted by the opine regulon influences the TraR-dependent quorum-sensing system has not been critically examined, nor has the role of TraM, if any, in quorum dependence been determined. In this study, we analyzed the influence of regulation by the conjugal opine on the expression of the quorum-sensing system of the nopaline-type Ti plasmid, pTiC58. We also examined the roles of the opine-responsive regulatory protein AccR and the antiactivator TraM in controlling Ti plasmid transfer. Our results indicate that these regulatory components, while not required for TraR-AAI-mediated autoinduction, are essential for the quorum-dependent character of Ti plasmid transfer.