Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR

Abstract

ABSTRACT Conjugal transfer of Ti plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this plasmid contains two copies of traR , and each is associated with a different opine-regulated operon. One copy, traR noc , is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traR noc abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd , an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traR acc , is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer. The association of traR noc with nox is unique, but the operon containing traR acc is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR , each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.