Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.

Abstract

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gail tumors.The opines also induce Ti plasmid-encoded genes required by the bacteria for' opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR.Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis During interactions between Agrobacterium tumefaciens and its plant hosts, the bacterium senses and responds to several plant-produced signals. For example, virulent agrobacteria use a two-component signal transduction system to sense small phenolic compounds released from wounded plant tissue. In response, the bacteria express Ti plasmid-encoded Vir functions, which, in turn, facilitate T-strand excision and transfer to susceptible plant cells (1). The resulting plant neoplasias synthesize unique low molecular weight carbon compounds, called opines, which are thought to provide a source of carbon for tumor-colonizing agrobacteria (2).The opines also act as signals. For example, the Ti plasmidencoded functions required for opine catabolism are specifically induced by their cognate substrates (3, 4). In addition, conjugal transfer of the Ti plasmid, which is normally repressed, is induced by a subclass of opines, the conjugal opines (5,6). A model proposing that conjugation and catabolism of the conjugal opines are coregulated emerged from the observation that mutants constitutive for conjugation are often derepressed for conjugal opine catabolism. Similarly, mutants selected for constitutive expression of these opine catabolic functions are generally transfer-constitutive (5, 7-9).For nopaline-type Ti plasmids such as pTiC58, conjugation is induced by the sugar phosphate opines, agrocinopines A and B (6). We report here that the coregulation of agrocinopine catabolism (Acc) and conjugal transfer (Tra) of pTiC58 is mediated by a repressor, AccR. This repressor is related to negative regulatory proteins that control sugar catabolic operons in several unrelated bacteria (10). Our findings provide a molecular framework for the coregulation model and illustrate the diversity of regulatory mechanisms that govern the interaction between Agrobacterium and its plant hosts. 11 MATERIALS AND METHODS Strains and Plasmids. A. tumefaciens strains used were C58 (11), NT1(pTiC58Trac) (12), C58ClCE (pWI1003) (8), NT1(pAgK84-A1) (13), and C58C1RS (rifampin resistant, streptomycin resistant) (8); Escherichia coli strains were DH5a, RR1, and S17-1...

Quorum Sensing but Not Autoinduction of Ti Plasmid Conjugal Transfer Requires Control by the Opine Regulon and the Antiactivator TraM

“…1) (1). As a consequence, strains harboring this Ti plasmid express traR constitutively, produce large amounts of AAI, and transfer the plasmid at high frequency in the absence of the conjugal opine (1,23,34).…”

“…The structure and functions of the acc, tra, and arc operons have been described (6,13,24,26,35). pTiC58 is wild type and conjugal transfer requires induction by agrocinopines A and B. pTiC58⌬accR contains a deletion mutation in accR resulting in the complete loss of repressor activity (1) and in the constitutive expression of acc and arc. This Ti plasmid is constitutive for transfer.…”

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

“…The strains of A. tumefaciens and the plasmids used in this study are listed in Table 1. Ti plasmids pCMA1 (traM), pTiC58⌬accR (accR), and pKMA1 (accR traM) are three transfer-constitutive [Tra(Con)] derivatives of pTiC58 (1,15). Agrobacterium strains were grown at 28°C on nutrient agar (Difco) and in L broth or AB minimal medium (6) containing 0.2% mannitol (ABM medium).…”

“…For example, transfer of pTiC58 is induced by the sugar phosphodiester opines agrocinopine A and agrocinopine B. In this plasmid, traR is a member of the five-gene arc operon, the expression of which is regulated by the opine-responsive repressor AccR (1). In the absence of the conjugative opines, the expression of arc is repressed.…”

Bases of biocontrol: Sequence predicts synthesis and mode of action of agrocin 84, the Trojan Horse antibiotic that controls crown gall

“…Moreover, the agrocinopines produced by the tumors induce the expression of the Ti plasmid genes coding for their uptake and catabolism, thereby insuring that the tumorigenic strains are maximally susceptible to agrocin 84 produced by strain K84 (6,7,9). Clearly, evolution has tailored this antibiotic to a metabolic system in pathogenic isolates guaranteed to be expressed at high levels in the habitat provided by crown gall tumors.…”