The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

Abstract

Ti plasmids ofAgrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally activeAgrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.The gram-negative phytopathogen Agrobacterium tumefaciens induces crown gall tumors on susceptible plant species by a novel gene-transfer process. Tumorigenicity is dependent upon a large plasmid, called Ti, present in the bacterium. During infection, a portion of the Ti plasmid called the T-region is excised and exported from the agrobacterial cell to a plant cell. Once the T-region is integrated into the plant nuclear genome, expression of transfer DNA (T-DNA)-encoded genes leads to the synthesis of phytohormones and unique nutritional compounds collectively named opines. The overproduction of the plant growth factors causes tumor formation. Opines are secreted from the tumors into the rhizosphere, where they become available as a nutritional source to A. tumefaciens strains carrying an appropriate Ti plasmid encoding functions for opine uptake and catabolism (39).In addition to carrying pathogenicity determinants, Ti plasmids also encode conjugal transfer functions which allow for Ti plasmid transmission to Ti plasmid-less agrobacteria (31). Ti plasmid conjugal transfer is normally repressed but is inducible by an opine subset, the conjugal opines (23, 38). Once a strain ofAgrobacterium has acquired a Ti plasmid, it is capable of utilizing opines as a sole nutritional substrate. Thus, the opines synthesized by the biologically engineered plant cells act as signals to s...

Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system

“…That component containing the oriT region should be transmissible from a strain harboring both elements. We chose pRK415 (IncP1␣) and pDSK519 (IncQ) as vectors for the binary constructs because the two are compatible with each other but neither is mobilized by the conjugal transfer system of pTiC58 (9).…”

“…This 6.6-kb region contains six identifiable genes arranged in two divergently transcribed units, the traAFB and traCDG operons (14). A 258-bp intergenic region separates traC and traA and contains the origin of transfer (oriT), as well as the promoters and other cis-acting elements required for regulated expression of the two tra operons (8,9). Predicted products from three of the six tra genes are related to proteins required for transfer of other conjugal and mobilizable plasmids (14).…”

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

“…(B) Gene structure of the conjugation control region and locations of mutations affecting regulation used in this study. 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.…”

Mutational Analysis of TraR

“…Digestions with restriction endonucleases were carried out as described by the manufacturers of the enzymes. Plasmid DNA was introduced into E. coli by calcium chloride-mediated transformation (18) and into A. tumefaciens by electroporation (21) or by biparental matings from E. coli S17-1 (27).…”