Characterization and mapping of the agrocinopine-agrocin 84 locus on the nopaline Ti plasmid pTiC58

Abstract

Overlapping segments of pTiC58 inserted into cosmid vectors were used to characterize the agrocinopineagrocin 84 locus from the nopaline/agrocinopine A and B Agrobacterium tumefaciens strain C58. All of the clones conferring agrocin 84 sensitivity on agrobacteria also conferred uptake of agrocin 84 and agrocinopines A and B. Transposon Tn3-HoHol insertion mutations of one such clone were generated that simultaneously abolished agrocin 84 sensitivity and transport of agrocinopines A and B and agrocin 84. Such insertions were found to cluster within a 4.4-kilobase region. Analysis of ,B-galactosidase activity in these insertion mutants suggested a single transcriptional unit regulated at the transcriptional level by agrocinopines A and B. The smallest DNA fragment subcloned from theiregion to confer all three activities was 8.5 kilobases long. This subclone was still properly regulated, indicating that the regulatory gene is closely linked to the locus. The data are consistent with a single operon encoding catabolism of agrocinopines A and B and conferring sensitivity to agrocin 84. Based on these results, we support the locus name acc, for agrocinopine catabolism.Classical nopaline strains of Agrobacterium tumefaciens such as C58 induce crown gall tumors that produce nopaline and agrocinopines A and B. These opines can be catabolized by the inciting bacteria by using functions encoded by the Ti plasmid (2,14,29,38,39). The structures for agrocinopines A and B were recently determined. Agrocinopine A consists of sucrose and L-arabinose linked by a phosphodiester bond from the 2-OH of arabinose to the 4-OH of the fructose moiety. The glucose moiety of agrocinopine A can be removed either enzymatically or chemically to produce agrocinopine B (30).Agrocinopine A -has roles in two phenomena associated with nopaline strains. First, this opine is the specific inducer of Ti plasmid conjugal transfer in strain C58 (12). Second, nopaline/agrocinopine A strains take up and are killed by the toxic adenine analog agrocin 84, produced by the biotype 2 Agrobacterium radiobacter strain K84 (13,21,28). Transport of this highly specific antiagrobacterial agent is inducible by agrocinopine A (14), suggesting a link between opine catabolism and agrocin 84 sensitivity.Several lines of evidence indicate that agrocin 84 and agrocinopines A and B are transported by the same uptake system. First, spontaneous mutations causing constitutive transfer of pTiC58 result in increased transport rates for both agrocinopine A and agrocin 84 (15). Such mutants are supersensitive to agrocin 84. Second, agrocin 84-resistant mutants of A. tumefaciens A208 no longer transport agrocinopine A (14). Third, a molar excess of agrocinopine A blocks the transport of agrocin 84 into another nopaline strain, K57A (14). A periplasmic protein fraction thought to be involved in transport has been identified from this strain which binds reversibly to agrocin 84 with high affinity (27). Finally, determinants for agrocin 84 sensitivity, some conjugal transfer functio...

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

“…Ti plasmid deletions were detected by separation of linear DNA from plasmid DNA on an agarose gel after lysis in wells (8). These deletions were analyzed by separate digestions of total bacterial DNA with EcoRI, HindIII, and BamHI, and probing by Southern hybridization with overlapping cosmid clones from the vir and T regions of pTiC58 (9). The ability to be complemented in trans for pathogenicity by cosmid clones containing wild-type virA or virG was examined as previously described (7).…”

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

“…Overlays of tested strains contain C58 (1), C58(pAgSmaG) (2), C58(pAgSmaD) (3), and C58(pAgAgnG) (4). (B) Agrobacterium strains harboring pAgK84-A1 (1 and 4) and the agnG mutant pAgK84-A1agnG::Sp without (2 and 5) or complemented with (3 and 6) pAgnG were grown in liquid medium and tested for accumulation of agrocin 84 in culture supernatants (1, 2, 3) and in cell-free lysates (4,5,6) with the supersensitive strain NT1(pTiC58⌬accR) as the indicator, all as described in Materials and Methods.…”

“…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.…”

“…In turn, the inducing bacteria catabolize the released opines using metabolic systems also coded for by the resident Ti or Ri plasmids. Strains susceptible to agrocin 84 harbor Ti or Ri plasmids that code for biosynthesis by the plant and catabolism by the bacteria of one such class of opines, a set of sugar phosphodiesters called agrocinopines (5)(6)(7).…”

“…Agrocin 84, a mimic of the agrocinopines, is taken up by the pathogens via the opine transporter coded for by the Ti plasmid (5)(6)(7)(8). 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).…”