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

Abstract

Agrobacterium radiobacter K84, used worldwide to biocontrol crown gall disease caused by Agrobacterium tumefaciens, produces an antiagrobacterial compound called agrocin 84. We report the nucleotide sequence of pAgK84, a 44.42-kb plasmid coding for production of this disubstituted adenine nucleotide antibiotic. pAgK84 encodes 36 ORFs, 17 of which (agn) code for synthesis of or immunity to agrocin 84. Two genes, agnB2 and agnA, encode aminoacyl tRNA synthetase homologues. We have shown that the toxic moiety of agrocin 84 inhibits cellular leucyl-tRNA synthetases and AgnB2, which confers immunity to the antibiotic, is a resistant form of this enzyme. AgnA, a truncated homologue of asparaginyl tRNA synthetase could catalyze the phosphoramidate bond between a precursor of the methyl pentanamide side group and the nucleotide. We propose previously undescribed chemistry, catalyzed by AgnB1, to generate the precursor necessary for this phosphoramidate linkage. AgnC7 is related to ribonucleotide reductases and could generate the 3'-deoxyarabinose moiety of the nucleoside. Bioinformatics suggest that agnC3, agnC4, and agnC6 contribute to maturation of the methyl pentanamide, whereas agnC2 may produce the glucofuranose side group bound to the adenine ring. AgnG is related to bacterial exporters. An agnG mutant accumulated agrocin 84 intracellularly but did not export the antibiotic. pAgK84 is transmissible and encodes genes for conjugative DNA processing but lacks a type IV secretion system, suggesting that pAgK84 transfers by mobilization. By sequence analysis, the deletion engineered into pAgK1026 removed the oriT and essential tra genes, confirming the enhanced environmental safety of this modified form of pAgK84.

Fig. 1.

Gene structure and physical map of pAgK84. Restriction endonuclease sites were derived from the nucleotide sequence. Locations and organizations of identified ORFs are shown as arrows color-coded as genes involved in: synthesis, export, or immunity to agrocin 84 (red); replication and conjugative transfer (yellow); and unknown function (open). Sites of Tn5 (13) and Tn3HoHo1 (15) insertions are shown. Horizontal bars on Tn3HoHo1 symbols indicate orientation of the lacZ reporter with respect to the target gene. Effect of each insertion in agn or tra genes on production of agrocin 84 or conjugative transfer is indicated by (−), no detectable antibiotic or transfer; (+/−), detectable but reduced production; and (+), wild-type production or transfer. The region of the plasmid absent from pAgBo542 is indicated by the outer arc. The structure of agrocin 84 (2, 3) is shown in the middle of the map.

Fig. 2.

The conjugative transfer determinants of pAgK84 and those on the linear chromosome of C58 are closely related. (A) The organization of the genes in the transfer regions of the two replicons are shown with identical fill patterns indicating significant amino acid sequence relatedness. Open arrows represent genes showing no significant relatedness. The region of pAgK84 deleted in the construction of pAgK1026 (24) is shown as a horizontal line above the maps. (B) Nucleotide sequences of oriT regions of RSF1010, pTF1, pTiC58, pAtC58, the linear chromosome (L.C.) of C58, and pAgK84 were aligned by using clustalx (38). Regions of sequence identity shared in the six segments are boxed. Arrows indicate inverted repeats. Filled triangles show experimentally determined nick sites.

Fig. 3.

agnG is required for agrocin 84 export. (A) Agrobacterium strains harboring pTiC58 were tested for influence of clones expressing agnG on susceptibility to agrocin 84 produced by strain NT1(pAgK84-A1) spotted in the center of the plate. 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(pTiC58ΔaccR) as the indicator, all as described in Materials and Methods.

Fig. 4.

Proposed roles of AgnB1, AgnA, and AgnC4 in the formation of the phosphoramidate bond with the nucleotide analogue and subsequent deamination of the methyl pentanamide precursor. See text for details.