The Journal of Antibiotics (2018) 71:438–446 https://doi.org/10.1038/s41429-017-0014-y ARTICLE Agrocin 108 is a 5'-cytidine nucleotide bacteriocin containing a carbocyclic phosphoryl-ascorbate group Christopher M. Elvin 1,2 ● Robert E. Asenstorfer 1 ● Maarten H. Ryder 1 ● Scott C. Donner 1,3 ● Graham P. Jones 1 ● Max E. Tate 1 Received: 9 August 2017 / Revised: 26 November 2017 / Accepted: 1 December 2017 / Published online: 18 January 2018 © Japan Antibiotics Research Association 2018 Abstract Agrocin 108 is a 3′-O-β-D-xylopyranosyl-cytidine-5′-O-phosphodiester of an ascorbate-carbocyclic cyclopentenone analogue, with bacteriocin-like properties. This bacteriocin exhibits orders of magnitude greater than the inhibition zone diameter towards the indicator strain than either ampicillin or streptomycin. It has been isolated from cultures of Rhizobium rhizogenes strain K108. The structure of the agrocin 108 without detail, has been previously published. We now report a detailed structure elucidation, including the hitherto undetermined residual 5′-phospho-diester fragment by a combination of 1D and 2D NMR studies at various pH values in H 2 O/D 2 O, high resolution MS, pKa determination, and chemical degradation. Introduction Agrocins are highly selective bacteriocins. Agrocin 84 (a fraudulent, N-6, O-5′-disubstituted adenine arabinofuranosyl nucleotide) together with agrocin 434 (an N-4-substituted cytidine nucleoside, with 3′-O-enol ether blocking group) are two representatives of a class of non-peptide bacteriocins that have been successfully used in combination for four decades [1], to effect biological control of the dicotyledonous plant cancer known as crown gall. This disease is induced by pathogenic strains of Agrobacterium tumefaciens and A. rhizogenes (now known as Rhizobium rhizogenes) which can carry a tumour-inducing (Ti) plasmid. An interesting feature of agrocins is that they have a toxic moiety and an uptake moiety, and certain bacterial permeases designed for specific nutrients allows the entry of the bacteriocin. Agrocin 84 produced by R. rhizogenes strain K84 is a fraudulent ade- nosine and only bacteria with a specific permease are sen- sitive to agrocin 84 [2]. This uptake system has been described as a Trojan Horse [3]. To protect against crown gall, both agrocin 84 and agrocin 434 are delivered via application of a live non-pathogenic R. rhizogenes (strain K1026) producer of these agrocins to the roots, cuttings or seeds of susceptible plants, prior to planting. We suggest that a thorough understanding of the mode of action [4] of these long-standing and remarkably effective nucleoside and nucleotide bacteriocin-like antibiotics will provide clues as how to avoid the build-up of resistant pathogens which is very much the characteristic of broad spectrum antibiotics. We now report the structure of yet another low- molecular weight nucleotide bacteriocin: agrocin 108. A proposed structure of agrocin 108 by ME Tate with neither stereo-chemistry nor structural information was first pub- lished in a review in 1995 [5]. We wish to provide further details regarding the structure of this compound. Experimental procedures Bacterial strains Rhizobium rhizogenes strain K108 and R. rhizogenes strain K46 (syn. TR101) were obtained from Professor Allen Kerr of the University of Adelaide, Waite Campus. Both the * Robert E. Asenstorfer robert.asenstorfer@adelaide.edu.au 1 School of Agriculture, Food and Wine, Waite Campus, The University of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia 2 CWAN Technologies Pty Ltd, 28 Dexter Street, Tennyson, QLD 4105, Australia 3 Department of Environment, Water and Natural Resource, Level 4, 81 Waymouth St, Adelaide, SA 5000, Australia Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41429-017-0014-y) contains supplementary material, which is available to authorised users. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1234567890