The structure of tagetitoxin.

Based on detailed analysis of newly acquired NMR data, we show that the previously revised structure of tagetitoxin is incorrect. A new structure of tagetitoxin is proposed which is consistent with the NMR and MS data.

Synthesis of amino acid conjugates to 2-imino-3-methylene-5-carboxypyrrolidine and 2-imino-3-methylene-6-carboxypiperidine.

The four stereomers of 2-imino-3-methylene-5-L(carboxy-L-valyl)pyrrolidine, a bacterial metabolite that is inhibitory to the fire blight bacterium Erwinia amylovora, were synthesised and compared for antibacterial activity. Several alternative amino acid conjugates with L,L-stereochemistry were also prepared, and the synthesis was extended to 3-methylenepiperidine-6-L-carboxylic acid and a selection of 2-imino-3-methylenepiperidine-6-L-carboxy-L-amino acid conjugates. All synthetic amino acid conjugates (L,L-stereomers) were inhibitory to the growth of E. amylovora. The likely participation of the conjugated iminomethylene moiety as a Michael acceptor is implicated.

An antibacterial pyrazole derivative from Burkholderia glumae, a bacterial pathogen of rice.

Burkholderia glumae, a bacterial pathogen on rice, produced compounds in liquid culture that, in agar diffusion assays, gave strong inhibitory action against Erwinia amylovora, the bacterium responsible for fire blight disease of apple and pear trees. Products were isolated from culture medium by cation exchange and then purified by bioassay-guided chromatographic methods. Two major products were obtained, one of which was not active when fully purified. Each product showed a single ninhydrin-staining spot on TLC and a single HPLC peak. The non-active product was deduced from NMR, MS, and chemical data, to be the tripeptide L-alanyl-L-homoserinyl-L-aspartate. The NMR data for the active product demonstrated that it contained the same tripeptide, but functionalised at the beta-carboxyl of the C-terminal aspartate, by a moiety that provided an additional 98 mass units to the parent tripeptide. Various data led to the interpretation that this moiety was a highly unusual oxygenated pyrazole structure, and thus the bioactive product was deduced to be 3-[L-alanyl-L-homoserinyl-L-aspartyl-beta-carboxy]-4-hydroxy-5-oxopyrazole. This compound was found to inhibit the growth of a number of different bacterial species.

A mutation in an exbD gene reduces tagetitoxin production by Pseudomonas syringae pv. tagetis.

A mutant of Pseudomonas syringae pv. tagetis EB037 with limited ability to produce tagetitoxin was isolated after transposon mutagenesis and the mutation was characterized. The mutation occurred in a gene with a high degree of sequence identity to exbD. exbD is contiguous with tonB and exbB upstream and with a gene for a TonB-dependent receptor downstream. Using reverse transcription - polymerase chain reaction with RNA from the wild-type and exbD mutant strains, we demonstrated that the mutation in exbD did not have a polar affect on the expression of downstream genes. The exbD mutant was able to grow well in conditions where iron is not freely available. Siderophore production by the exbD mutant was similar to that of the wild-type strain. We conclude that the mutation in exbD disrupts tagetitoxin production without compromising iron metabolism. The results indicate that tagetitoxin export by P. syringae pv. tagetis involves an efflux pump that requires a functional TonB system that is not essential for normal iron metabolism.

Antibacterial iminopyrrolidines from Burkholderia plantarii, a bacterial pathogen of rice.

Burkholderia plantarii, a bacterial pathogen on rice, produced compounds in liquid culture that gave strong inhibitory action against Erwinia amylovora, the bacterium responsible for fire blight disease of apple and pear trees. The active component was hydrophilic and ionic, and was fractionated extensively by passage through SP, DEAE, and LH20 Sephadexes. Final purification was achieved by reverse phase chromatography on C(18)-bonded silica. Biological activity was associated with two compounds detectable by HPLC (UV lambda 218 nm). A third related compound was non-active. The chemical structures of the pure compounds were deduced by NMR and MS, and corroborated by chemical synthesis which also verified the stereochemical assignments. Thus, the two active compounds are reported herewith as 2-imino-3-methylene-5-L(carboxy-L-valyl)-pyrrolidine, and 2-imino-3-methylene-5-L(carboxy-L-threoninyl)-pyrrolidine. The non-active component was deduced to be 2-imino-5-L(carboxy-L-threoninyl)-pyrrolidine.

The phytotoxin coronatine and methyl jasmonate impact multiple phytohormone pathways in tomato.

Coronatine (COR) is a phytotoxin produced by several pathovars of Pseudomonas syringae and consists of coronafacic acid (CFA), an analog of methyl jasmonic acid (MeJA), and coronamic acid (CMA), which resembles 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor to ethylene. An understanding of how COR functions, is perceived by different plant tissues, and the extent to which it mimics MeJA remain unclear. In this study, COR and related compounds were examined with respect to structure and function. The results indicate that conjugation of CFA to an amino acid is required for optimal activity in tomato, including chlorosis, changes in chloroplast structure, cell wall thickening, accumulation of proteinase inhibitors, induction of anthocyanins, and root growth inhibition. cDNA microarrays were utilized to understand the molecular processes that are regulated by MeJA, COR, CFA and CMA in tomato leaves. A comparison of COR- and MeJA-regulated transcriptomes revealed that COR regulated 35% of the MeJA-induced genes. There was significant overlap in the number of COR and CFA-regulated genes with CFA impacting the expression of 39.4% of the COR-regulated genes. Taken together, the results of biological assays, ultrastructural studies, and gene expression profiling demonstrate that: (1) the intact COR molecule impacts signaling in tomato via the jasmonic acid, ethylene, and auxin pathways; (2) CMA does not function as a structural analog of ACC; (3) COR has a broader range of functions than either CFA or CMA; and (4) COR and MeJA share similar, but not identical activities and impact multiple phytohormone pathways in tomato.

Kinetic analysis of the L-ornithine transcarbamoylase from Pseudomonas savastanoi pv. phaseolicola that is resistant to the transition state analogue (R)-N delta-(N'-sulfodiaminophosphinyl)-L-ornithine.

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).

Chlorosis-inducing products from Pseudomonas syringae pathovars: new N-coronafacoyl compounds.

Liquid cultures of Pseudomonas syringae pv. tomato strains that also produced the phytotoxin coronatine, were found to have a new chlorosis-inducing activity, not previously described. Bioassay-guided fractionation and HPLC analysis revealed two new peaks that were chlorosis-inducing on leaves of bean plants. Mass spectrometry and NMR analyses of the compounds led to the derivation of their structures as coronafacoyl-L-serine and coronafacoyl-L-threonine, respectively. The amino acid C-2 configurations were determined by GC analysis following hydrolysis with 6M HCl. Both compounds have more polar and more acidic properties than coronatine, norcoronatine, and other known coronafacoyl conjugates with non-polar amino acids. Investigation of Pseudomonas syringae pv. glycinea strains that are known to be prolific producers of the coronatine family of compounds revealed the production of coronafacoylserine and coronafacoylthreonine also.