A naturally-occurring structural analogue of the phytotoxin coronatine

Chlorosis-inducing compounds in liquid cultures of the phytopathogenic bacterium Pseudomonas syringae pv. atropurpurea have been investigated. In addition to coronatine as previously reported, a new compound was discovered. This gave a mass spectral fragmentation pattern which indicated that it was, like coronatine, an amide of coronafacic acid. Acid-hydrolysis of the new toxin liberated the amino acid valine. This observation, together with mass spectral and NMR data, established the structure of the new toxin as N-coronafacoylvaline. Some implications to biosynthesis are discussed. Along with the two chlorosis-inducing compounds, the biologically inactive coronafacic acid was also isolated from the growth medium.     

Coronatine analogues produced by Xanthomonas campestris pv Phormiicola

Liquid cultures of Xanthomonas campestris pv phormiicola were found to contain two analogues of coronatine lacking the cyclopropane ring structure, and no trace of either coronatine or norcoronatine. The two compounds were isolated and fully characterised by NMR, MS, hydrolysis and GC of hydrolysis products, as N-coronafacoyl- -valine and N-coronafacoyl- -isoleucine. A survey of 12 strains from 10 other X. campestris pathovars did not locate another source of production of these compounds, whereas all three strains of X. campestris pv phormiicola examined produced comparable levels of both compounds. This is the first report of phytotoxins biosynthetically derived from coronafacic acid outside of the genus Pseudomonas. The implications of these findings to the biosynthesis of the cyclopropane ring structure of coronatine are discussed.     

Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv. glycinea.

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The coronatine synthesis genes in PG4180 were previously shown to reside on a 90-kb plasmid designated p4180A. In the present study, clones containing a 34-kb region of p4180A were saturated with Tn5, and 71 unique mutations were recombined into p4180A by marker exchange. The effect of each mutation on coronatine synthesis was determined by analyzing the organic acids produced by the mutants by reverse-phase high-performance liquid chromatography. The organic acids of selected mutants were derivatized to their methyl esters and analyzed by gas chromatography and gas chromatography-mass spectrometry. Mutations in a 20.5-kb region of p4180A completely blocked the synthesis of coronafacic acid and coronatine. Mutations within a 4.4-kb region of p4180A prevented the formation of coronatine but allowed for production of coronafacic acid, coronafacoylvaline, coronafacoylisoleucine, and coronafacoylalloisoleucine. The phenotypes of selected mutants were further confirmed in feeding experiments in which coronafacic acid or coronamic acid was added to the culture media. The results of this study allow us to speculate on the likely sequence of steps in the later stages of coronatine biosynthesis.     

Plasmid-mediated production of the phytotoxin coronatine in Pseudomonas syringae pv. tomato.

Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were previously generated by using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants either were missing pPT23A (29 mutants) or contained deletions in this plasmid (4 mutants). These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce coronatine. A bioassay for coronatine suggested that PS61(pPT23A) transconjugants were able to make this phytotoxin. In a chemical analysis, organic acids were isolated from PT23.2, PS61, and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The derivatized organic acids extracted from PT23.2 and PS61(pPT23A) contained peaks that corresponded to coronafacic acid, coronafacoylvaline, and coronatine, but these were absent in the extracts from the wild-type strain PS61. The identification of these components was confirmed by combined gas chromatography-mass spectrophotometry. Therefore, the acquisition of pPT23A by PS61 resulted in biosynthesis of coronafacic acid, coronafacoylvaline, and coronatine, clearly demonstrating the involvement of pPT23A in coronatine production in P. syringae pv. tomato.     

Azido-Coronatine: A Useful Platform for “Click Chemistry”-Mediated Probe Synthesis for Bioorganic Studies

We report on the development of azide-coronatine as a useful platform for azide alkyne cycloaddition (“click chemistry”)-mediated synthesis of molecular probes. (+)-Azido-coronatine was synthesized in 10 steps with 11% yield using improved synthesis of coronafacic acid, in which the highly exo-selective Diels-Alder reaction (endo:exo > 1:25) is the key step. Azido coronatine was as effective as the original coronatine in a stomatal opening assay, and was easily modified to a fluorescein isothiocyanate (FITC)-labeled probe with high yield.     

Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.     

Triterpenoids and glycosides from Geum japonicum

Two new glucosides and two known ester glucosides have been isolated from Geum japonicum. The two new glucosides were isolated by formation of their acetates and were identified as glucosides of 2-isopropyl-5-methylhydroquinone by chemical and spectral studies. The two known ester glucosides were identified as niga-ichigoside F1 and suavissimoside F1 by direct comparison with authentic samples. 2α,19α-Dihydroxyursolic acid and the known glycoside, gein, were also isolated from the same plant, in addition to a mixture of 2α-hydroxyursolic acid and 2α-hydroxyoleanolic acid.     

Biosynthesis of azetidine-2-carboxylic acid from methionine in Nicotiana tabacum

The administration of dl-methionine-[1¹⁴C] to Nicotia tabacum resulted in the formation of radioactive azetidine-2-carboxylic acid (isolated by dilution) which was specifically labelled on its carboxyl group. This result and other evidence strongly indicates that this imino acid is a normal component of tobacco.     

Depsidone constituents from the quintaria group of Nephroma species

A new lichen depsidone was isolated, in the form of its triacetate derivative from the acetylated extracts of Nephroma antarcticum and has been demonstrated to be hypoconstictic acid-triacetate. Two related depsidones, hypostictic acid hyposalazinic acid, were isolated from N. australe.     

Sesquiterpenoid metabolites from Stereum complicatum

A new sesquiterpene antibiotic, complicatic acid, isolated from cultures of Stereum complicatum (Fr.)Fr. has been shown to be dehydrohirsutic acid C. Hirsutic acid C was also isolated from the same fungus. [2-¹⁴C]-MVA was incorporated into both metabolites and complicatic acid has been shown to be formed from hirsutic acid C both in vivo and in vitro.     

Iridoid glucosides from Utricularia australis and Pinguicula vulgaris (lentibulariaceae)

Utricularia australis contained 6-deoxycatalpol, a new iridoid glucoside, besides aucubin, gardoside and mussaenosidic acid. From Pinguicula vulgaris was isolated catalpol, globularin and 10-(Z)-cinnamoyl-catalpol, the latter being a new compound. Thus, the iridoids found in Lentibulariaceae belong to structural types which are common in Scrophulariaceae and related families.     

The biosynthesis of abscisic acid in Cercospora rosicola

1′-Deoxyabscisic acid (1′-deoxy-ABA) has been isolated from cultures of Cercospora rosicola which are actively synthesizing abscisic acid (ABA)     

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a β-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.     

Triterpenoid and phenolic constituents of Colubrina granulosa

Ether extracts of Colubrina granulosa heartwood yield the polyphenolic coumaranone maesopsin and 3 pentacyclic triterpene acids of the lup-20(29)-en-28-oic acid group, alphitolic, betulinic and ceanothic acids. Two new compounds, granulosic and colubrinic acids, which belong to a very small class of A ring contracted triterpenes were also isolated. The co-occurrence of maesopsin, alphitolic, betulinic and ceanothic acids suggests that C. granulosa (from Panama) may be closely related or identical to the Australian species, Alphitonia whitei, in which these relatively rare compounds also occur.     

A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni

A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component.     

A herbicidal fatty acid produced by Lyngbya aestuarii

A herbicidal component isolated from ethanolic extracts of Lyngbya aestuarii was identified as 2,5-dimethyldodecanoic acid. It inhibited the growth of Lemna minor at concentrations higher than 200 ng/ml. Growth inhibition was strongly pH dependent.     

N-Coronafacoyl-L-isoleucine and N-coronafacoyl-L-alloisoleucine,potential biosynthetic intermediates of the phytotoxin coronatine

Two new naturally-occurring analogues of the phytotoxin coronatine have been isolated from liquid cultures of Pseudomonas syringae pv. glycinea. These have been identified as N-coronafacoyl-L-isoleucine and N-coronafacoyl-L-alloisoleucine by mass spectrometry and by studies of the products of acid hydrolysis of the two compounds. The compounds were purified as a mixture of ca 2:1 composition, but the two parent components were not preparatively separated. The possible significance of the two compounds, to the biosynthesis of coronatine, is discussed.     

The triterpenoid acids of Schinus molle

From the acidic fraction of the oleoresin obtained from the berries of Schinus molle was isolated isomasticadienonic, isomasticadienonalic, masticadienonic and 3-epi-isomasticadienolic acids. In addition the new 3-epi-isomasticadienolalic acid was isolated. Both isomasticadienonalic and 3-epi-isomasticadienolalic acids had an aldehydic group attached to C-20. The absolute configuration of this centre was established. The PMR study of the isolated compounds, of their reduction products and O-acetyl derivatives is reported.     

New organic bases from amazonian Banisteriopsis caapi

Three new bases were isolated from Banisteriopsis caapi; they are harmine N-oxide, harmic acid methyl ester (methyl 7-methoxy-β-carboline 1-carboxylate) and harmalinic acid (7-methoxy-3,4-dihydro-β-carboline 1-carboxylic acid).