Investigation of the mechanisms underlying the high acetylene-reducing activity exhibited by the soil bacterial community from BC2 horizon in the permafrost zone of the East Siberian larch forest bed.

AIMS: This study addressed a microbial composition of East Siberian larch forest bed soil and focused on major nitrogen-fixing bacterial consortia showing the highest capacity to induce acetylene reduction. METHODS AND RESULTS: The ability of a soil microbial consortium to effectively reduce acetylene was investigated using gellan gum medium. In parallel, each microbial component in the medium was analysed by a 16S rRNA gene-targeting denaturing gradient gel electrophoresis (DGGE), together with an attempt to isolate pure culture of the culturable eubacteria. Six strains of the isolated bacteria were characterized as diazotrophs, and Burkholderia xenovorans was found to be one of the major nitrogen fixer in the bacterial community cultured from the BC2 horizon soil microbiota. Co-culturing of either a diazotrophic Pseudomonas sp. with a nondiazotrophic Luteibacter sp. or a diazotrophic Luteibacter sp. with a nondiazotrophic bacterium B. xenovorans resulted in a marked elevation in acetylene reduction over that observed in the pure culture of each diazotroph. CONCLUSIONS: A promotion of acetylene reduction in mixed bacterial cultures, particularly when the cultures included Luteibacter, was clearly observed due to an increase in the bacterial cell population. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study, which showed that some unculturable bacteria including anaerobic Clostridium sp. that can provide major contribution to nitrogen fixation, may provide key information useful in identifying the 'missing link' in the nitrogen cycle in the cryogenic soil ecosystem of boreal forests.

Capability of wild Rosa rugosa and its varieties and hybrids to produce sesquiterpene components in leaf glandular trichomes.

The sesquiterpene contents in leaves of wild Rosa rugosa and of sixty-one hybrid rugosas were quantitatively measured by a GC analysis. In this group of samples, the greater the number of glandular trichomes the hybrid rugosas possessed on their leaves, the larger the amount of sesquiterpenes they accumulated. In contrast, those having no leaf glandular hairs contained only a trace amount of sesquiterpene components. The concentrations of bisaborosaol A (1) and carota-1,4-dienaldehyde (2) as representative sesquiterpenes of R. rugosa were positively correlated with the density of the glandular trichomes. Furthermore, an approximately regular correlation was observed between the concentrations of 1 and 2 in most of the sesquiterpene-producing hybrid rugosas, regardless of their productivity. This suggests that a major part of these hybrid rugosas have inherited from R. rugosa the ability to produce two skeletally different sesquiterpenes in parallel with a phenotype to develop leaf glandular trichomes. This investigation also led to discovering 1-dominant (e.g., Amelie Gravereaux and Purple Pavement), 2-dominant (e.g., David Thompson), and other-dominant (e.g., Martin Frobisher) types of sesquiterpene-producing hybrid rugosas.

Induction of 4-hydroxycinnamate decarboxylase in Klebsiella oxytoca cells exposed to substrates and non-substrate 4-hydroxycinnamate analogs.

The 4-hydroxycinnamate decarboxylase (4-HCD)-inducing activity of several substrate analogs toward Klebsiella oxytoca was investigated. Four E-cinnamate-class compounds, E-4-hydroxycinnamic acid (1), caffeic acid (2), ferulic acid (3) and E-2,4-dihydroxycinnamic acid (4), all of which were accepted as substrates, all of which were accepted as substrates of 4-HCD, enable K. oxytoca cells to induce the decarboxylase at a 2.0 mM concentration, while five non-substrate compounds of the E-cinnamate class so far tested were completely inactive. However, 6-hydroxy-2-naphthoic acid (11) and 7-hydroxycoumarin 3-carboxylic acid (14), both of which are non-cinnamate-class analogs of the substrate, acted as strong 4-HCD inducers, even at a 0.5 mM concentration. The 4-HCD-inducing activities of compounds 11 and 14 at 0.5 mM were 10-12-fold higher than that of substrate 1. Compound 11 maintained its 4-HCD-inducing activity toward cultured cells through the late-log and stationary phases, unlike 1 that induced 4-HCD only in the early log phase. SDS-PAGE electrophoresis of protein mixtures from the cultured cells exposed to any 4-HCD inducer indicated that the 21.5 kDa protein was always present.

Activation of isoflavone biosynthesis in excised cotyledons of Lupinus seedlings by jasmonoids and excess light.

Exogenous jasmonic acid (JA) and methyl jasmonate (MJ) induced accumulation of isoflavone constituents in cotyledons prepared from imbibed seeds of white lupin (Lupinus albus L.). Exogenous 0.2 mM MJ enhanced the levels of 7-O-(6"-O-malonyl)glucosylgenistein and 7-O-glucosylgenistein in the cotyledons of etiolated seedlings that had been incubated in the dark for 48 h. Regarding isoflavone induced by excision and slicing in the cotyledons as background level, the effect of light was 2- to 3-fold higher than that of 0.2 mM MJ. Cotyledons exposed to MJ along with a 24-h light period displayed a higher level of isoflavone accumulation than that of light alone. Total molar amounts of isoflavone accumulated in the cotyledons treated with MJ under continuous light were approximately the sum of those induced by MJ alone and light alone, respectively. The additive-like effect of MJ and light on isoflavone accumulation in lupin tissues suggested the presence of two different signaling systems independently responsible for those two stimuli. Excised cotyledons from etiolated yellow lupin (L. luteus L. cv. Topaz) seedlings also supported this hypothesis. The cotyledons could accumulate both an isoflavone and a flavone, and MJ selectively increased some of the isoflavone constituents, whereas light enhanced the levels of both. The selective accumulation mechanism of isoflavonoids in cotyledons, in which jasmonoids are involved, clearly differed from that activated by light.

(+)-4-epi-alpha-bisabolol as a major sesquiterepene constituent in the leaves of two Rosa rugosa hybrids, Martin Frobisher and Vanguard.

During an investigation of the sesquiterpene phases and contents in leaves of several Rosa rugosa hybrids (hybrid rugosas), Martin Frobisher and Vanguard were found to accumulate a large amount of (+)-4-epi-alphabisabolol (1) as a single constituent. Although glandular trichomes of Martin Frobisher on the leaves are dense, this R. rugosa hybrid produces none of the carota-1,4dienaldehyde (2) or bisaborosaol A (3) that are both found as representative sesquiterpenes of the carotane and bisabolane classes, respectively, in a glandular trichome exudate of wild-type R. rugosa. Compound 1 was also apparent as a nearly single constituent detectable by GC in the leaf constituents of Vanguard possesses sparse glandular trichomes on the leaf. Martin Frobisher and Vanguard had likely lost their capability to form carotane-type sesquiterpenes and had also lost their activity to oxygenate the C-7 allyl methyl carbon of compound 1 to convert 3. The presence of (+)-4-epi-alphabisabolol-accumulating R. rugosa hybrids is significant when considering the sesquiterpene biogenesis of Rosa rugosa.

Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease.

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 microg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.

Accumulation of Isohemigossypolone and Its Related Compounds in the Inner Bark and Heartwood of Diseased Pachira aquatica.

Isohemigossypolone (1) and 2-O-methylisohemigossypolone (2), major fungitoxins of Pachira aquatica, were found to accumulate locally in the outer bark of the swollen trunk, whereas the inner bark and heartwood contained only a trace amount of them. From P. aquatica that was infected with a phytopathogenic bacterium, we detected significant amounts of 1 and 2 from browned inner tissues of the swollen trunk. According to a quantitative analysis by a gas-chromatograph, the concentration of 1 in the diseased inner tissues was calculated to be approximately 780 µg/g f.w., which was the same level as that in the outer bark of healthy individuals. These findings suggest that the inner tissues inducibly produced and accumulated antifungals 1 and 2 during infection events, as do many plants with phytoalexins. 11-Nor-2-O-methylisohemigossypolone (3), showing approximately equivalent fungitoxic activity to that of 1 and 2, was also isolated from the infected inner tissues. We screened pathogenic bacteria from the infected tissue, and isolated a rod-shaped bacterium that was tentatively identified as Pseudomonas sp. which promoted tissue-browning on sectioned disks of P. aquatica trunks.

Factors responsible for inhibiting the motility of zoospores of the phytopathogenic fungus Aphanomyces cochlioides isolated from the non-host plant Portulaca oleracea.

In a survey of plant secondary metabolites regulating the behaviour of Aphanomyces cochlioides zoospores, we found that root extracts of Portulaca oleracea inhibited zoospore motility. Bioassay-directed fractionation of Portulaca constituents revealed that the inhibitory activity was dependent on the interaction of two chemically different factors. These were identified as a phenolic compound, N-trans-feruloyltyramine, which by itself was active as a zoospore stimulant, and an acidic compound, 1-linoleoyl-2-lysophosphatidic acid monomethyl ester, which had zoospore-repellent activity. When Chromosorb W AW particles coated with a mixture of these pure compounds were bioassayed in Petri dishes, the inhibitory effect on zoospore motility was identical with that caused by root tip or root extracts of P. oleracea. Inhibited zoospores rapidly settled to the bottom of the Petri dishes where they initially encysted, and then germinated within 1-2 h. This is the first report of factors which inhibit zoospore motility without killing or bursting the zoospores.

Stereochemically specific proton transfer in decarboxylation of 4-hydroxycinnamic acids by 4-hydroxycinnamate decarboxylase from Klebsiella oxytoca.

The stereochemical specificity in the decarboxylation of E-4-hydroxycinnamic acid catalyzed by E-4-hydroxycinnamate decarboxylase (4-HCD) of Klebsiella oxytoca was investigated. Unlike the pyrolytic decarboxylation of 8-deuterated E-4-hydroxycinnamic acid to yield an equimolecular mixture of 8-Z- and 8-E-deuterated 4-hydroxystyrenes, treating 8-deuterated E-4-hydroxycinnamic acid with the enzyme in H2O-based buffer yielded 8-Z-deuterated 4-hydroxystyrene selectively. The specific E-orientation in catalysis and the substrate specificity requiring 4-OH in the substrates suggest that decarboxylation by K. oxytoca 4-HCD occurs via a para-quinone methide intermediate. Stereoselective protonation and the liberation of CO2 by an intermediary molecule are most likely the key reaction steps in the stereochemical specificity of the newly incorporated hydrogen.

Pyromeconic acid and its glucosidic derivatives from leaves of Erigeron annuus, and the siderophile activity of pyromeconic acid.

3'-O-Caffeylerigeroside (pyromeconic acid 3-O-beta-D-glucoside 3'-O-caffeyl ester) was obtained from the leaves of Erigeron annuus as a new pyromeconic acid derivative, and its structure was elucidated. Together with the gamma-pyrone derivative, pyromeconic acid (3-hydroxy-4H-pyran-4-one) and its beta-glucoside (erigeroside) were also isolated from the aerial parts of E. annuus. The siderophile activity of pyromeconic acid was also studied.

An enzymatic formation of 13-oxo-trideca-9,11-dienoic acid from 13-hydroperoxylinolenic acid by a homolytic hydroperoxide lyase in elicitor-treated soybean cotyledons.

An activity of homolytic hydroperoxide lyase (HPLS) catalyzing the specific cleavage of 13-hydroperoxylinolenic acid to form a volatile compound and 13-oxo acid was found in the enzyme extract from soybean cotyledons. 2-Penten-1-ol was characterized as a volatile metabolite by gas chromatography-mass spectrometry analysis. The methyl ester derivatives of reaction products were separated and isolated by a normal-phase high-performance liquid chromatography, and identified to be 13-oxo-trideca-9(Z),11(E)-dienoic acid methyl ester and its geometric isomer possessed 9(E),11(E) moiety by the analyses of high resolution mass spectrometry and nuclear magnetic resonance spectrometry. An activity of homolytic HPLS found in soybean cotyledons was evidently enhanced by elicitation. The products of 13-oxo-tridecadienoic acid with alpha, beta, gamma, delta-unsaturation were shown to be antifungal substances by a chromatographic bioassay. The metabolites via lipoxygenase and HPLS pathways may have physiological roles in the resistant action of host plants.

Cloning of a DNA Fragment Carrying the 4-Hydroxycinnamate Decarboxylase (pofK) Gene from Klebsiella oxytoca, and Its Constitutive Expression in Escherichia coli JM109 Cells.

The 4-hydroxycinnamate decarboxylase gene (pofK gene) was cloned from Klebsiella oxytoca, an epiphytic bacterium able to decarboxylate hydroxycinnamic acids to styrene derivatives, in Escherichia coli JM109. Colonies of the enzyme activity-positive transformants were screened by a selection assay combined with an antifungal test using Cladosporium herbarum IHU9262 as the bio-indicator. Two positive transformants constitutively producing 4-hydroxycinnamate decarboxylase were obtained. One of the transformants had a recombinant plasmid designated as pTCD100 in which a 9.6 kb HindIII segment carrying pofK gene was contained. As the expression of the pofK gene in K. oxytoca was substrate-inducible, it was most likely that the pofK gene expression in E. coli cells was free from any regulation by a pofK gene repressor postulated in K. oxytoca cells. The decarboxylase synthesized in E. coli cells showed almost the same specific activity as that of K. oxytoca induced by the substrate.

Decarboxylative Conversion of Hydroxycinnamic Acids by Klebsiella oxytoca and Erwinia uredovora, Epiphytic Bacteria of Polymnia sonchifolia Leaf, Possibly Associated with Formation of Microflora on the Damaged Leaves.

Two bacteria, Klebsiella oxytoca and Erwinia uredovora, which constituted epiphytic microftora on yacon (Polymnia sonchifolia) leaves, converted hydroxycinnamic acids into hydroxystyrenes decarboxylatively. Hydroxycinnamate decarboxylase was extracted as crude protein from the bacterial cells, and was substrate-inducible. This decarboxylation was for the bacteria a detoxification of hydroxycinnamic acids of plants, but the metabolites were toxic to other test bacteria and fungi, including some phytopathogens. The possible ecological role of these epiphytic bacteria on the host-plant was discussed. from the viewpoint of their chemical interaction via the styrene derivatives.