Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Degradation of Microcystin-RR by Sphingomonas sp. CBA4 isolated from San Roque reservoir (Córdoba – Argentina)

We report the aerobic biodegradation of Microcystin-RR (MC-RR) by a bacterial strain isolated from San Roque reservoir (Córdoba – Argentina). This bacterium was identified as Sphingomonas sp. (CBA4) on the basis of 16S rDNA sequencing. The isolated strain was capable of degrading completely MC-RR (200 μg l⁻¹) within 36 h. We have found evidence that MC-RR biodegradation pathway by this Sphingomonas sp. strain would start by demethylating MC-RR, affording an intermediate product, which is finally biodegraded by this strain within 72 h. Our results confirm that certain environmental bacteria, living in the same habitat as toxic cyanobacteria, have the capability to perform complete biodegradation of MC, leading to natural bioremediation of waterbodies. The bacterium reported here presents genetic homologies with other strains that degrade MC-LR. However, initial demethylation of MC-RR has been not described previously, raising questions on the probable presence of different biodegradation pathways for different MC variants.     

Purification and characterization of pterocarpan hydroxylase,a flavoprotein monooxygenase from the fungus Ascochyta rabiei involved in pterocarpan phytoalexin metabolism

Crude protein extracts from the chickpea (Cicer arietinum) pathogenic fungus Ascochyta rabiei catalyze the hydroxylation of the pterocarpan phytoalexins medicarpin and maackiain to the corresponding 1a-hydroxy-1,4-diene-3-one derivatives. The enzyme reaction depends on NAD(P)H and molecular oxygen. Low amounts of FAD are necessary for maximal enzyme activity. The pterocarpan hydroxylase is a new flavoprotein monooxygenase with a molecular weight of 58 kDa in SDS-PAGE. The soluble enzyme can utilize NADH and NADPH with similar values for K _m and V _max respectively. The pterocarpan hydroxylase and a pterocarpan reductase (M _r 29 kDa; Höhl and Barz 1987) are constitutively expressed by A. rabiei isolates.Abbreviations AAS atomic absorption spectroscopy - BCS bathocuproindisulfonate - BSA bovine serum albumin - FAD flavin-adenine dinucleotide - FMN flavin-mononucleotide - M _r molecular weight - PAGE polyacrylamide gelelectrophoresis - pda pisatin demethylating ability - SDS sodium dodecylsulfate - Tris tris(hydroxymethyl)aminomethane     

Isolation of a phytoalexin-detoxification gene from the plant pathogenic fungus Nectria haematococca by detecting its expression in Aspergillus nidulans

Detoxification of the pea phytoalexin pisatin via demethylation, mediated by a cytochrome P-450 monooxygenase, is thought to be important for pathogenicity of the fungus Nectria haematococca on pea. To isolate a fungal gene encoding pisatin demethylating activity (pda), we transformed Aspergillus nidulans with a genomic library of N. haematococca DNA constructed in a cosmid which carried the A. nidulans trpC gene. Transformants were selected for Trp+ and then screened for pda. One transformant among 1250 tested was Pda+ and was less sensitive to pisatin in culture than Pda- A. nidulans. The cosmid containing the gene (PDA) conferring this activity was recovered by phage lambda packaging of transformant genomic DNA. When A. nidulans was transformed with the cloned cosmid, 98% of the Trp+ transformants were Pda+. RNA blots probed with a 3.35 kb subclone carrying PDA indicated that the gene is expressed constitutively in A. nidulans but is inducible by pisatin in N. haematococca.     

Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450

The fungus Nectria haematococca, a pathogen of garden pea (Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The K_m for pisatin was relatively low, less than 5 μm. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 mm) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.     

A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA

Summary A single exposure of germinated rice seeds (Oryza sativa) to either of the DNA demethylating agents 5-azacytidine (azaC) or 5-azadeoxycytidine (azadC) induced dwarf plants. At maturity, seeds treated with azaC exhibited normal morphological characteristics in comparison with untreated controls except that their height (total stem length) was reduced by about 15%. The M₁ progeny, obtained by self-fertilization of an azaC-induced dwarf plant, segregated into dwarf (35%) and apparently tall types (65%). The M₂ progenies, obtained by self-fertilization of dwarf M₁ plants, were also dwarf, while those from tall M₁ plants were only tall. Genomic DNA isolated from mature leaves of azaC-treated seeds showed about a 16% reduction in the 5-methylcytosine (m⁵C) content in comparison with DNA from untreated samples. A similar reduction in the m⁵C content was also observed in the M₁ and M₂ progenies. Thus, both undermethylation and dwarfism induced by azaC treatment were heritable. The results suggest that azaC induced demethylation of genomic DNA, which caused an altered pattern of gene expression and consequently a reduction in plant stem length.     

Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Dynamics of Free Pisatin Elicitor Accumulation in Infection-droplets of Monilinia fructicola on Pea Endocarp*

Rapid elicitor accumulation in the infection-droplet of the pea-M. fructicola interaction began between 2 and 3 h after inoculation. Rapid accumulation of pisatin began between 2 and 3 h, however, low levels (0.06–0.1 μg/ml) were detected in the infection-droplet as early as 1–2 h following inoculation with the fungus. The pisatin concentration reached levels inhibitory to the fungus between 6 and 12 h (ca. 1–5 μg/ml) and the ED₅₀ value of 10 μg pisatin/ml for mycelial growth of M. fructicola was attained after 14 h. Elicitor activity in infection-droplets after 4 and 18 h was a function of inoculum concentration and pisatin accumulation in diffusate after 40 h was a function of elicitor concentration (linear doseresponse curve). However, when elicitor was applied at zero time, the rate of pisatin accumulation over the first 12 h was indistinguishable from that which occurred when M. fructicola conidia were applied to the endocarp and elicitor accumulated with time. The initial rate of pisatin accumulation therefore appears to be dependent on the pea pod and independent of any time delays associated with conidial germination and elicitor accumulation. However, the final pisatin concentration which accumulated in the infection-droplet was dependent on the, dose' of elicitor irrespective of the nature and timing of the elicitor treatment. The presence of elicitor activity was demonstrated in the interaction in space and time where and when these molecules could function as elicitors of pisatin in vivo.     

一株林可霉素降解菌的筛选鉴定及其降解机制

【背景】抗生素污染越来越引起人们的关注。利用微生物处理抗生素污染被认为是一种环境友好型的方法。【目的】筛选林可霉素高效降解菌并研究其降解机制。【方法】经形态学观察、生理生化鉴定和16S rRNA基因测序分析进行鉴定;通过PCR技术和质谱分析技术对该菌抗性基因和降解产物等进行分析。【结果】从林可霉素菌渣堆肥样本中获得一株高效降解林可霉素的假单胞菌(Pseudomonas RST-1),该菌在林可霉素浓度为3.0 g/L的牛肉膏蛋白胨培养基上培养40 h后,林可霉素降解率高达57.3%。该菌含有intI1、sul1、sul2等抗性基因,降解产物为去甲基林可霉素和2-丙基-N-甲基脯氨酸。【结论】菌株RST-1具有高效降解林可霉素的能力,推测可能的降解机制为去甲基化和酰胺键水解作用,该菌株降解特性及降解机制研究为林可霉素降解工程菌及其高效降解菌剂的研制奠定了基础。     

Loss of sulfide adaptation ability in a thermophilic Oscillatoria

A spontaneous variant incapable of anoxygenic photosynthesis was derived from a fully competent strain of Oscillatoria amphigramulata which was originally isolated from a high sulfide-containing hot spring of New Zealand. Although the variant (Oa-2) acquired a slight ability to photosynthesize in the presence of 0.3–0.4 mM sulfide, this was only after a 24 h exposure to sulfide and represented oxygenic photosynthesis only. Unlike the parent strain, the incompetent variant never grew in the presence of sulfide >0.05 mM, nor was there any relief of the inhibition by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of CO₂ photoincorporation when sulfide was present. The variant strain has retained all of these characteristics over a 4 year period with monthyl transfers in non-sulfide medium. The wild type, under identical conditions, has retained all of its competence with respect to sulfide.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Rapid induction of genomic demethylation and T-DNA gene expression in plant cells by 5-azacytosine derivatives

We have optimized conditions for demethylation of the genome and induction of a silent, hypermethylated T-DNA gene (ipt) by 5-azacytosine (5-azaCyt) derivatives in a suspension culture of tobacco cells. In this system, 5-azacytidine (5-azaC) is more effective in causing genomic demethylation and ipt gene induction than 5-azaCyt or 5-azadeoxycytidine (5-azadC). A single treatment with 2.5 M 5-azaC resulted in a maximal level of ipt gene induction without inhibiting cell growth. However, we could not reduce the level of genomic methylation below approximately 2/3 of that found in untreated controls, even after extensive 5-azaC treatment. Furthermore, remethylation of the genome occurred after removal of 5-azaC. The use of 5-azaC as an inducer of silent plant genes is discussed, along with differences in the response of plant and animal genomes to demethylating agents.Abbreviations C cytidine - Cyt cytosine - 5-azaCyt 5-azacytosine - 5-azaC 5-azacytidine - 5-azadC 5-azadeoxycytidine - m⁵Cyt 5-methylcytosine     

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.     

A comparison of the requirements for various carbon and nitrogen sources and vitamins in some Frankia isolates

Summary It was established that anAlnus glutinosa isolate (LDAgp 1) is able to utilize mono- and disaccharides and shows a limited growth ability on arabinose and starch. This contrasts with an isolate fromAlnus viridis (AvcI 1) andComptonia peregrina (CpI1), which apparently lack glycolytic pathway activity. These latter isolates can utilize some tricarboxylic acids in contrast to LDAgp1. Volatile fatty acids or their salts, such as propionic acid and acetate, were utilized by all three isolates. Besides a general ability to utilize inorganic nitrogen sources, some amino acids and urea, selected isolates showed a limitedability to utilize adenine and uracil. A simple, synthetic medium based on propionic acid as the energy source was developed. On this medium some isolates showed growth stimulation in the presence of biotin. The metabolic aspects of the utilization of carbon and nitrogen sources, as well as some ecological consequences are discussed.     

SHORT COMMUNICATIONSTEROID HORMONE (HYDROCORTISONE, OESTRADIOL AND TESTOSTERONE) UPTAKE, STORAGE OR INDUCED SYNTHESIS INTETRAHYMENA

After cyclodextrin-coated 10⁻⁶ steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P₄₅₀aromatase.     

Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms

Effects of tourmaline ceramic balls on growth and metabolism of Saccharomyces cerevisiae, Lactobacillus acidophilus and Aspergillus oryzae were studied. Treatments with 3, 6, 9 or 12 g of tourmaline ceramic balls in a 50 ml culture showed significant stimulation of the growth of the three microorganisms. In optimal treatments with 12 g of tourmaline balls, the growth of S. cerevisiae, L. acidophilus, and A. oryzae was increased by 34, 32 and 10%, respectively. After 72 h fermentation of S. cerevisiae, total carbohydrate content in the culture medium was decreased by 65% and ethanol production was increased by 150%. Total carbohydrate content was decreased by 80% and the pH value was decreased by 0.3, as a result of organic acid production in the medium of L. acidophilus after 72 h fermentation. In the case of A. oryzae, enzyme activities of protease and amylase were increased by 90 and 31%, respectively, after 96 h fermentation. Results indicated that tourmaline stimulates initiation of growth in the early lag stage and increases production of metabolites at a later stage of fermentation. The strong stimulatory effect of tourmaline on growth, utilization of substrates and production of metabolites in the three microorganisms suggests a potential application in the fermentation industry.     

Conditions for the production of jadomycin B byStreptomyces venezuelae ISP5230: Effects of heat shock,ethanol treatment and phage infection

Summary The novel benzoxazolophenanthridine antibiotic, jadomycin B, is produced byStreptomyces venezuelae ISP5230 following a 42 °C heat shock or exposure to ethanol. To further characterize these unusual culture conditions, studies were carried out using different media, varying nutrient concentrations, initial pH, and time of application of heat or ethanol stress. Highest titers of jadomycin B accumulated 48 h afterS. venezuelae ISP5230 was inoculated into ad-galactose-l-isoleucine production medium (pH 7.5) which was supplemented with ethanol (6%, v/v) between 6 and 13 h. Cultures supplemented with ethanol later than 17 h post inoculation into the production medium produced little or no jadomycin B. Among other heat-shock inducing treatments examined, infection with phage SV1 was associated with increased jadomycin B production. Although jadomycin B titers showed little change with variations in the concentration of phosphate in the production medium, the nature of the nitrogen source was found to be important. Different colored pigments, presumed to be jadomycin B analogs, were formed when other amino acids replacedl-isoleucine in the medium as the sole nitrogen source. Increased jadomycin B titers accompanied increasedl-isoleucine andd-galactose concentrations in the production medium.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.     

Development of tolerance against toxic cyanobacteria in Daphnia

We tested whether previous exposure to a toxic strain of cyanobacteria (Microcystis) affects survival, growth, and reproduction of a common herbivore, Daphnia magna. Samples from three natural populations of D. magna were each divided into two parts; one part was fed a mixture of toxic Microcystis and the non-toxic green alga Scenedesmus whereas the other part was fed only Scenedesmus. After four weeks, we compared the ability of these two populations to withstand the toxic Microcystis by assessing survivorship, growth, and reproduction. We found that the ability of D. magna to cope successfully with toxic Microcystis is improved if the animals have experienced previous exposure to toxic Microcystis. This suggests that the toxin may less affect the D. magna populations that are repeatedly exposed to toxic cyanobacteria in their natural habitat than populations lacking prior exposure. Since the ability to tolerate toxins is manifested in both improved survival and larger size of the animals, it may have considerable impact on zooplankton community composition in fresh-waters.