Serpula lacrymans reacts with a general,unspecialized chemical response during interaction with mycoparasitic Trichoderma spp. and bacteria

Serpula lacrymans causes structural damage in timber via cellulose and hemicellulose decomposition. Serpula lacrymans shares its habitat with bacteria and fungi, interactions that often result in stress and changed behaviour of the partners. A typical response to environmental stress is the production of bioactive metabolites and pigments. The diversity and function of these metabolites in inter- and intra-kingdom interactions, is largely elusive. Using dual culture approaches and secondary metabolite fingerprinting with high performance thin layer chromatography (HPTLC) it was demonstrated that stress induces an unspecific vs. Similar metabolite patterns were identified when S. lacrymans was co-cultured with different bacteria and mycoparasitic fungi (Trichoderma spp.). By analysing the metabolites produced along the gradient from the contact zone to more remote parts of the mycelium, we show a systemic reaction of S. lacrymans and that direct contact with other microbes is not a necessity to induce secondary metabolites.     

Influence of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> mycelia culture conditions on antimicrobial metabolite production

Enokipodins A, B, C, and D are α-cuparene-type sesquiterpenoids antimicrobial metabolites produced in the stationary stage of Flammulina velutipes mycelia development in malt extract broth. This study assessed the influence of nutritional and environmental factors on F. velutipes mycelia culture for the production of these metabolites. The mycelia growth and antimicrobial activity were assessed by determining dry matter and the diffusion in agar method, respectively. The best F. velutipes mycelia growth was observed in dextrose potato broth, and greater antimicrobial metabolite production occurred in complete Pontecorvo’s culture medium. Environmental modifications, such as a rise in temperature from 25° to 37°C on the 15th day of F. velutipes mycelia culture in malt extract and peptone broth, also optimized antimicrobial metabolite production. The metabolites produced in these treatments were correlated with the enokipodins A and B in thin-layer chromatography (TLC) and the antifungal activity test by TLC bioautography. This study showed that there was no correlation between biomass production and antimicrobial metabolite production, but there may be a correlation between culture medium composition and enokipodins biosynthesis.     

MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides

The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface.     

Production and Excretion of Nod Metabolites by Rhizobium leguminosarum bv. trifolii Are Disrupted by the Same Environmental Factors That Reduce Nodulation in the Field

Lipooligosaccharides (Nod metabolites) have been shown to be essential for the successful nodulation of legumes. In strains of Rhizobium leguminosarum bv. trifolii, Nod metabolites were detected predominantly within the cell and to a lesser extent in the periplasmic space and the growth medium. The production, and in particular the excretion, of Nod metabolites was restricted by a range of environmental conditions which are associated with poor nodulation in the field. Lowering the medium pH from 7.0 to 5.0, reducing the phosphate concentration from 1 mM to 5 μM KH₂PO₄, and lowering the incubation temperature from 28 to 18°C affected the number and relative concentrations of the Nod metabolites made. The form and concentration of the nitrogen source affected the relative concentrations of the Nod metabolites produced and excreted. KNO₃ concentrations of >10 mM did not affect cell growth rate but substantially reduced the number of Nod metabolites released. Environmental stresses differentially altered Nod metabolite production and excretion in the same strain carrying different introduced nod regions. Strain ANU845(pWLH1) produced and excreted comparatively fewer Nod metabolites at pH 5.0 and at 18°C than strain ANU845(pRI4003). The excretion but not the production of Nod metabolites by strain ANU845(pRtO32) was dependent on the presence of both nodI and nodJ. Tn5-induced nodI and nodJ mutants did not accumulate any metabolites either outside the cell or within the outer membrane or periplasmic space. Recognition that Nod metabolite accumulation is a complex system of production and excretion, with each component responding differently to changes in environmental conditions, has many consequences, both at the molecular level and in the field. The ability of different strains to produce and release Nod metabolites is likely to be a major determinant of nodule occupancy and should be considered when screening strains suitable for adverse environments.     

Impact of a Transposon Insertion in phzF2 on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa

In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a Pseudomonas aeruginosa phenazine biosynthetic gene, phzF2. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with Aspergillus fumigatus by influencing triacetylfusarinine production.     

Isolation and characterization of active metabolites of tryptophan-pyrolysate mutagen,TRP-P-2, formed by rat liver microsomes

The mutagenic compound derived from the pyrolysis of tryptophan, 3-amino-1-methyl-5H-pyrido-[4,3b]indole (Trp-P-2) was metabolized by rat liver microsomes to more than four metabolites, separable by high performance liquid chromatography. Among these metabolites, two metabolites, M-3 and M-4 were directly active in increasing the frequency of mutation in Salmonella typhimurium TA98. Treatments of rats with polychlorinated biphenyl (PCB) or 3-methylcholanthrene dramatically induced the activity of liver microsomes to form these active metabolites, while treatment with phenobarbital was without effect. A major active metabolite (M-3) formed the pentacyano-ammine ferroate, which is known to be formed by reaction of sodium pentacyano-ammine ferroate with some hydroxylamines. Further this metabolite was oxidized to the minor active metabolite (M-4) with potassium ferricyanide or γ-manganese dioxide, and was reduced back to Trp-P-2 with titanium trichloride. These results indicated that the major active metabolite of Trp-P-2, which is formed by cytochrome P-450, is the 3-hydroxyamino derivative.     

Enhanced thermo-tolerance in transgenic potato (Solanum tuberosum L.) overexpressing hydrogen peroxide-producing germin-like protein (GLP)

Germins and germin-like proteins (GLPs) were reported to participate in plant response to biotic and abiotic stresses involving hydrogen peroxide (H₂O₂) production, but their role in mitigating heat stress is poorly understood. Here, we investigated the ability of a Solanum tuberosum L. GLP (StGLP) gene isolated from the yeast cDNA library generated from heat-stressed potato plants and characterized its role in generating innate and/or acquired thermo-tolerance to potato via genetic transformation. The transgenic plants exhibited enhanced tolerance to gradual heat stress (GHS) compared with sudden heat shock (SHS) in terms of maximal cell viability, minimal ion leakage and reduced chlorophyll breakdown. Further, three StGLP transgenic lines (G9, G12 and G15) exhibited enhanced production of H₂O₂, which was either reduced or blocked by inhibitors of H₂O₂ under normal and heat stress conditions. This tolerance was mediated by up-regulation of antioxidant enzymes (catalase, ascorbate peroxidase and glutathione reductase) and other heat stress-responsive genes (StHSP70, StHSP20 and StHSP90) in transgenic potato plants. These results demonstrate that H₂O₂ produced by over-expression of StGLP in transgenic potato plants triggered the reactive oxygen species (ROS) scavenging signaling pathways controlling antioxidant and heat stress-responsive genes in these plants imparting tolerance to heat stress.     

Biological Validations of Fecal Glucocorticoid,Testosterone, and Progesterone Metabolite Measurements in Captive Stumptail Macaques (<Emphasis Type="Italic">Macaca arctoides</Emphasis>)

Measuring hormone metabolites in fecal samples allows the noninvasive assessment of some steroid hormones in primates. However, noninvasive hormone assays need analytical and biological validation owing to variation in hormone metabolism and excretion between the sexes and across species. We aimed to validate the measurement of fecal glucocorticoid (fGC), testosterone (fT), and progesterone (fP) metabolites in 15 captive stumptail macaques (Macaca arctoides). We collected fecal samples before and after we induced a stress response by restraining and injecting the subjects with saline solution. We then measured hormone metabolites using a methanol extraction technique and ¹²⁵I radioimmunoassay kits. We analyzed the change in glucocorticoid production before and after the stressor, as well as sexual and social rank differences. For fT metabolite levels we investigated variation with sex, age, and social rank, and for fP metabolite levels, we tested for sexual and cycle phase differences. We found a significant increase in fGC metabolite levels 22–25 h poststressor in both sexes. The increase was greater in high-ranking than in low-ranking individuals. Levels of fT metabolites were higher in males than in females, correlated positively with rank only in males, and correlated negatively with age in both sexes. fP metabolite levels were higher in females than in males, and were higher during the luteal phase than in the follicular phase. These findings indicate that our assays reliably detected hormonal changes related to stress (fGC) and detected differences between social and sexual categories (fT, fP) in stumptail macaques.     

Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.     

In vitro grown potato microtubers are a suitable system for the study of primary carbohydrate metabolism

The aim of this work was to determine the suitability of tissue culture microtubers for the study of primary carbohydrate metabolism in potato plants (Solanum tuberosum L. cv. Desirée). We have determined the levels of key enzymes and intermediates in the pathways of sucrose metabolism, starch metabolism and glycolysis in wild type microtubers. Comparison of the metabolite levels in microtubers with a range of published studies on soil-grown developing tubers showed that the two systems were similar both in the absolute levels and in the ratios between metabolites, despite some differences in the maximum catalytic activities of some glycolytic enzymes. We conclude that in vitro grown microtubers are an adequate model system for studying primary carbohydrate metabolism in developing potato tubers.     

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml⁻¹ for two of the three test organisms).     

Metabolic processes involved with sugar alcohol and secondary metabolite production in the hyperaccumulator lichen Diploschistes muscorum reveal its complex adaptation strategy against heavy-metal stress

The synthesis of various unique secondary metabolites by lichens is the result of mutualistic symbiotic association between the mycobiont and autotrophic photobiont. The function of these compounds and causal factors for their production are not fully understood. This paper examines the effect of heavy-metal bioaccumulation and physiological parameters related to photosynthesis and carbon metabolism on the production of lichen substances in hyperaccumulator Diploschistes muscorum. The obtained model of secondary metabolite concentrations in the thalli demonstrates that the carbon source provided by the photobiont and associated polyols produced by the mycobiont have positive impact on the production; on the contrary, the increased intracellular load of heavy metals and excessive loss of cell membrane integrity adversely affected secondary metabolite contents. Additionally, the production of secondary metabolites appears to be more dependent on intracellular metal concentrations than on soil pollution level. To compensate for metal stress, both efficient functioning of algal component and sufficient production of secondary metabolites are required. The balanced physiological functioning of mycobiont and photobiont constitutes the complex protective mechanism to alleviate the harmful effects of heavy metal stress on primary and secondary metabolism of lichens.     

Chemical Derivatization of Metabolite Mass Profiling of the Recretohalophyte <Emphasis Type="Italic">Aeluropus lagopoides</Emphasis> Revealing Salt Stress Tolerance Mechanism

Plants are the primary producers of food for human being. Their intracellular environment alternation is influenced by abiotic stress factors such as drought, heat and soil salinity. Aeluropus lagopoides is a strong halophyte that grows with ease under high saline muddy banks of creeks of Gujarat, India. To study the response of salinity on metabolite changes in Aeluropus, three treatments, i.e. control, salinity and recovery, were selected for both shoot and root tissue. The cytosolic metabolite state was analysed by molecular chemical derivatization gas chromatography mass profiling. During saline treatment, significant increase of compatible solutes in shoot and root tissue was observed as compared to control. Subsequently, metabolic concentration decreased under recovery conditions. The metabolites like amino acids, organic acids and polyols were significantly detected in both shoot and root of Aeluropus under salinity. The metabolites like proline, aspartic acid, glycine, succinic acid and glycolic acid were significantly upregulated under stress. The salicylic acid was found to play a role in maintaining the polyols level by its down-regulation during salinity. The principle component analysis of all detected metabolites in both shoot and root showed that metabolites expressed under salinity (component 1) were highly variable, while metabolites expressed under recovery (component 2) were comparatively less variable as compared to control. The evolved intracellular compartmentalization of amino acids, organic acids and polyols in A. lagopoides can be a hallmark to sustaining at high salinity stress.     

Chemical and physiological characterization of taxa in theFusarium sambucinum complex

Forty-one isolates ofFusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains ofF. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remainingF. sambucinum strains produced T-2 toxin, TB1 and TB2.Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB.Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.     

Metabolism of 4-Chloronitrobenzene by the Yeast Rhodosporidium sp

The yeast Rhodosporidium sp. metabolized 4-chloronitrobenzene by a reductive pathway to give 4-chloroacetanilide and 4-chloro-2-hydroxyacetanilide as the major final metabolites. The intermediate production of 4-chloronitrosobenzene, 4-chlorophenylhydroxylamine, and 4-chloroaniline was demonstrated by high-pressure liquid chromatography. Additional studies with selected metabolites established that the metabolite 4-chloro-2-hydroxyacetanilide was produced by an initial Bamberger rearrangement of the hydroxylamine metabolite, followed by acetylation. Direct C hydroxylation of the aromatic ring was not observed in this species. No hydroxamic acid production was detected, even though significant concentrations of the nitroso and hydroxylamine precursors to this functional group were observed.     

Metabolomic Profiling of Faecal Extracts from Cryptosporidium parvum Infection in Experimental Mouse Models

Cryptosporidiosis is a gastrointestinal disease in humans and animals caused by infection with the protozoan parasite Cryptosporidium. In healthy individuals, the disease manifests mainly as acute self-limiting diarrhoea, but may be chronic and life threatening for those with compromised immune systems. Control and treatment of the disease is challenged by the lack of sensitive diagnostic tools and broad-spectrum chemotherapy. Metabolomics, or metabolite profiling, is an emerging field of study, which enables characterisation of the end products of regulatory processes in a biological system. Analysis of changes in metabolite patterns reflects changes in biochemical regulation, production and control, and may contribute to understanding the effects of Cryptosporidium infection in the host environment. In the present study, metabolomic analysis of faecal samples from experimentally infected mice was carried out to assess metabolite profiles pertaining to the infection. Gas-chromatography mass spectrometry (GC-MS) carried out on faecal samples from a group of C. parvum infected mice and a group of uninfected control mice detected a mean total of 220 compounds. Multivariate analyses showed distinct differences between the profiles of C. parvum infected mice and uninfected control mice,identifying a total of 40 compounds, or metabolites that contributed most to the variance between the two groups. These metabolites consisted of amino acids (n = 17), carbohydrates (n = 8), lipids (n = 7), organic acids (n = 3) and other various metabolites (n = 5), which showed significant differences in levels of metabolite abundance between the infected and uninfected mice groups (p < 0.05). The metabolites detected in this study as well as the differences in abundance between the C. parvum infected and the uninfected control mice, highlights the effects of the infection on intestinal permeability and the fate of the metabolites as a result of nutrient scavenging by the parasite to supplement its streamlined metabolism.