RESUMEN
Reactive oxygen species (ROS) are among the most severe types of cellular stressors with the ability to damage essential cellular biomolecules. Excess levels of ROS are correlated with multiple pathophysiological conditions including neurodegeneration, diabetes, atherosclerosis, and cancer. Failure to regulate the severely imbalanced levels of ROS can ultimately lead to cell death, highlighting the importance of investigating the molecular mechanisms involved in the detoxification procedures that counteract the effects of these compounds in living organisms. One of the most abundant forms of ROS is H2 O2 , mainly produced by the electron transport chain in the mitochondria. Numerous genes have been identified as essential to the process of cellular detoxification. Yeast YAP1, which is homologous to mammalian AP-1 type transcriptional factors, has a key role in oxidative detoxification by upregulating the expression of antioxidant genes in yeast. The current study reveals novel functions for COX5A and NPR3 in H2 O2 -induced stress by demonstrating that their deletions result in a sensitive phenotype. Our follow-up investigations indicate that COX5A and NPR3 regulate the expression of YAP1 through an alternative mode of translation initiation. These novel gene functions expand our understanding of the regulation of gene expression and defense mechanism of yeast against oxidative stress.
Asunto(s)
Aterosclerosis , Proteínas de Saccharomyces cerevisiae , Animales , Saccharomyces cerevisiae/genética , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno , Antioxidantes , Mamíferos , Factores de Transcripción/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Lithium chloride (LiCl) has been widely researched and utilized as a therapeutic option for bipolar disorder (BD). Several pathways, including cell signaling and signal transduction pathways in mammalian cells, are shown to be regulated by LiCl. LiCl can negatively control the expression and activity of PGM2, a phosphoglucomutase that influences sugar metabolism in yeast. In the presence of galactose, when yeast cells are challenged by LiCl, the phosphoglucomutase activity of PGM2p is decreased, causing an increase in the concentration of toxic galactose metabolism intermediates that result in cell sensitivity. Here, we report that the null yeast mutant strains DBP7∆ and YRF1-6∆ exhibit increased LiCl sensitivity on galactose-containing media. Additionally, we demonstrate that DBP7 and YRF1-6 modulate the translational level of PGM2 mRNA, and the observed alteration in translation seems to be associated with the 5'-untranslated region (UTR) of PGM2 mRNA. Furthermore, we observe that DBP7 and YRF1-6 influence, to varying degrees, the translation of other mRNAs that carry different 5'-UTR secondary structures.
Asunto(s)
Cloruro de Litio , Proteínas de Saccharomyces cerevisiae , Cloruro de Litio/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , Galactosa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ARN Helicasas DEAD-box/metabolismoRESUMEN
Lithium chloride (LiCl) is a widely used and extensively researched drug for the treatment of bipolar disorder (BD). As a result, LiCl has been the subject of research studying its toxicity, mode of action, and downstream cellular responses. LiCl has been shown to influence cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3 in mammalian cells. LiCl's significant downstream effects on the translational pathway necessitate further investigation. In yeast, LiCl is found to lower the activity and alter the expression of PGM2, a gene encoding a sugar-metabolism enzyme phosphoglucomutase. When phosphoglucomutase activity is reduced in the presence of galactose, intermediates of galactose metabolism aggregate, causing cell sensitivity to LiCl. In this study, we identified that deleting the genes PEX11 and RIM20 increases yeast LiCl sensitivity. We further show that PEX11 and RIM20 regulate the expression of PGM2 mRNA at the translation level. The observed alteration of translation seems to target the structured 5'-untranslated region (5'-UTR) of the PGM2 mRNA.
Asunto(s)
Cloruro de Litio , Proteínas de la Membrana , Peroxinas , Proteínas de Saccharomyces cerevisiae , Galactosa , Cloruro de Litio/farmacología , Proteínas de la Membrana/genética , Peroxinas/genética , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Regiones no TraducidasRESUMEN
Vegetative incompatibility (VI) is a form of non-self allorecognition in filamentous fungi that restricts conspecific hyphal fusion and the formation of heterokaryons. In the chestnut pathogenic fungus, Cryphonectria parasitica, VI is controlled by six vic loci and has been of particular interest because it impedes the spread of hypoviruses and thus biocontrol strategies. We use nuclear magnetic resonance and high-resolution mass spectrometry to characterize alterations in the metabolome of C. parasitica over an eight-day time course of vic3 incompatibility. Our findings support transcriptomic data that indicated remodeling of secondary metabolite profiles occurs during vic3 -associated VI. VI-associated secondary metabolites include novel forms of calbistrin, decumbenone B, a sulfoxygenated farnesyl S-cysteine analog, lysophosphatidylcholines, and an as-yet unidentified group of lipid disaccharides. The farnesyl S-cysteine analog is structurally similar to pheromones predicted to be produced during VI and is here named 'crypheromonin'. Mass features associated with C. parasitica secondary metabolites skyrin, rugulosin and cryphonectric acid were also detected but were not VI specific. Partitioning of VI-associated secondary metabolites was observed, with crypheromonins and most calbistrins accumulating in the growth medium over time, whereas lysophosphatidylcholines, lipid disaccharide-associated mass features and other calbistrin-associated mass features peaked at distinct time points in the mycelium. Secondary metabolite biosynthetic gene clusters and potential biological roles associated with the detected secondary metabolites are discussed.
Asunto(s)
Ascomicetos , Virus ARN , Ascomicetos/genética , Metabolómica , MicelioRESUMEN
The fate, toxicity and bioaccumulation of copper oxide nanoparticles (nCuO) was investigated in soil, with and without biosolids amendment, through chronic exposures using the earthworm, Eisenia andrei, and the collembolan, Folsomia candida. The effects of copper sulphate (CuSO4) were included so as to compare the behavior of nCuO to a readily soluble counterpart. The fate of nCuO was evaluated through characterization of dissolved and nano-particulate fractions (via single particle ICP-MS) as well as extractable Cu2+ throughout the duration of select tests. Neither Cu form was particularly toxic to F. candida, but effects on E. andrei reproduction were significant in all treatments (IC50 range: 98 - 149 mg Cu kg-1 dry soil). There were no significant differences in toxicity between the Cu forms, nor in extractable Cu2+ activities, indicative that particle dissolution within the soil and, subsequent activity of Cu2+ was likely the primary mode of toxicity in the nCuO exposures. The presence of biosolids did not significantly alter toxicity of nCuO, but did affect Cu2+ activity over time. Bioaccumulation of total Cu in E. andrei when exposed to nCuO (kinetic bioaccumulation factor (BAFk): 0.80 with biosolids and 0.81 without) was lower than exposure to CuSO4 (BAFk: 2.31 with biosolids and 1.12 without). Enhanced dark-field hyperspectral imaging showed accumulation of nCuO along the epidermis and gut of E. andrei, with trace amounts observed in muscle and chloragogenous tissue, providing evidence of nCuO translocation within the organism. The present study demonstrates that the current risk assessment approach for trace metals in the environment, based on substance solubility and bioavailability of the dissolved free ion, are applicable for nCuO exposure to soil invertebrates, but that the rate of particle dissolution in different soil environments is an important factor for consideration.
Asunto(s)
Biosólidos , Sulfato de Cobre/toxicidad , Cobre/toxicidad , Invertebrados/fisiología , Contaminantes del Suelo/toxicidad , Animales , Artrópodos , Bioacumulación , Disponibilidad Biológica , Nanopartículas , Oligoquetos/efectos de los fármacos , Óxidos/farmacología , Suelo , Contaminantes del Suelo/análisisRESUMEN
Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L50 = 5; N50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.
Asunto(s)
Ascomicetos , Fagaceae , Virus Fúngicos , América del Norte , Enfermedades de las PlantasRESUMEN
Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome, has killed millions of bats across eastern North America and continues to threaten new bat populations. The spread and persistence of P. destructans has likely been worsened by the ability of this fungus to grow as a saprotroph in the hibernaculum environment. Reducing the environmental growth of P. destructans may improve bat survival. Volatile organic compounds (VOCs) are attractive candidates to target environmental P. destructans, as they can permeate through textured environments that may be difficult to thoroughly contact with other control mechanisms. We tested in hibernaculum sediment the performance of VOCs that were previously shown to inhibit P. destructans growth in agar cultures and examined the inhibition kinetics and specificity of these compounds. Three VOCs, 2-methyl-1-butanol, 2-methyl-1-propanol, and 1-pentanol, were fungicidal towards P. destructans in hibernaculum sediment, fast-acting, and had greater effects against P. destructans than other Pseudogymnoascus species. Our results suggest that use of these VOCs may be considered further as an effective management strategy to reduce the environmental exposure of bats to P. destructans in hibernacula.
Asunto(s)
Ascomicetos/efectos de los fármacos , Fungicidas Industriales/farmacología , Sedimentos Geológicos/microbiología , Compuestos Orgánicos Volátiles/farmacología , Animales , Ascomicetos/fisiología , Quirópteros/microbiología , Enfermedades Nasales/microbiología , Enfermedades Nasales/veterinariaRESUMEN
Nisin is a class I polycyclic bacteriocin produced by the bacterium Lactococcus lactis, which is used extensively as a food additive to inhibit the growth of foodborne Gram-positive bacteria. Nisin also inhibits growth of Gram-negative bacteria when combined with membrane-disrupting chelators such as citric acid. To gain insight into nisin's mode of action, we analyzed chemical-genetic interactions and identified nisin-sensitive Escherichia coli strains in the Keio library of knockout mutants. The most sensitive mutants fell into two main groups. The first group accords with the previously proposed mode of action based on studies with Gram-positive bacteria, whereby nisin interacts with factors involved in cell wall, membrane, envelope biogenesis. We identified an additional, novel mode of action for nisin based on the second group of sensitive mutants that involves cell cycle and DNA replication, recombination, and repair. Further analyses supported these two distinct modes of action.
Asunto(s)
Antibacterianos/farmacología , Conservantes de Alimentos/farmacología , Lactococcus lactis/química , Nisina/farmacología , Bacterias/metabolismo , Pared Celular/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Escherichia coli/efectos de los fármacos , Técnicas de Inactivación de Genes , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacosRESUMEN
For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5'-Untranslated region (5'-UTR) of PGM2 mRNA in yeast.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Antimaníacos/farmacología , Cloruro de Litio/farmacología , Biosíntesis de Proteínas/genética , ARN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Regiones no Traducidas 5' , Aminoacil-ARNt Sintetasas/genética , Antimaníacos/uso terapéutico , Trastorno Bipolar/tratamiento farmacológico , Trastorno Bipolar/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Cloruro de Litio/uso terapéutico , Organismos Modificados Genéticamente , Fosfoglucomutasa/antagonistas & inhibidores , Fosfoglucomutasa/metabolismo , ARN Helicasas/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genéticaRESUMEN
Individuals of the basidiomycete fungus Armillaria are well known for their ability to spread from woody substrate to substrate on the forest floor through the growth of rhizomorphs. Here, we made 248 collections of A. gallica in one locality in Michigan's Upper Peninsula. To identify individuals, we genotyped collections with molecular markers and somatic compatibility testing. We found several different individuals in proximity to one another, but one genetic individual stood out as exceptionally large, covering hundreds of tree root systems over approximately 75 hectares of the forest floor. Based on observed growth rates of the fungus, we estimate the minimum age of the large individual as 2500 years. With whole-genome sequencing and variant discovery, we also found that mutation had occurred within the somatic cells of the individual, reflecting its historical pattern of growth from a single point. The overall rate of mutation over the 90 mb genome, however, was extremely low. This same individual was first discovered in the late 1980s, but its full spatial extent and internal mutation dynamic was unknown at that time. The large individual of A. gallica has been remarkably resistant to genomic change as it has persisted in place.
Asunto(s)
Armillaria/genética , Evolución Clonal , Inestabilidad Genómica , Genotipo , Armillaria/crecimiento & desarrollo , ADN de Hongos/análisis , Michigan , MutaciónRESUMEN
Balancing selection has been inferred in diverse organisms for nonself recognition genes, including those involved in immunity, mating compatibility, and vegetative incompatibility. Although selective forces maintaining polymorphisms are known for genes involved in immunity and mating, mechanisms of balancing selection for vegetative incompatibility genes in fungi are being debated. We hypothesized that allorecognition and its consequent inhibition of virus transmission contribute to the maintenance of polymorphisms in vegetative incompatibility loci (vic) in the chestnut blight fungus, Cryphonectria parasitica. Balancing selection was demonstrated at two loci, vic2 and vic6, by trans-species polymorphisms in C. parasitica, C. radicalis, and C. japonica and signatures of positive selection in gene sequences. In addition, more than half (31 of 54) of allele frequency estimates at six vic loci in nine field populations of C. parasitica from Asia and the eastern US were not significantly different from 0.5, as expected at equilibrium for two alleles per locus under balancing selection. At three vic loci, deviations from 0.5 were predicted based on the effects of heteroallelism on virus transmission. Twenty-five of 27 allele frequency estimates were greater than or equal to 0.5 for the allele that confers significantly stronger inhibition of virus transmission at three loci with asymmetric transmission. These results are consistent with the allorecognition hypothesis that vegetative incompatibility genes are under selection because of their role in reducing infection by viruses.
Asunto(s)
Frecuencia de los Genes , Polimorfismo Genético , Saccharomycetales/genética , Selección Genética , Saccharomycetales/clasificación , Especificidad de la EspecieRESUMEN
CONTEXT: Plants of the genus Echinacea (Asteraceae) are among the most popular herbal supplements on the market today. Recent studies indicate there are potential new applications and emerging markets for this natural health product (NHP). OBJECTIVE: This review aims to synthesize recent developments in Echinacea biotechnology and to identify promising applications for these advances in the industry. METHODS: A comprehensive survey of peer-reviewed publications was carried out, focusing on Echinacea biotechnology and impacts on phytochemistry. This article primarily covers research findings since 2007 and builds on earlier reviews on the biotechnology of Echinacea. RESULTS: Bioreactors, genetic engineering and controlled biotic or abiotic elicitation have the potential to significantly improve the yield, consistency and overall quality of Echinacea products. Using these technologies, a variety of new applications for Echinacea can be realized, such as the use of seed oil and antimicrobial and immune boosting feed additives for livestock. CONCLUSIONS: New applications can take advantage of the well-established popularity of Echinacea as a NHP. Echinacea presents a myriad of potential health benefits, including anti-inflammatory, anxiolytic and antibiotic activities that have yet to be fully translated into new applications. The distinct chemistry and bioactivity of different Echinacea species and organs, moreover, can lead to interesting and diverse commercial opportunities.
Asunto(s)
Biotecnología/tendencias , Echinacea , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Transferencia de Tecnología , Animales , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Biotecnología/métodos , Predicción , Humanos , Inflamación/tratamiento farmacológico , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Fitoquímicos/uso terapéutico , Extractos Vegetales/farmacologíaRESUMEN
Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-α-L-rhamnopyranosyl-(1 â 2)-ß-D-galactopyranoside (1) and new saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-ß-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 µM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 µM).
Asunto(s)
Antifúngicos/farmacología , Cestrum/química , Hongos/efectos de los fármacos , Extractos Vegetales/farmacología , Saponinas/farmacología , Espirostanos/farmacología , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Candida albicans/efectos de los fármacos , Cryptococcus neoformans/efectos de los fármacos , Etnicidad , Fusarium/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Medicina Tradicional , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fitoterapia , Extractos Vegetales/química , Hojas de la Planta/química , Plantas Medicinales , Saccharomyces cerevisiae/efectos de los fármacos , Saponinas/química , Saponinas/aislamiento & purificación , Solanaceae , Espirostanos/química , Espirostanos/aislamiento & purificaciónRESUMEN
All organisms are faced with environmental uncertainty. Bet-hedging theory expects unpredictable selection to result in the evolution of traits that maximize the geometric-mean fitness even though such traits appear to be detrimental over the shorter term. Despite the centrality of fitness measures to evolutionary analysis, no direct test of the geometric-mean fitness principle exists. Here, we directly distinguish between predictions of competing fitness maximization principles by testing Cohen's 1966 classic bet-hedging model using the fungus Neurospora crassa. The simple prediction is that propagule dormancy will evolve in proportion to the frequency of 'bad' years, whereas the prediction of the alternative arithmetic-mean principle is the evolution of zero dormancy as long as the expectation of a bad year is less than 0.5. Ascospore dormancy fraction in N. crassa was allowed to evolve under five experimental selection regimes that differed in the frequency of unpredictable 'bad years'. Results were consistent with bet-hedging theory: final dormancy fraction in 12 genetic lineages across 88 independently evolving samples was proportional to the frequency of bad years, and evolved both upwards and downwards as predicted from a range of starting dormancy fractions. These findings suggest that selection results in adaptation to variable rather than to expected environments.
Asunto(s)
Evolución Biológica , Ambiente , Aptitud Genética , Neurospora crassa/fisiología , Adaptación Fisiológica , África , Haití , Neurospora crassa/genética , Estados UnidosRESUMEN
PURPOSE: To study the effect of functional foods on human cytochrome P450 (CYP) and the gut bacterial microflora that may potentially affect drug metabolism and ultimately affect human health and wellness. METHODS: This study examined a variety of food plants from the Apiaceae, Fabaceae, and Lamiaceae families for their inhibitory potential on cytochrome 2D6-, 3A4-, 3A5-, and 3A7-mediated metabolism. The antimicrobial effects of these samples were also investigated with 7 selected bacterial surrogate species to determine potential effects on the gut microflora. RESULTS: The highest CYP inhibitory activities, based upon visual examination, were observed from extracts of celery seed, cumin, fennel seed, basil, oregano, and rosemary belonging to the Apiaceae and Lamiaceae families, respectively. Likewise, the strongest antimicrobial activities were also observed in the Apiaceae and Lamiaceae. No significant antimicrobial and CYP inhibition was observed in the Fabaceae extracts. CONCLUSION: Results demonstrated the possible risk of food-drug interactions from spice and herb plants may affect drug disposition and safety.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Alimentos Funcionales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Apiaceae/química , Apium/química , Cuminum/química , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Foeniculum/química , Humanos , Lamiaceae/química , Pruebas de Sensibilidad Microbiana , Ocimum basilicum/química , Origanum/química , Semillas/química , Relación Estructura-ActividadRESUMEN
Little is known about the fungal metabolism of nC10 and nC11 fatty acids and their conversion into lipids. A mixed batch culture of soil fungi, T. koningii and P. janthinellum, was grown on undecanoic acid (UDA), a mixture of UDA and potato dextrose broth (UDA+PDB), and PDB alone to examine their metabolic conversion during growth. We quantified seven intracellular and extracellular lipid classes using Iatroscan thin-layer chromatography with flame ionization detection (TLC-FID). Gas chromatography with flame ionization detection (GC-FID) was used to quantify 42 individual fatty acids. Per 150 mL culture, the mixed fungal culture grown on UDA+PDB produced the highest amount of intracellular (531 mg) and extracellular (14.7 mg) lipids during the exponential phase. The content of total intracellular lipids represented 25% of the total biomass-carbon, or 10% of the total biomass dry weight produced. Fatty acids made up the largest class of intracellular lipids (457 mg/150 mL culture) and they were synthesized at a rate of 2.4 mg/h during the exponential phase, and decomposed at a rate of 1.8 mg/h during the stationary phase, when UDA+PDB was the carbon source. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and vaccenic acid (C18:1) accounted for >80% of the total intracellular fatty acids. During exponential growth on UDA+PDB, hydrocarbons were the largest pool of all extracellular lipids (6.5 mg), and intracellularly they were synthesized at a rate of 64 µg/h. The mixed fungal species culture of T. koningii and P. janthinellum produced many lipids for potential use as industrial feedstocks or bioproducts in biorefineries.
Asunto(s)
Ácidos Grasos/metabolismo , Lípidos/biosíntesis , Penicillium/metabolismo , Trichoderma/metabolismo , Carbono/metabolismo , Medios de Cultivo , Ácidos Decanoicos/metabolismo , Ácidos Grasos/análisis , Glucosa/metabolismo , Microbiología Industrial/métodos , Lípidos/químicaRESUMEN
The capacity of two soil fungi, Trichoderma koningii and Penicillium janthinellum, to oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and store intracellular lipids during growth is unknown. This article reports for the first time the metabolism of decanoic acid (DA, C10:0), undecanoic acid (UDA, n-C11:0), a mixture of the acids (UDA+DA) and a mixture of UDA+ potato dextrose broth (PDB) by T. koningii and P. janthinellum and their mixed culture. A control PDB complex substrate was used as a substrate control treatment. The fungal cultures were assayed for their capacity to: (1) oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and (2) store lipids intracellularly during growth. On all four fatty acid substrates, the mixed T. koningii and P. janthinellum culture produced more biomass and CO2 than the individual fungal cultures. Per 150 mL culture, the mixed species culture grown on UDA+PDB and on PDB alone produced the most biomass (7,567 mg and 11,425 mg, respectively). When grown in DA, the mixed species culture produced the least amount of biomass (6,400 mg), a quantity that was lower than those obtained in UDA (7,550 mg) or UDA+DA (7,270 mg). Amounts of CO2 produced ranged from 210 mg under DA to 618 mg under PDB, and these amounts were highly correlated with biomass (r(2) = 0.99). Fluorescence microscopy of stained lipids in the mixed fungal cell cultures growing during the exponential phase demonstrated larger fungal cells and higher accumulation of lipids in membranes and storage bodies than those observed during the lag and stationary phases. T. koningii and P. janthinellum grown on n-C10:0 and n-C11:0 fatty acids produced lower amounts of biomass and CO2, but stored higher amounts of intracellular lipids, than when grown on PDB alone.
Asunto(s)
Dióxido de Carbono/metabolismo , Ácidos Decanoicos/metabolismo , Ácidos Grasos/metabolismo , Penicillium/metabolismo , Trichoderma/metabolismo , Técnicas de Cultivo Celular por Lotes , Biomasa , Carbono/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Microscopía Fluorescente , Oxidación-Reducción , Penicillium/crecimiento & desarrollo , Microbiología del Suelo , Trichoderma/crecimiento & desarrolloRESUMEN
Maintaining cellular homeostasis in the face of stress conditions is vital for the overall well-being of an organism. Reactive oxygen species (ROS) are among the most potent cellular stressors and can disrupt the internal redox balance, giving rise to oxidative stress. Elevated levels of ROS can severely affect biomolecules and have been associated with a range of pathophysiological conditions. In response to oxidative stress, yeast activator protein-1 (Yap1p) undergoes post-translation modification that results in its nuclear accumulation. YAP1 has a key role in oxidative detoxification by promoting transcription of numerous antioxidant genes. In this study, we identified previously undescribed functions for NCE102, CDA2, and BCS1 in YAP1 expression in response to oxidative stress induced by hydrogen peroxide (H2O2). Deletion mutant strains for these candidates demonstrated increased sensitivity to H2O2. Our follow-up investigation linked the activity of these genes to YAP1 expression at the level of translation. Under oxidative stress, global cap-dependent translation is inhibited, prompting stress-responsive genes like YAP1 to employ alternative modes of translation. We provide evidence that NCE102, CDA2, and BCS1 contribute to cap-independent translation of YAP1 under oxidative stress.
RESUMEN
BACKGROUND: The ability to distinguish nonself from self is a fundamental characteristic of biological systems. In the filamentous fungus Neurospora crassa, multiple incompatibility genes mediate nonself recognition during vegetative growth. One of these genes, un-24, encodes both nonself recognition function and the large subunit of a type I ribonucleotide reductase, an evolutionarily conserved enzyme that is essential for the conversion of NDP precursors into dNDPs for use in DNA synthesis. Previous studies have shown that co-expression of the two allelic forms of un-24, Oakridge (OR) and Panama (PA), in the same cell results in cell death. RESULTS: We identify a 135 amino acid nonself recognition domain in the C-terminus region of UN-24 that confers an incompatibility-like phenotype when expressed in the yeast, Saccharomyces cerevisiae. Low-level expression of this domain results in several cytological and phenotypic characteristics consistent with an incompatibility reaction in filamentous fungi. These incompatibility phenotypes are correlated with the presence of a non-reducible complex consisting of the PA incompatibility domain and Rnr1p, a large subunit of ribonucleotide reductase in yeast. When the PA incompatibility domain is switched to high-level expression, the incompatibility phenotype transitions to wild-type concomitant with the appearance of a complex containing the PA incompatibility domain and Ssa1p, an Hsp70 homolog. CONCLUSIONS: Results from this study provide insights into the mechanism and control of vegetative nonself recognition mediated by ribonucleotide reductase in N. crassa, thus establishing the yeast system as a powerful tool to study fungal nonself recognition. Our work shows that heat shock proteins may function to deactivate vegetative incompatibility systems, as required for entry into the sexual cycle. Finally, our results suggest that variations on the PA incompatibility domain may serve as novel and specific antimicrobial peptides.
Asunto(s)
Neurospora crassa/fisiología , Ribonucleótido Reductasas/metabolismo , Neurospora crassa/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Ribonucleótido Reductasas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The antifungal mode of action of thymol was investigated by a chemical-genetic profile analysis. Growth of each of ~4700 haploid Saccharomyces cerevisiae gene deletion mutants was monitored on medium with a subinhibitory concentration (50 µg/ml) of thymol and compared to growth on non-thymol control medium. This analysis revealed that, of the 76 deletion mutants with the greatest degree of susceptibility to thymol, 29% had deletions in genes involved in telomere length maintenance. A telomere restriction fragment (TRF) length assay showed that yeast exposed to a subinhibitory concentration of thymol for 15 days had telomere size reductions of 13-20% compared to non-thymol controls. By accelerating telomere shortening, thymol may increase the rate of cell senescence and apoptosis. Furthermore, real-time RT-PCR analysis revealed approximately two-fold reductions in EST2 mRNA but no change in TLC1 RNA in thymol-treated S. cerevisiae relative to untreated cells. EST2 encodes the essential reverse transcriptase subunit of telomerase that uses TLC1 RNA as a template during addition of TG(1-3) repeats to maintain telomere ends. This study provides compelling evidence that a primary mode of thymol antifungal activity is through inhibition of transcription of EST2 and thus telomerase activity.