RESUMEN
Phosphorus in the form of phosphate (Pi) is an essential element for metabolic processes, including lipid metabolism. In yeast, the inositol polyphosphate kinase vip1 mediated synthesis of inositol heptakisphosphate (IP7) regulates the phosphate-responsive (PHO) signaling pathway, which plays an important role in response to Pi stress. The role of vip1 in Pi stress and lipid metabolism of Candida albicans has not yet been studied. We found that when vip1Δ/Δ was grown in glucose medium, if Pi was supplemented in the medium or mitochondrial Pi transporter was overexpressed in the strain, the lipid droplet (LD) content was reduced and membrane damage was alleviated. However, further studies showed that neither the addition of Pi nor the overexpression of the Pi transporter affected the energy balance of vip1Δ/Δ. In addition, the LD content of vip1Δ/Δ grown in Pi limitation medium PNMC was lower than that grown in SC, and the metabolic activity of vip1Δ/Δ grown in PNMC was also lower than that grown in SC medium. This suggests that the increase in Pi demand by a high energy metabolic rate is the cause of LD accumulation in vip1Δ/Δ. In addition, in the vip1Δ/Δ strains, the core transcription factor PHO4 in the PHO pathway was transported to the vacuole and degraded, which reduced the pathway activity. However, this does not mean that knocking out vip1 completely blocks the activation of the PHO pathway, because the LD content of vip1Δ/Δ grown in the medium with ß-glycerol phosphate as the Pi source was significantly reduced. In summary, the increased Pi demand and the decreased PHO pathway activity in vip1Δ/Δ ultimately lead to LD accumulation and cell membrane damage.
Asunto(s)
Metabolismo Energético/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Candida albicans/metabolismo , Membrana Celular/metabolismo , Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/genética , Fosfatos de Inositol , Gotas Lipídicas/metabolismo , Fosfatos/metabolismo , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/fisiología , Transducción de Señal , Factores de Transcripción/metabolismo , Vacuolas/metabolismoRESUMEN
Several recent studies have shown that the concept of proteome constraint, i.e., the need for the cell to balance allocation of its proteome between different cellular processes, is essential for ensuring proper cell function. However, there have been no attempts to elucidate how cells' maximum capacity to grow depends on protein availability for different cellular processes. To experimentally address this, we cultivated Saccharomyces cerevisiae in bioreactors with or without amino acid supplementation and performed quantitative proteomics to analyze global changes in proteome allocation, during both anaerobic and aerobic growth on glucose. Analysis of the proteomic data implies that proteome mass is mainly reallocated from amino acid biosynthetic processes into translation, which enables an increased growth rate during supplementation. Similar findings were obtained from both aerobic and anaerobic cultivations. Our findings show that cells can increase their growth rate through increasing its proteome allocation toward the protein translational machinery.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Aminoácidos/biosíntesis , Aminoácidos/metabolismo , Fenómenos Bioquímicos , Fenómenos Biológicos , Perfilación de la Expresión Génica/métodos , Regulación Fúngica de la Expresión Génica/fisiología , Glucosa/metabolismo , Proteoma/metabolismo , Proteómica , Ribosomas/metabolismo , Ribosomas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Auricularia cornea is a widely cultivated edible fungus with substantial nutritive value. This study aimed to enrich the multifunctional bionutrient element selenium in A. cornea to improve its quality and explore the accumulation of selenium in the fungus using high-throughput RNA-Seq technology. In general, the treatment group with a 100 µg/g supply of selenium outperformed the other treatment groups in terms of high yield, rich crude polysaccharides and a high total selenium concentration. Additional evidences demonstrated the budding and mature phases were two typical growth stages of A. cornea and were important for the accumulation of selenium. Therefore, the budding and mature phase tissues of A. cornea in the treatment group with a 100 µg/g supply of selenium were used for transcriptome analysis and compared to those of a control group that lacked additional selenium. A total of 2.56 × 105 unigenes from A. cornea transcriptome were assembled and annotated to five frequently used databases including NR, GO, KEGG, eggNOG and SwissProt. GO and KEGG pathway analysis revealed that genes involved in metabolic process and translation were up-expressed at the budding stage in response to selenium supplementation, including amino acid metabolism, lipid metabolism, ribosome. In addition, the differential gene expression patterns of A. cornea suggested that the up-expressed genes were more likely to be detected at the budding stage than at the mature stage. These results provide insights into the transcriptional response of A. cornea to selenium accumulation.
Asunto(s)
Agaricales/genética , Selenio/metabolismo , Agaricales/metabolismo , Basidiomycota/genética , Perfilación de la Expresión Génica/métodos , Regulación Fúngica de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Transcriptoma/genéticaRESUMEN
Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.
Asunto(s)
Aspergillus fumigatus/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Aspergillus nidulans/genética , Proteínas Bacterianas/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Hierro/metabolismo , Leucina/biosíntesis , Leucina/genética , Nitrógeno/metabolismo , Proteostasis , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , VirulenciaRESUMEN
All eukaryotic organisms rely on iron as an essential micronutrient for life because it participates as a redox-active cofactor in multiple biological processes. However, excess iron can generate reactive oxygen species that damage cellular macromolecules. The low solubility of ferric iron under physiological conditions increases the prevalence of iron deficiency anemia. A common strategy to treat iron deficiency consists of dietary iron supplementation. The baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, but also as a feed supplement. In response to iron deficiency, the yeast Aft1 transcription factor activates cellular iron acquisition. However, when constitutively active, Aft1 inhibits growth probably due to iron toxicity. In this report, we have studied the consequences of using hyperactive AFT1 alleles, including AFT1-1UP, to increase yeast iron accumulation. We first characterized the iron sensitivity of cells expressing different constitutively active AFT1 alleles. We rescued the high iron sensitivity conferred by the AFT1 alleles by deleting the sphingolipid signaling kinase YPK1. We observed that the deletion of YPK1 exerts different effects on iron accumulation depending on the AFT1 allele and the environmental iron. Moreover, we determined that the impairment of the high-affinity iron transport system partially rescues the high iron toxicity of AFT1-1UP-expressing cells. Finally, we observed that AFT1-1UP inhibits oxygen consumption through activation of the RNA-binding protein Cth2. Deletion of CTH2 partially rescues the AFT1-1UP negative respiratory effect. Collectively, these results contribute to understand how the Aft1 transcription factor functions and the multiple consequences derived from its constitutive activation.
Asunto(s)
Hierro/metabolismo , Saccharomyces cerevisiae/metabolismo , Alelos , Regulación Fúngica de la Expresión Génica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética/genéticaRESUMEN
In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.
Asunto(s)
Proteínas Bacterianas/genética , Ácidos Grasos Insaturados/metabolismo , Fermentación/genética , Glucosa/metabolismo , Kluyveromyces/metabolismo , Consumo de Oxígeno/genética , Factores de Transcripción/genética , Antifúngicos/farmacología , Antimicina A/farmacología , Cardiolipinas/metabolismo , Hipoxia de la Célula/fisiología , Respuesta al Choque por Frío/fisiología , Ergosterol/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Glucólisis/genética , Kluyveromyces/efectos de los fármacos , Kluyveromyces/genética , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Transcripción Genética/genéticaRESUMEN
Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Glicósido Hidrolasas/genética , Micotoxinas/genética , Rhizoctonia/genética , Solanum tuberosum/microbiología , Técnicas de Hibridación Sustractiva/métodos , Secuencia de Aminoácidos , Bacillus subtilis/fisiología , Secuencia de Bases , ADN Complementario/genética , Regulación hacia Abajo , Proteínas Fúngicas/genética , Biblioteca de Genes , Genoma Fúngico/genética , Glicósido Hidrolasas/metabolismo , Interacciones Huésped-Patógeno , Datos de Secuencia Molecular , Micotoxinas/metabolismo , Filogenia , Enfermedades de las Plantas/microbiología , Rhizoctonia/citología , Rhizoctonia/enzimología , Análisis de Secuencia de ADN , Stachybotrys/fisiología , Regulación hacia ArribaRESUMEN
We have previously shown that copper supplementation extends the replicative life span of Saccharomyces cerevisiae when grown under conditions forcing cells to respire. We now show that copper's effect on life span is through Fet3p, a copper containing enzyme responsible for high affinity transport of iron into yeast cells. Life span extensions can also be obtained by supplementing the growth medium with 1mM ferric chloride. Extension by high iron levels is still dependent on the presence of Fet3p. Life span extension by iron or copper requires growth on media containing glycerol as the sole carbon source, which forces yeast to respire. Yeast grown on glucose containing media supplemented with iron show no extension of life span. The iron associated with cells grown in media supplemented with copper or iron is 1.4-1.8 times that of cells grown without copper or iron supplementation. As with copper supplementation, iron supplementation partially rescues the life span of superoxide dismutase mutants. Cells grown with copper supplementation display decreased production of superoxide as measured by dihydroethidium staining.
Asunto(s)
Proteínas de Transporte de Catión/genética , Respiración de la Célula/genética , Cobre/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Hierro/farmacología , Mitocondrias/metabolismo , Animales , Transporte Biológico/genética , Proteínas de Transporte de Catión/metabolismo , Respiración de la Célula/fisiología , Cobre/farmacología , Medios de Cultivo , Glicerol/farmacología , Hierro/metabolismo , Esperanza de Vida , Mitocondrias/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+/idi-::hph), crtE (crtE+/crtE-::hph), crtYB (crtYB+/crtYB-::hph), crtI (crtI+/crtI-::hph) and crtS (crtS+/crtS-::hph) and homozygote mutants crtYB (crtYB-::hph/crtYB-::hph), crtI (crtI-::hph/crtI-::hph) and crtS (crtS-::hph/crtS-::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.
Asunto(s)
Basidiomycota/genética , Regulación Fúngica de la Expresión Génica/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Genes Fúngicos/genética , Reacción en Cadena de la Polimerasa , ARN de Hongos/genética , Xantófilas/biosíntesis , Xantófilas/genéticaRESUMEN
The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.
Asunto(s)
Basidiomycota/genética , Regulación Fúngica de la Expresión Génica/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Genes Fúngicos/genética , Reacción en Cadena de la Polimerasa , ARN de Hongos/genética , Xantófilas/biosíntesis , Xantófilas/genéticaRESUMEN
AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. CONCLUSIONS: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.
Asunto(s)
Microbiología Industrial/métodos , Riboflavina/biosíntesis , Saccharomycetales/aislamiento & purificación , Medios de Cultivo , Fermentación , Proteínas Fúngicas/análisis , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Ingeniería Genética/métodos , Mutación , Oxidorreductasas/metabolismo , Aceites de Plantas/metabolismo , Proteoma/análisis , Saccharomycetales/genética , Saccharomycetales/metabolismo , Esporas Fúngicas/aislamiento & purificación , Succinatos/metabolismoRESUMEN
The biological role of ExgA (Exg1), a secretory beta-1,3-exoglucanase of Aspergillus oryzae, and the expression pattern of the exgA (exg1) gene were analyzed. The exgA disruptant and the exgA-overexpressing mutant were constructed, and phenotypes of both mutants were compared. Higher mycelial growth rate and conidiation efficiency were observed for the exgA-overexpressing mutant than for the exgA disruptant when beta-1,3-glucan was supplied as sole carbon source. On the other hand, no difference in phenotype was observed between them in the presence or absence of the inhibitors of cell wall beta-glucan remodeling when grown with glucose. exgA Expression was induced in growth on solid surfaces such as filter membrane and onion inner skin. A combination of poor nutrition and mycelial attachment to a hydrophobic solid surface appears to be an inducing factor for exgA expression. These data suggest that ExgA plays a role in beta-glucan utilization, but is not much involved in cell wall beta-glucan remodeling.
Asunto(s)
Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Glucano 1,3-beta-Glucosidasa/genética , Glucanos/metabolismo , Aspergillus oryzae/crecimiento & desarrollo , Pared Celular/química , Pared Celular/ultraestructura , Medios de Cultivo , ADN Complementario/biosíntesis , ADN Complementario/genética , ADN de Hongos/genética , Filtración , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/fisiología , Micelio/enzimología , Micelio/crecimiento & desarrollo , Fenómenos Fisiológicos de la Nutrición , Cebollas/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Propiedades de Superficie , Transformación GenéticaRESUMEN
Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.
Asunto(s)
Botrytis/patogenicidad , Proteínas Fúngicas/análisis , Genes Fúngicos/fisiología , Proteoma/análisis , Factores de Virulencia/aislamiento & purificación , Botrytis/química , Botrytis/genética , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica/genética , Proteoma/química , InvestigaciónRESUMEN
Candida albicans biofilms are structured microbial communities with high levels of drug resistance. Farnesol, a quorum-sensing molecule that inhibits hyphal formation in C. albicans, has been found to prevent biofilm formation by C. albicans. There is limited information, however, about the molecular mechanism of farnesol against biofilm formation. We used cDNA microarray analysis to identify the changes in the gene expression profile of a C. albicans biofilm inhibited by farnesol. Confocal scanning laser microscopy was used to visualize and confirm normal and farnesol-inhibited biofilms. A total of 274 genes were identified as responsive, with 104 genes up-regulated and 170 genes down-regulated. Independent reverse transcription-PCR analysis was used to confirm the important changes detected by microarray analysis. In addition to hyphal formation-associated genes (e.g., TUP1, CRK1, and PDE2), a number of other genes with roles related to drug resistance (e.g., FCR1 and PDR16), cell wall maintenance (e.g., CHT2 and CHT3), and iron transport (e.g., FTR2) were responsive, as were several genes encoding heat shock proteins (e.g., HSP70, HSP90, HSP104, CaMSI3, and SSA2). Further study of these differentially regulated genes is warranted to evaluate how they may be involved in C. albicans biofilm formation. Consistent with the down-regulation of the cell surface hydrophobicity-associated gene (CSH1), the water-hydrocarbon two-phase assay showed a decrease in cell surface hydrophobicity in the farnesol-treated group compared to that in the control group. Our data provide new insight into the molecular mechanism of farnesol against C. albicans biofilm formation.
Asunto(s)
Biopelículas , Candida albicans/metabolismo , ADN Complementario/genética , ADN de Hongos/genética , Farnesol/farmacología , Regulación Fúngica de la Expresión Génica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Candida albicans/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Medios de Cultivo , Sondas de ADN , ADN Complementario/biosíntesis , ADN de Hongos/biosíntesis , Farmacorresistencia Fúngica , Proteínas de Choque Térmico/metabolismo , Hibridación in Situ , Microscopía Confocal , Proteínas de Transferencia de Fosfolípidos/genética , ARN de Hongos/biosíntesis , ARN de Hongos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Manganese peroxidase (MnP) is a major, extracellular component of the lignin-degrading system produced by the wood-rotting basidiomycetous fungus Phanerochaete chrysosporium. The transcription of MnP-encoding genes (mnps) in P. chrysosporium occurs as a secondary metabolic event, triggered by nutrient-nitrogen limitation. In addition, mnp expression occurs only under Mn2+ supplementation. Using a reporter system based on the enhanced green fluorescent protein gene (egfp), we have characterized the P. chrysosporium mnp1 promoter by examining the effects of deletion, replacement, and translocation mutations on mnp1 promoter-directed egfp expression. The 1,528-bp mnp1 promoter fragment drives egfp expression only under Mn2+-sufficient, nitrogen-limiting conditions, as required for endogenous MnP production. However, deletion of a 48-bp fragment, residing 521 bp upstream of the translation start codon in the mnp1 promoter, or replacement of this fragment with an unrelated sequence resulted in egfp expression under nitrogen limitation, both in the absence and presence of exogenous Mn2+. Translocation of the 48-bp fragment to a site 120 bp downstream of its original location resulted in Mn2+-dependent egfp expression under conditions similar to those observed with the wild-type mnp1 promoter. These results suggest that the 48-bp fragment contains at least one Mn2+-responsive cis element. Additional promoter-deletion experiments suggested that the Mn2+ element(s) is located within the 33-bp sequence at the 3' end of the 48-bp fragment. This is the first promoter sequence containing a Mn2+-responsive element(s) to be characterized in any eukaryotic organism.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Manganeso/metabolismo , Peroxidasas/genética , Phanerochaete/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Clonación Molecular , Inducción Enzimática , Genes Fúngicos/genética , Proteínas Fluorescentes Verdes , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Peroxidasas/metabolismo , Phanerochaete/metabolismoRESUMEN
The zinc-responsive transcriptional activator Zap1 regulates the expression of both high- and low-affinity zinc uptake permeases encoded by the ZRT1 and ZRT2 genes. Zap1 mediates this response by binding to zinc-responsive elements (ZREs) located within the promoter regions of each gene. ZRT2 has a remarkably different expression profile in response to zinc compared to ZRT1. While ZRT1 is maximally induced during zinc limitation, ZRT2 is repressed in low zinc but remains induced upon zinc supplementation. In this study, we determined the mechanism underlying this paradoxical Zap1-dependent regulation of ZRT2. We demonstrate that a nonconsensus ZRE (ZRT2 ZRE3), which overlaps with one of the ZRT2 transcriptional start sites, is essential for repression of ZRT2 in low zinc and that Zap1 binds to ZRT2 ZRE3 with a low affinity. The low-affinity ZRE is also essential for the ZRT2 expression profile. These results indicate that the unusual pattern of ZRT2 regulation among Zap1 target genes involves the antagonistic effect of Zap1 binding to a low-affinity ZRE repressor site and high-affinity ZREs required for activation.
Asunto(s)
Proteínas de Transporte de Catión/genética , Regulación Fúngica de la Expresión Génica/genética , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TATA Box/genética , Transactivadores/metabolismo , Sitios de Unión , Regiones Promotoras Genéticas/genética , Unión Proteica , Elementos de Respuesta/genética , Factores de Transcripción , Sitio de Iniciación de la Transcripción , Transcripción Genética/genética , Zinc/deficiencia , Zinc/farmacologíaRESUMEN
In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the "filamentous" polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K(+) channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K(+) channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K(+) efflux. NcTOKA channel gating was sensitive to extracellular K(+) such that channel activation was dependent on the reversal potential for K(+). However, expression of NcTOKA was able to overcome the K(+) auxotrophy of a yeast mutant missing the K(+) uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K(+) influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K(+) currents at potentials negative of E(K). NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca(2+), verapamil, quinine, and TEA(+) but was insensitive to Cs(+), 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.
Asunto(s)
Membrana Celular/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Neurospora crassa/metabolismo , Canales de Potasio/aislamiento & purificación , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Membrana Celular/genética , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Potenciales de la Membrana/genética , Datos de Secuencia Molecular , Mutación/genética , Neurospora crassa/genética , Potasio/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal candidiasis (OPC) in AIDS. The development of azoles, such as fluconazole, for the treatment of OPC has proven effective except in cases where C. albicans develops resistance to fluconazole during the course of treatment. In the present study, we used microarray technology to examine differences in gene expression from a fluconazole-susceptible and a fluconazole-resistant well-characterized, clinically obtained matched set of C. albicans isolates to identify genes which are differentially expressed in association with azole resistance. Among genes found to be differentially expressed were those involved in amino acid and carbohydrate metabolism; cell stress, cell wall maintenance; lipid, fatty acid, and sterol metabolism; and small molecule transport. In addition to CDR1, which has previously been demonstrated to be associated with azole resistance, the drug resistance gene RTA3, the ergosterol biosynthesis gene ERG2, and the cell stress genes CRD2, GPX1, and IFD5 were found to be upregulated. Several genes, such as the mitochondrial aldehyde dehydrogenase gene ALD5, the glycosylphosphatidylinositol synthesis gene GPI1, and the iron transport genes FET34 and FTR2 were found to be downregulated. Further study of these differentially regulated genes is warranted to evaluate how they may be involved in azole resistance. In addition to these novel findings, we demonstrate the utility of microarray analysis for studying the molecular mechanisms of drug resistance in pathogenic organisms.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , ADN Complementario/metabolismo , ADN de Hongos/metabolismo , Fluconazol/farmacología , Regulación Fúngica de la Expresión Génica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Metabolismo de los Hidratos de Carbono , Pared Celular/metabolismo , Sondas de ADN , ADN Complementario/genética , ADN de Hongos/genética , Farmacorresistencia Fúngica , Ergosterol/biosíntesis , Hierro/metabolismo , Pruebas de Sensibilidad Microbiana , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4-fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two-fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4-fold decrease in transcript level and only one of these, PCK1, was decreased greater than two-fold Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound.
Asunto(s)
Conservantes de Alimentos/farmacología , Proteoma/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/biosíntesis , Saccharomyces cerevisiae/genética , Ácido Sórbico/farmacología , ADN Complementario/química , ADN de Hongos/química , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/fisiología , Procesamiento de Imagen Asistido por Computador , Focalización Isoeléctrica , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta/genética , Sistemas de Lectura Abierta/fisiología , Proteoma/fisiología , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Regulación hacia ArribaRESUMEN
The ino2Delta, ino4Delta, opi1Delta, and sin3Delta mutations all affect expression of INO1, a structural gene for inositol-1-phosphate synthase. These same mutations affect other genes of phospholipid biosynthesis that, like INO1, contain the repeated element UAS(INO) (consensus 5' CATGTGAAAT 3'). In this study, we evaluated the effects of these four mutations, singly and in all possible combinations, on growth and expression of INO1. All strains carrying an ino2Delta or ino4Delta mutation, or both, failed to grow in medium lacking inositol. However, when grown in liquid culture in medium containing limiting amounts of inositol, the opi1Delta ino4Delta strain exhibited a level of INO1 expression comparable to, or higher than, the wild-type strain growing under the same conditions. Furthermore, INO1 expression in the opi1Delta ino4Delta strain was repressed in cells grown in medium fully supplemented with both inositol and choline. Similar results were obtained using the opi1Delta ino2Delta ino4Delta strain. Regulation of INO1 was also observed in the absence of the SIN3 gene product. Therefore, while Opi1p, Sin3p, and the Ino2p/Ino4p complex all affect the overall level of INO1 expression in an antagonistic manner, they do not appear to be responsible for transmitting the signal that leads to repression of INO1 in response to inositol. Various models for Opi1p function were tested and no evidence for binding of Opi1p to UAS(INO), or to Ino2p or Ino4p, was obtained.