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The Difference in Calcium Levels in Aspergillus nidulans Grown on Glucose or Pectin  [PDF]
Janice Aparecida Rafael, Suraia Said
Advances in Microbiology (AiM) , 2012, DOI: 10.4236/aim.2012.22016
Abstract: Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca2+ levels maintenance and consequently by the nuclear division in A. nidulans.
Identification of Glucose Transporters in Aspergillus nidulans  [PDF]
Thaila Fernanda dos Reis, Jo?o Filipe Menino, Vinícius Leite Pedro Bom, Neil Andrew Brown, Ana Cristina Colabardini, Marcela Savoldi, Maria Helena S. Goldman, Fernando Rodrigues, Gustavo Henrique Goldman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081412
Abstract: To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.
A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover  [cached]
Saykhedkar Sayali,Ray Anamika,Ayoubi-Canaan Patricia,Hartson Steven D
Biotechnology for Biofuels , 2012, DOI: 10.1186/1754-6834-5-52
Abstract: Background Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study. Results A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance. Conclusions Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.
GmcA Is a Putative Glucose-Methanol-Choline Oxidoreductase Required for the Induction of Asexual Development in Aspergillus nidulans  [PDF]
Oier Etxebeste, Erika Herrero-García, Marc S. Cortese, Aitor Garzia, Elixabet Oiartzabal-Arano, Vivian de los Ríos, Unai Ugalde, Eduardo A. Espeso
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040292
Abstract: Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation.
High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes  [PDF]
Josep V. Forment, Michel Flipphi, Luisa Ventura, Ramón González, Daniel Ramón, Andrew P. MacCabe
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0094662
Abstract: Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation.
The nucleation of microtubules in Aspergillus nidulans germlings
Andrade-Monteiro, Cristina de;Martinez-Rossi, Nilce M.;
Genetics and Molecular Biology , 1999, DOI: 10.1590/S1415-47571999000300004
Abstract: microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. in fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. we used the antimicrotubule drug benomyl in block/release experiments to depolymerize and repolymerize microtubules in aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. twenty seconds after release from benomyl short microtubules were formed from several bright (immunofluorescent) dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in a. nidulans germlings. since nuclear movement is dependent on microtubules in a. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants) have some obvious microtubule defect. cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. however, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. this suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.
The nucleation of microtubules in Aspergillus nidulans germlings  [cached]
Andrade-Monteiro Cristina de,Martinez-Rossi Nilce M.
Genetics and Molecular Biology , 1999,
Abstract: Microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. In fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. We used the antimicrotubule drug Benomyl in block/release experiments to depolymerize and repolymerize microtubules in Aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. Twenty seconds after release from Benomyl short microtubules were formed from several bright (immunofluorescent) dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in A. nidulans germlings. Since nuclear movement is dependent on microtubules in A. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants) have some obvious microtubule defect. Cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. However, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. This suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.
L-Rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake
Juan A Tamayo-Ramos, Michel Flipphi, Ester Pardo, Paloma Manzanares, Margarita Orejas
Microbial Cell Factories , 2012, DOI: 10.1186/1475-2859-11-26
Abstract: Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (rhaE). The cDNA of rhaE was expressed in Saccharomyces cerevisiae and yielded pNP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse A. nidulans strains cultivated under different growth conditions indicate that rhaA and rhaE are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these rha genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression.The A. nidulans rhaE gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of rhaE and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, A. nidulans α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR.The degradation of plant cell wall polysaccharides (i.e. cellulose, hemicellulose and pectins), and the subsequent utilization of their components as carbon sources is a key and highly regulated event when filamentous fungi grow on these substrates or infect plants. A number of those plant-derived substrates contain the neutral sugar L-rh
Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans
Ferreira, Adlane V. B.;
Revista de Microbiologia , 1998, DOI: 10.1590/S0001-37141998000400009
Abstract: intra and extracellular nuclease production by strains of aspergillus niger and aspergillus nidulans was estimated using a modified dnase test agar and cell-free extract assays. differences in the production of nucleases by a. niger and a. nidulans were observed. these observations suggest that the dnase test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. the assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.
Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans  [cached]
Ferreira Adlane V. B.
Revista de Microbiologia , 1998,
Abstract: Intra and extracellular nuclease production by strains of Aspergillus niger and Aspergillus nidulans was estimated using a modified DNAse test agar and cell-free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.
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