Poster Category 4:


Fungal Physiology and Biochemistry



Components of the saga complex are involved in acetate repression in Aspergillus nidulans

Paraskevi (Vivian) Georgakopoulos, Robin A. Lockington, Joan M. Kelly

School of Molecular and Biomedical Sciences, University of Adelaide

Acetate, in A. nidulans, is a repressing carbon source that leads to similar levels of CreA mediated repression as glucose.  acdX was identified in a mutation screen in Aspergillus nidulans to identify genes involved in acetate repression but not in glucose repression. The conservation of the amino acid sequence of AcdX of A. nidulans and Spt8 of Saccharomyces cerevisiae suggests that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex may have a role in acetate repression in A. nidulans, since Spt8 is a component of the SAGA complex.

The SAGA complex is highly conserved from yeast to humans. In yeast it is involved mostly in the regulation of highly regulated genes that respond to environmental stresses, such as metabolic starvation, DNA damage and heat. SAGA in yeast has been shown to have positive and negative functions on transcription.  Bioinformatic analysis indicates that the components of the SAGA complex are also present in
A. nidulans.

We report results of experiments undertaken to confirm the existence of the SAGA complex in
A. nidulans and to determine whether AcdX is a component of the complex.  The A. nidulans homologue of the S. cerevisiae SAGA complex Spt3, designated SptC, was N terminally tagged with the TAP tag to allow the purification of the SAGA complex.  From these results it is evident that AcdX is a component of a multiprotein complex and that it co-immunoprecipitates with SptC, providing further evidence that the SAGA complex exists in A. nidulans, and includes AcdX.




Self-protection against a potent weapon: the dothistromin toxin story

Rosie Bradshaw[1] Zhilun Feng[1] Arne Schwelm[1] Yongzhi Yang[2] Shuguang Zhang[1]

1Massey University, 2Qinghai Academy, Xining, China


Dothistromin is a non-host selective toxin that is toxic to most types of cells. It is a close chemical relative of the aflatoxins and has similarities in both genetics and biochemistry to these compounds.  Dothistroma septosporum, a serious pine needle pathogen, is a prolific producer of dothistromin. In planta, dothistromin that accumulates in necrotic disease lesions is visible due to its red colouration and gives rise to the common name of ‘red-band needle blight’.  Studies with dothistromin-deficient mutants of D. septosporum revealed that dothistromin is not required for pathogenicity to the susceptible host Pinus radiata.  The current hypothesis is that dothistromin has a role in competition against other microorganisms that are known to reside in pine needles, and in vitro studies with pine needle endophytes support this hypothesis.  The purpose of the current work was to determine how D. septosporum is able to protect itself against its own potent toxin.  A gene (dotC) adjacent to known dothistromin biosynthetic genes is predicted to encode a Major Facilitator Superfamily (MFS) transporter. To determine whether the DotC protein is a membrane-bound transporter that can pump dothistromin out of the cell, dotC-deficient mutants, complemented mutants and strains containing DotC-GFP fusions were studied.  As predicted, dotC mutants secreted less dothistromin than wild type cells and the DotC-GFP fusions indicated a membrane location.  However the results also suggested that DotC has an important role in regulating dothistromin biosynthesis and that compartmentalization within the cell may be an important mechanism for self-protection against dothistromin.


SidL, an acetyltransferase involved in biosynthesis of the intracellular siderophore ferricrocin in Aspergillus fumigatus

Michael Blatzer, Schrettl Markus, Hubertus Haas

Medical University Innsbruck - Biozentrum


Virtually all organisms require iron as indispensable cofactor for various metabolic processes. The opportunistic fungal pathogen Aspergillus fumigatus produces two major siderophores (low molecular-mass ferric iron chelators): it excretes triacetylfusarinine C for iron uptake and accumulates ferricrocin for intracellular iron storage. Biosynthesis of both triacetylfusarinine C and ferricrocin has previously been shown to be crucial for virulence of A. fumigatus.

Here, we report the functional characterization of a new component of the fungal siderophore biosynthetic machinery Afu1g04450, termed SidL. SidL is conserved in siderophore-producing but not non-siderophore producing ascomycetes. The C-terminal half of SidL shows similarity to acetylases involved in bacterial siderophore biosynthesis, e.g.
Escherichia coli IucB (a hydroxylysine acetylase required for aerobactin biosynthesis) and PvdY (a hydroxyornithine acetylase required for pyoverdin biosynthesis), and the hydroxyornithine:anhydromevalonyl coenzyme A-transacylase SidF that is essential for triacetylfusarine C biosynthesis. Deletion of sidL in A. fumigatus reduced ferricrocin biosynthesis during iron starvation and blocked ferricirocin biosynthesis during iron-replete growth. Furthermore, sidL-deficiency blocked conidial ferricrocin accumulation under strict iron-replete conditions but not when mycelia were transferred from iron-depleted to iron-replete conditions before sporulation. In contrast, SidL-deficiency had no effect on triacetylfusarinine C production. The expression of sidL was affected neither by iron availability nor the iron regulator SreA.

Taken together, these data show that SidL is a constitutively expressed hydroxyornithine acetylase involved in ferricrocin biosynthesis. Moreover, the data indicate the existence of a second hydroxyornithine acetylase, the activity of which is induced by iron starvation. This study identified a novel component of the fungal siderophore biosynthetic machinery and revealed unexpected complexity. 




GliT protects Aspergillus fumigatus against the harmful effects of gliotoxin

Markus Schrettl[2] Stephen Carberry[1] Kevin Kavanagh[1] Hubertus Haas[2] Aine Nolan[1] Sean Doyle[1]

1Department of Biology and National Institute for Cellular Biotechnology, National University of Ireland Maynooth, Co. Kildare, Ireland, 2Biocenter, Division of Molecular Biology, Innsbruck Medical University, Austria

Gliotoxin, an epipolythiodioxopiperazine (ETP)-type toxin (326 Da) containing an essential disulphide bridge, plays a major role in mediating the virulence of the human pathogen, Aspergillus fumigatus. Gliotoxin toxicity in mammalian cells is generally enabled by direct inactivation of essential protein thiols as well as redox cycling, leading to hydrogen peroxide formation. In A. fumigatus, enzymes involved in gliotoxin biosynthesis are located within a coordinately expressed, multi-gene cluster. Here we report the functional characterisation of a putative thioredoxin reductase encoded by gliT within this gene cluster. Expression of gliT is subject to regulation by the transcriptional activator GliZ and gliotoxin. Deletion of gliT is detrimental for growth only in the presence of exogenously added gliotoxin, which can be cured by supplementation with reduced glutathione. GliT is localised in the cytoplasm and in the nucleus. GliT is not essential for virulence of A. fumigatus in larvae of the greater wax-moth Galleria mellonella. The potential autoprotective role of GliT was investigated further by heterologous expression of gliT in Aspergillus nidulans. And indeed, GliT conferred resistance to gliotoxin, making it a valuable tool for transformation of fungi lacking an ortholog of gliT.


Involvement of Trichoderma reesei (Hypocrea jecorina) G-alpha protein GNA1 during mycoparasitsm against Pythium ultimum

Roberto do Nascimento Silva[1] Andrei Stecca Steindorff[1] Cirano José Ulhoa[1] Carlos Roberto Félix[2]

1Federal University of Goias, 2University of Brasilia


Trichoderma reesei (Hypocrea jecorina) is widely used in industry and its potential for use in agriculture as a biocontrol agent against phytophatogenic fungi has just started. We have investigated the involvement of G proteins during mycoparasitism against plant pathogens. Here we described the role of GNA1, a G-alpha protein which belongs to alfai group in Cell Wall Degrading Enzymes (CWDEs) production by T. reesei during antagonism against Pythium ultimum. For that, two mutants were used: Δgna1 and gna1QL (constitutively activated version of GNA1). The gna1QL mutant, like the parental TU-6, inhibited the growth of   P. ultimum in plate confrontation assay and grew faster than the parental TU-6 while the Δgna1 did not grow over P. ultimum. Scanning electron microscopy showed that the gna1QL mutant promoted more morphological alterations of P. ultimum cell wall than the parental TU-6 while the Δgna1 caused no effects. The mutant Δgna1 produced less CWDEs than gna1QL and TU-6. The gna1QL mutant showed a better performance in production of CWDEs such as endochitinase, N-Acetyl-β-D-glucosaminidase (NAGase), β-1,3-glucanase, protease, lipase and acid phosphatase, after 72 hours of incubation. However, the parental TU-6 showed higher cellulase activity than gna1QL and Δgna1. The intracellular content of cAMP in the strains after 72 hours of incubation was: gna1QL (79.85 ± 12), Δgna1 (268.65 ± 8.5) and TU-6 (109.70 ± 9.2) pmol/mg protein. We therefore suggest that the production of some CWDEs during mycoparasitism by T. reesei against P. ultimum can be mediated by GNA1 activity or cAMP levels.





Galacturonic acid catabolism in Botrytis cinerea

Lisha Zhang,  Jan van Kan

Wageningen University, Laboratory of Phytopathology


D-galacturonic acid (GalA) is the major component of pectin, which can be degraded by plant pathogens; GalA potentially is an important carbon source for microorganisms living on decaying plant material. For bacteria, a catabolic pathway of GalA has been described, which consists of five enzymes converting GalA to pyruvate and glyceraldehyde-3-phosphate. A different catabolic pathway is proposed in filamentous fungi.  In Hypocrea jecorina, GalA is converted to pyruvate and glycerol via D-galacturonate reductase, L-galactonate dehydratase, 2-keto-3-deoxy-L-galactonate aldolase, and glycerol dehydrogenase.

Botrytis cinerea genome contains a D-galacturonate reductase gene (BcgaaA), a  L-galactonate dehydratase gene (BcgaaB), and a 2-keto-3-deoxy-L-galactonate aldolase gene (BcgaaC). The three genes were cloned into a protein expression vector and the enzymatic activity determined for each gene separately. The heterologous simultaneous expression of BcgaaA, BcgaaB, and BcgaaC in an E. coli ΔuxaC mutant which cannot grow on GalA is performed to determine whether the catabolic pathway from B. cinerea can restore the growth deficiency in E.coli. Targeted gene replacement of BcgaaC or both BcgaaA and BcgaaC resulted in ΔgaaC mutants and ΔgaaAC double knock-out mutants that displayed significantly reduced growth when D-galacturonic acid was used as the sole carbon source. The mutants showed similar virulence as the wild-type stain B05.10 on tomato leaves, indicating that GalA is not the main carbon source for B. cinerea growth during infection on tomato leaves. The virulence will be tested on other pectin-rich plants and tissues.




Regulation of D-galactose metabolism in Aspergillus nidulans

Birgit S. Gruben1, Ulla Christensen2, Igor Nikolaev2, Ronald P. de Vries1,3

1Microbiology, Utrecht University, Utrecht, The Netherlands; 2 Danisco-Genencor, Leiden, The Netherlands; 3CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands

D-Galactose is present in hemicelluloses and pectin which are constituents of the plant cell wall. In pectin, β-1,4-linked D-galactose residues are present in galactan or in arabinogalactan side chains. In hemicelluloses, D-galactose residues are present in the form of side residues which are β-linked in to xyloglucan and xylan, but α-linked in to galactoglucomannan.

For the degradation of these structures by filamentous fungi, several enzyme classes are required depending on the linkage. These classes are α- and β-galactosidases and endo- and exogalactanases.

Aspergillus nidulans, a saprobic filamentous fungus, is able to use D-galactose efficiently as a carbon source. A. nidulans can convert D-galactose through two pathways: the common Leloir pathway as well as the recently described alternative D-galactose utilization pathway.

Recently two regulators, GalR and GalX were identified that control D-galactose metabolism in
A. nidulans. The interaction of these regulators, their control of the various genes of the two D-galactose utilization pathways as well as genes encoding extracellular galactose releasing enzymes will be discussed.







Genetic Basis for the 3-ADON and 15-ADON Trichothecene Chemotypes in Fusarium graminearum

Nancy Alexander[1] Susan McCormick[1] Cees Waalwijk[2] Robert Proctor[1]

1 National Center for Agricultural Utilization Research, Peoria, IL, 2Plant Research International B.V,, Wageningen, the Netherlands

In some regions of the world, most strains of the wheat head blight pathogen Fusarium graminearum have one of two trichothecene mycotoxin production profiles (chemotypes), which are designated as 3-ADON and 15-ADON.  In a defined medium, strains with the 3-ADON chemotype produce a trichothecene (3-acetyldeoxynivalenol) with an acetate at carbon atom 3 (C3) but not at C15, whereas 15-ADON strains produce a trichothecene (15-acetyldeoxynivalenol) with an acetate at C15 but not at C3.  Despite this, strains with both chemotypes possess enzymatic activities necessary for production of trichothecenes with acetates at both C3 and C15, e.g. 3,15-diacetyldeoxynivalenol (3,15-diADON), suggesting the chemical modification responsible for the two chemotypes occurs near the end of the trichothecene biosynthetic pathway.  Polymorphisms in the trichothecene biosynthetic genes TRI3, which encodes a C15 acetyltransferase, and TRI12, which encodes a transport protein, are used as genetic markers to distinguish between strains with 3-ADON and 15-ADON chemotypes.  However, a causal relationship between TRI3/TRI12 and the chemotypes has not been demonstrated.  Sequence analysis has revealed marked differences in the coding sequence of the esterase gene TRI8 in 3-ADON versus 15-ADON strains.  To determine whether these differences can affect trichothecene chemotype, we examined the activity of TRI8 as well as TRI3.  The data indicate that differences in activity of the TRI8 esterase, rather than the TRI3 acetyltransferase, are the basis of 3-ADON and 15-ADON chemotypes in F. graminearum.




Tools for exploration of Aspergillus gene, protein, and sequence information at the Aspergillus Genome Database (AspGD)

Martha B. Arnaud[1] Jonathan Binkley[1] Marcus C. Chibucos[2] Maria C. Costanzo[1] Jonathan Crabtree[2] Diane O. Inglis[1] Joshua Orvis[2] Prachi Shah[1] Marek S. Skrzypek[1] Gail Binkley[1] Stuart R. Miyasato[1] Jennifer R. Wortman[2] Gavin Sherlock[1]

1Department of Genetics, Stanford University School of Medicine, Stanford, CA, 2Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD

The Aspergillus Genome Database (AspGD) is an online genomic resource designed to facilitate research on Aspergilli and on other medically and economically important fungal pathogens.   We provide an online reference for Aspergillus genomics and molecular biology, with up-to-date, high-quality information curated from the scientific literature, as well as web-based research tools for exploration and analysis of these data.  The Sybil Comparative Genomics tool at AspGD displays alignments of the genomic regions encoding clusters of homologous proteins from ten Aspergillus genomes (A. nidulans, A. fumigatus, A. flavus, A. oryzae, A. niger, A. clavatus, A. terreus, and Neosartorya fischeri), as well as various displays for exploration of syntenic regions among these organisms.  The GBrowse Genome Browser supports navigation and searching of genes and chromosomal regions of the A. nidulans FGSC A4 and A. fumigatus Af293 genomes, and will be extended to other Aspergilli in the future.  Additional tools are available for search and retrieval of A. nidulans sequence and gene and protein information that has been curated from the scientific literature. The suite of sequence analysis tools includes BLAST, pattern matching, restriction mapping, and primer design.  In addition, AspGD offers keyword-based and gene-property-based searches, Gene Ontology (GO)-based analysis of gene lists by function and localization, bulk data query and retrieval, and downloadable files.  While AspGD curation has initially focused on A. nidulans, we will begin curation of the scientific literature on A. fumigatus and other Aspergillus species in 2010 and will provide the full suite of AspGD tools for each of these species in the future.  We also provide tools for community interaction, including a colleague registry by which Aspergillus research community members may share contact information and research interests to facilitate collaboration, and a list of Aspergillus research laboratories.  Our mission is to be responsive to the needs of the research community, and we welcome your feedback and suggestions, at  All of the data in AspGD are freely available to the public from  AspGD is supported by grant RO1 AI077599 from the NIAID at the NIH.





The antifungal protein AFP from Aspergillus giganteus inhibits the viability of Saccharomyces cerevisiae strains deficient in cell wall integrity

Jean-Paul Ouedraogo[1], Silke Hagen[1], Anja Spielvogel[1], Ulf Stahl[1], Vera Meyer[1,2]

1Berlin University of Technology, Institute of Biotechnology, Department of Microbiology and Genetics, Berlin, Germany
2Leiden University, Institute of Biology, Molecular Microbiology and Biotechnology, Leiden, The Netherlands


The antifungal protein AFP, secreted by Aspergillus giganteus is able to inhibit the growth of a variety of filamentous fungi (e.g. A. fumigatus, A. niger, Fusarium oxysporum) by causing membrane invaginations and membrane permeabilisations. However, the protein does not negatively affect the growth of yeast, bacteria or mammalian cell types. Our recent work demonstrated that AFP inhibits chitin synthase activities in sensitive fungi which is counteracted by increased activities of the cell wall integrity pathway (CWIP).

In order to understand the resistance mechanism of yeast strains, we have screened S. cerevisiae mutants deleted for the components of the CWIP and chitin synthesis. Most of the ~ 70 screened strains remained AFP-resistant, except the knock out mutants Δwsc1, Δtor1, Δvps34 and Δchs1, which became moderate-sensitive towards AFP. The plasma membranes of these mutants became readily permeabilized by AFP. Interestingly, the presence of AFP provoked increased chitin synthesis in these strains, an observation which we also made for the AFP-resistant filamentous fungus Penicillium chrysogenum and the moderate-sensitive mutant of F. oxysporum ΔchsV.

The obtained results stipulate the hypothesis that moderate-sensitive and resistant filamentous fungi counteract AFP inhibitory effects by strongly increasing their chitin levels, thereby making the cell walls presumably more rigid. However, this response does not occur in AFP-sensitive fungi. Apparently, the classical CWIP is not sufficient to counteract AFP inhibitory effects and seems not to be involved in increased chitin synthesis. Our findings give rise to the assumption that fungal strains which only use the classical CWIP are AFP-sensitive.




Photoreception and the Neurospora circadian clock

Maria Olmedo, D. Lenssen, M. Merrow

Department of Chronobiology, University of Groningen

The circadian clock creates a temporal structure within cells in animals, plants, fungi and cyanobacteria. One aspect of the clock is a self-sustained oscillation with a period of ~24h in constant conditions. However, circadian clocks in nature are rarely subjected to constant conditions. They are normally exposed to a rhythmic environment, where signals (zeitgebers) such as light and temperature entrain the oscillations to the 24 h day. Blue-light responses in Neurospora require the wc-1 and wc-2 genes and these proteins also regulate the circadian clock. However, the Neurospora genome contains additional genes coding for putative photoreceptors, including a cryptochrome, an opsin, and two phytochrome genes. We obtained knockout mutants for the genes coding for putative photoreceptors from the Neurospora Functional Genomics Project and screened them in a variety of protocols for circadian rhythm and entrainment. When assayed under constant conditions Δcry, Δphy-1, Δphy-2, Δnop-1 and Δorp-1 strains have the same free running period relative to the wild-type control. In cycling conditions, however, all of the knockouts show differences in phase of entrainment compared to the wild-type strain. The most extreme phenotype in our study is the Δcry, Δphy-2, matA strain, which is arrhythmic under all conditions tested. In summary, our results show that additional photoreceptors play a role in entraining the Neurospora circadian clock. We conclude that Neurospora indeed models circadian clocks of in other organisms with respect to light input, because they also show striking contributions of input pathway components on circadian clock behaviour.


Chitinases of Aspergillus niger upregulated during autolysis

Jolanda van Munster[3] B.M. Nitsche[1] R.M. van der Kaaij[2] A.F.J. Ram[1] L. Dijkhuizen[3] M.J.E.C. van der Maarel[3]

1Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2TNO Quality of Life, Dep. Food and Biotechnology Ingredients, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
3Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Groningen University, Kerklaan 30, 9751 NN Haren, The Netherlands.

The filamentous fungus
Aspergillus niger is well known for its capacity to secrete high amounts of proteins and metabolites, and is therefore used in industry for the production of enzymes and chemicals.
The mycelium of this fungus is highly differentiated. After stationary growth phase, part of the mycelium is degraded in a process called autolysis. Autolysis is characterized by hyphal fragmentation, loss of biomass, ammonia release and the production of enzymes such as proteases and glycoside hydrolases. These glycoside hydrolases could function in degradation of cell wall polymers such as chitin. However, knowledge about the exact mechanism of autolysis is currently limited.

During industrial fermentation processes, autolysis can cause the productive biomass to decrease, causing reduced product yield. A better understanding of autolysis can contribute to the formation of strategies to increase efficiency of fermentations.

In order to increase understanding of the dynamics of the fungal mycelium, a consortium of academic and industrial partners investigates autolysis and differentiation in
Aspergillus niger. One goal of this project is the identification and characterization of glycoside hydrolases that are involved in autolysis.
By using microarrays to monitor transcription levels during growth, we have identified genes that are upregulated during the autolytic phase compared to exponential growth phase. Four of these genes belong to glycoside hydrolyse family 18, which consists mainly of (putative) chitinases. In order to investigate the properties of these enzymes we performed heterologous gene expression in
E. coli with subsequent purification using affinity tags. The activity of purified proteins is investigated.





Curation of Aspergillus gene and protein information at the Aspergillus Genome Database (AspGD)

Diane Inglis[1] Martha B. Arnaud[1] Jon Binkley[1] Maria C. Costanzo[1] Marcus C. Chibucos[2] Jonathan Crabtree[2] Joshua Orvis[2] Prachi Shah[1] Marek S. Skrzypek[1] Gail Binkley[1] Stuart R. Miyasato[1] Jennifer Wortman[2] Gavin Sherlock[1]

1Department of Genetics, Stanford University School of Medicine, Stanford, CA
2Institute for Genomic Sciences, University of Maryland School of Medicine, Baltimore MD

The Aspergillus Genome Database (AspGD; is a web-based genomics resource for researchers studying the genetics and molecular biology of an important group of fungal microorganisms, the aspergilli. AspGD provides high-quality manual curation of the experimental scientific literature, including gene names, general descriptions, phenotype data, and Gene Ontology (GO) annotations, as well as tools for exploring these data. AspGD is based on the framework of the Saccharomyces Genome Database (SGD) and Candida Genome Database (CGD), two genomic resources with which many users within the fungal research community are already familiar. The manual annotation of gene information, phenotype data and GO annotations is the focus of this presentation. Initially, we have focused on the manual curation of genomic information for Aspergillus nidulans, the best-characterized species of the group. We will expand our efforts to include curation of A. fumigatus, A. flavus, A. oryzae, A. niger, A. clavatus, A. terreus, and Neosartorya fischeri genomes. The curation process for gene-specific information entails screening of the published literature to identify the relevant journal articles, and manual evaluation of each paper by trained scientific curators, who extract the information for collection into the database using structured vocabularies (such as GO and our phenotype description system) as well as free-text descriptions. The curated information for each gene appears in brief on its Locus Summary page, which links out to details pages that provide additional information, including phenotype and GO details, a history of sequence and annotation that affect the gene, and a comprehensive list of references. We welcome questions, requests for data, comments, or suggestions to help us better serve the needs of the research community, and encourage researchers to contact us at  AspGD is supported by grant RO1 AI077599 from the NIAID at the NIH.



Identification of the putative glycerol transporter of the halophilic black yeast Hortaea werneckii

Tina Kogej, Cene Gostincar, Nina Gunde-Cimerman

University of Ljubljana, Biotechnical faculty, Department of Biology

Purpose: Hortaea werneckii is an ascomycetous halophilic black yeast naturally inhabiting hypersaline waters of solar salterns. It is remarkable for its growth at a wide range of NaCl concentrations (0 – 5.2 M) and is a novel eukaryotic organism for studying cellular responses to extremely elevated environmental salinity. We have found previously that H. werneckii adapts to high NaCl concentration by accumulating glycerol and erythritol. The purpose of this study was to search for and identify a glycerol transporter of the halophilic black yeast Hortaea werneckii. Methods: The partial gene sequence was obtained by suppression subtractive hybridization and extended by genome walking. The complete cDNA sequence was obtained by SMARTer rapid amplification of cDNA ends. The gene was cloned and sequenced, and the putative protein was characterized in silico. Results: In the halophilic black yeast Hortaea werneckii, we have identified a gene encoding a putative protein with a considerable degree of similarity to a number of uncharacterized sugar transporters, and also to Stl1p, a well-characterized member of the sugar transporter family, the glycerol/H+ symporter of the plasma membrane in Saccharomyces cerevisiae. We have named it HwSTL1. HwSTL1 consists of 1631 bp, encodes a protein of 543 aa with a calculated MW of 59417. Conclusions: We have identified, cloned and characterized the gene HwSTL1 encoding a glycerol-transporter-like protein of H. werneckii. In our further studies we will conclude whether the protein encoded by the HwSTL1 indeed functions as a glycerol transporter protein in H. werneckii.

glycerol transport, Hortaea werneckii, MFS superfamily, osmoadaptation, STL1-like transporter,


Enzymatic characterization of six recombinant serine-type carboxypeptidases of Aspergillus oryzae RIB40

Hiroto Morita[1] Ayako Okamoto[1] Youhei Yamagata[1] Ken-Ichi Kusumoto[2] Yoshinao Koide[3] Hiroki Ishida[4] Michio Takeuchi[1]

1Tokyo University of Agriculture and Technology, 2National Food Research Institute, 3Amano enzyme Inc., 4Gekkeikan Sake Co. Ltd

Serine-type carboxypeptidase (SCP) is an exopeptidase that has Ser, His, and Asp residues as a catalytic triad construct and can sequentially release C-terminal amino acid residues of peptides and proteins. In the genome of Aspergillus oryzae RIB40, 12 genes have been predicted to encode SCPs. However, the carboxypeptidase activities of the gene products have not yet been confirmed experimentally. Therefore, we have constructed those gene overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Here, we report enzymatic character of six of the gene products. The recombinant proteins were able to release amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be SCPs. The enzymes were stable at lower than 40-55°C, at low and neutral pH. The optimum pHs of the enzymes except for AOCP16 were around pH 4. That of AOCP16 was pH5.5. The substrate specificities of each enzyme for N-acyl-peptides were different. Result of transcriptional analysis of these genes suggested differences in transcriptional regulation between these genes. The enzymatic properties of AOCP6 and AOCP9 were different from those of any reported SCP. AOCP4 and AOCP13 had similar enzymatic properties to carboxypeptidases O1 and O2 and carboxypeptidase O from A. oryzae IAM2640. Results of sequence analysis of DNA and N-terminal amino acid sequences showed AOCP4 correspond to carboxypeptidases O1 and O2, and AOCP13 correspond to carboxypeptidase O.


This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences.



Characterization of newly found intracellular metallo-carboxypeptidases by genome sequencing of A. oryzae

Youhei Yamagata[1] Hiroshi Maeda[1] Ken-ichi Kusumoto[2] Yoshinao Koide[3] Hiroki Ishida[4] Michio Takeuchi[1]

1Dep. of Agriscience & Bioscience. Tokyo University of Agriculture & Technology, Tokyo, Japan
2NFRI, Ibaraki, Japan, 3Amano Enzyme Inc., Gifu, Japan, 4Gekkeikan Sake Company Ltd., Kyoto, Japan

Aspergillus oryzae is one of industrial microorganism as using for the Japanese traditional fermented food. The genome project of A. oryzae clarified that there were 134 genes coding proteolytic enzymes. We have been trying to characterize all proteolytic enzymes.

We found that A. oryzae had twelve genes for metallo-carboxypeptidases. Nine of them do not have the signal peptide, and it is presumed that these enzymes would be localized intracellularly. The enzymes were all classified in M20 super-family. As four of the enzyme genes were translated under the liquid culture condition, the genes were cloned and expressed in E. coli. The two purified enzymes (AOEXE305 and AOEXE306) showed maximum activity at alkaline pH in spite of those are intracellular enzymes. It was shown that acidic amino acid was favorable in P1’ site for AOEXE305. AOEXE306 showed wider substrate specificity than AOEXE305. The enzyme could cleavage between even Xaa-Pro bonds but did not favor acidic amino aicd in S1’ in substarates. The results might indicate that fungal intracellular metallo-carboxypeptidases had different roll in fungal cells.

This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).



Targeted functional proteomics: A putative translation elongation factor with glutathione S-transferase activity protects Aspergillus fumigatus against oxidative stress

Grainne O' Keeffe, Christoph Jöchl, Sean Doyle

NUI Maynooth

Aspergillus fumigatus is an opportunistic pathogen predominantly affecting immunocompromised individuals, resulting in pulmonary illnesses such as Invasive Aspergillosis. Sequencing of the genome has led to an increased understanding of the organism; however the functions of many genes remain unknown. A putative translation elongation factor 1Bγ (EF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione s-transferase activity, in A. fumigatus [1]. Normally, EF1Bγ plays a key role in the elongation step of protein synthesis. Our hypothesis is that elfA may also play a role in regulating the cellular redox state adjacent to the ribosome during protein synthesis. Consequently, elfA was disrupted in A. fumigatus ATCC46645 (wild-type) using a bipartite construct containing overlapping fragments of a pyrithiamine resistance gene (ptrA). The elfA mutant (ΔelfA) was complemented using a hygromycin resistance marker (hph). Southern Blot analysis was used to confirm the generation of ΔelfA and the complemented strain. RT-PCR confirmed the expression of elfA in wild-type and complemented strains, and absence of expression in ΔelfA. The availability of the mutant has facilitated phenotypic analysis of elfA functionality. A. fumigatus wild-type and ΔelfA were grown on AMM plates with the oxidant H2O2 (1 - 5 mM), voriconazole (0.25 - 1 µg/ml), and the thiol-reactive reagent, 4,4’-dithiodipyridine (3 - 7.5 µM). At 37°C, the elfA mutant was significantly more sensitive (p=0.0003) to H2O2 than wild-type. However, ΔelfA was significantly less sensitive (p=0.0251) to voriconazole than wild-type. At 37°C, the ΔelfA was significantly more sensitive (p=0.0056) to 4,4’-dithiodipyridine than wild-type.  These results implicate elfA in the oxidative stress response in A. fumigatus and also strongly indicate that elfA may play a role in the sensitisation of A. fumigatus to voriconazole. Global proteomic studies are currently underway using 2D-PAGE and MALDI-MS to explore alterations in the proteome consequential to elfA disruption with a view to gaining further insight into the function of elfA in A. fumigatus.

1. Carberry, S, et al. (2006), Biochem Biophys Res Commun, 341, 1096-1104.




The monomeric gtpase rheb: putative link between the amino acid metabolism regulation and the development control of phytopathogenic fungi

Géraldine MEY, Heber Gamboa-Meléndez, Bénédicte Gayrin, Tim Rollenske, Marie-Josèphe Gagey, Geneviève Billon-Grand, Michel Droux

Functional Genomics of Phytopathogenic Fungi, UMR 5240 CNRS-UCBL-INSA-Bayer CropScience

How phytopathogenic fungi fulfill their nutritional needs during the interaction with their hosts remains poorly documented. They may first mobilize their storage compounds during the early stages of the infection process. Then they may use the simple compounds, released by the subsequent host constituent enzymatic degradation, to complete their development in planta. The metabolism of amino acids may thus be redirected and adapted to the different stages of the plant infection strategy. Our studies focus on the amino-acid triggered regulation mechanisms required to successfully achieve plant infections. The monomeric GTPase Rheb (Ras Homologue Enriched in Brain) was known to activate the kinase TOR and to be involved in the regulation of arginine and lysine uptake in yeasts. Botrytis cinerea Rheb orthologue was inactivated using different approaches (RNA interference, promoter replacement). The assay of the amino acid content, using reversed chromatography (HPLC), highlighted the implication of Rheb in the control of amino acid metabolism. The putative involvement of Rheb in the amino acid uptake control was suggested with complementation experiments of a S. cerevisiae mutant strain. Rheb importance for B. cinerea development was analyzed using different model host systems and the microscopic observations of the early differentiation stages. A comparative study of Rheb functions in other phytopathogenic models is being performed by expressing Rheb hyper- and hypo-active mutant forms in Magnaporthe grisea. The results obtained with both fungi will give insights on the molecular mechanisms controlling amino acid uptake/metabolism and their requirement for the parasitic development of fungal plant pathogens.





Analysis of the histone deacetylases disruptants of Aspergillus oryzae

Moriyuki Kawauchi[1] Mika Nishiura[1] Kazuhiro Iwashita[2] Osamu yamada[2]

1Hiroshima univ., 2NRIB

Aspergillus oryzae is ubiquitous filamentous fungi in nature and has been used in a number of industries such as Japanese traditional fermented foods and pharmaceutical products.  In nature and industries, A. oryzae adapt to various environmental conditions by global change of transcriptional regulation.  In the previous study, we revealed that the expression of histone acetylation related genes were affected by the growth phase and alteration of growing environment, such as culture conditions and stress exposing.  In general, histone acetylation plays the fundamental roles for the genes expression, and closely relates to growth, morphology, differentiation and stress responses.  Recently, several reports indicate that histone acetylation also plays important roles in filamentous fungi.  In this context, we focused on histone acetylation related genes, particularly histone deacetylases (HDACs).  We attempted to disrupt 11 HDACs homologue of A. oryzae and 10 of them were disrupted.  However, in the deletion of RPD3 homologue, only the heterokaryon transformants had been isolated.  This result suggests that RPD3 homologue is essential in A. oryzae.  The phenotypes of 10 HDACs disruptants were observed in submerged and plate culture with/without several stress conditions.  As the results, four disruptants showed considerable growth and developmental defects in these conditions, especially, HOS2 homologue null mutant showed significant decline of growth in submerged culture.

In microscopic analysis, two disruptants showed abnormal hyphal branches and aberrant distribution of hyphae on the interface of medium and air.  This study has indicated that HDACs play important roles in the growth and stress adaptation of
A. oryzae.


Clitocypin and macrocypins cover different mushroom defences

Jerica Sabotic[2] Sreedhar Kilaru[1] Jože Brzin[2] Andy Bailey[1] Gary Foster[1] Janko Kos[2]

1School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, United Kingdom
2Department of Biotechnology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

Mycocypins, clitocypin and macrocypins, are cysteine protease inhibitors isolated from basidiomycetes clouded agaric (Clitocybe nebularis) and parasol mushroom (Macrolepiota procera). Two new families of protease inhibitors in the MEROPS classification have been established, I48 for clitocypin and I85 for macrocypins, based on their unique primary sequences and biochemical characteristics. Mycocypins are exceptionally stable proteins, exhibiting high thermal and broad pH stability. The physiological function of the two mycocypin families is proposed to be defence against pathogen infection and/or predation by insects or other pests, analogously to the phytocystatins that are involved in plant defence by inhibiting exogenous cysteine proteases during herbivory or infection. Sequence diversity of clitocypin genes is limited to 18 discreet positions that have no influence on its inhibitory activity. On the other hand, the sequence diversity of macrocypin genes is higher and includes amino acid sites of positive selection. The variations in inhibitory profile between different members of the macrocypin family reveal different specificities and strengths of inhibition of cysteine proteases of different evolutionary families, and even a serine protease. These findings together suggest an adaptation process and the selection of appropriate inhibitor isoforms providing effective defence. Analysis of expression regulation of mycocypins using different mycocypin promoters and green fluorescent protein as the reporter gene showed different patterns of expression during fruiting body development for clitocypin and macrocypins. In view of their proposed defensive roles different expression profiles suggest different target organisms as both families of mycocypins are present in each mushroom.





Identification of gene carD, encoding the aldehyde dehydrogenase responsible for neurosporaxanthin biosynthesis in Fusarium fujikuroi

Violeta Díaz Sánchez[1] Alejandro F. Estrada[1] Salim Al-Babili[2] Javier Avalos[1]

1Universidad de Sevilla, 2University of Freiburg


The gibberellin producing fungus Fusarium fujikuroi, is also used as a model for genetic and biochemical analysis of carotenoid biosynthesis. Its major carotenoid product is neurosporaxanthin (NX), an acidic apocarotenoid formerly discovered in N. crassa. The NX precursor beta‑apo‑4’‑carotenal is produced by F. fujikuroi through the activity of the enzymes encoded by genes carRA (cyclase and phytoene synthase), carB (phytoene desaturase), and carT (torulene cleaving oxygenase). The enzyme responsible for the oxidation of the aldehyde group of beta‑apo‑4’‑carotenal to yield NX, has not been described in this fungus. Based on our former results with ylo-1 in N. crassa, we have cloned the F. fujikuroi gene carD, coding for an aldehyde dehydrogenase putatively responsible of this enzymatic reaction. Crude protein extracts from an E. coli strain expressing a carD cDNA version were able to convert beta‑apo‑4’‑carotenal into the corresponding apocarotenoic acid, confirming the expected enzymatic activity. CarD was also active on shorter carotenoids, including acyclic ones, such as 8’-lycopenal, indicating the irrelevance of the cycled end of the molecule for substrate recognition. Also, we expressed the enzyme in a beta-apo-4’-carotenal producing E. coli strain and we got NX production in vivo. In contrast to other car genes, real-time RT-PCR analyses of carD mRNA levels showed a light-independent expression. However, the mRNA levels were increased in a carotenoid overproducing mutant, indicating common regulatory mechanisms for all the car genes in this fungus. Phenotypic effect of targeted carD disruption, currently in progress, will be reported.



Development and applications of smart screening platforms

Ellen L. Lagendijk[1] Benjamin M. Nitsche[1] Vera Meyer[2] Cees A.M.J.J. van den Hondel[1] Arthur F.J. Ram[1]

1Leiden University, Institute of Biology Leiden, Section of Molecular Microbiology & Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2Berlin University of Technology, Institute of Biotechnology, Department Microbiology and Genetics, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany

Increased resistance to currently used antifungal compounds and the fact that these agents are often harmful to man and environment have resulted in a growing demand for new antifungals, which selectively act on cellular processes that are unique to fungi. To meet this demand, we have established a luciferin/luciferase based reporter system for high-throughput screening of extracts obtained from natural sources. This system allows us to identify compounds that specifically target fungal cell wall biosynthesis. It has been well established in the yeast Saccharomyces cerevisiae that cell wall synthesis is a highly dynamic process, which is orchestrated by the cell wall integrity (CWI) pathway. Such CWI pathway is also present in the filamentous fungus Aspergillus niger and probably widespread among fungi. We have previously shown that transcription of the A. niger agsA gene, encoding an alpha-1,3-glucan synthase, is strongly and specifically up-regulated in response to cell wall stress (Damveld, 2005b; Meyer, 2007). We have also shown that the induced expression of the agsA gene is mediated via the RlmA transcription factor and its putative RlmA binding site within the agsA promoter (Damveld, 2005a). We are here presenting results for a new cell wall stress responsive A. niger reporter strain obtained by cloning the  agsA promoter in front of a codon optimized luciferase gene (Morgan, 2003). The performance of the system was verified by screening several antifungal compounds with a known mode of action and isolation of a antifungal compound from teakwood sawdust. The specific response dynamics of the new reporter system will allow us to identify new putative antifungal compounds from extracts of the whole biodiversity.




Utilization of exogenous heme by Aspergillus niger

Angelique Franken[2] B. Christien Lokman[1] Arthur F.J. Ram[2] Cees A.M.J.J. van den Hondel[1,2] Sandra de Weert[2]

1HAN Biocentre, Laan van Scheut 2, 6525 EM Nijmegen, The Netherlands
2Institute of Biology Leiden, Leiden University, Molecular Microbiology & Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands

The incorporation of heme as a cofactor is a putative limiting factor in the overproduction of heme-containing fungal peroxidases in Aspergillus species. Addition of hemin to growth medium has been reported to improve the production of peroxidase. However, hemin uptake and the effect of hemin addition on the transcriptional regulation of the heme biosynthesis pathway genes have hardly been studied in Aspergillus. To gain more insight into the heme biosynthesis pathway, the genes encoding the eight different enzymes in the pathway were identified in the A. niger genome. Individual deletion of four genes in the pathway (hemA, hemB, hemF or hemH) showed that all four are essential. Growth of the hemA deletion mutant could be restored by addition of 5’-aminolevulinic acid (ALA). Supplementation with hemin alone did not restore growth. The inability to grow directly on hemin is likely due to the lack of siroheme. Deletion strains of hemF and hemH, located after this branch point in the heme biosynthesis pathway, could be partially rescued by the addition of hemin. Growth of these mutants can be improved by additional supplementation of Tween80. A detailed characterization of the deletion strains is currently ongoing. These results strongly indicate that A. niger is capable of sequestering heme from its environment and utilize this heme for cellular processes.


A glutathione S-Transferase, GliG, may mediate thiol incorporation in gliotoxin biosynthesis and is not involved in auto-protection against gliotoxin.

Carol Davis[1] Stephen Carberry[1] Markus Schrettl[1] Dermot Brougham[2] Kevin Kavanagh[1] John Stephens[1] Sean Doyle[1]

1NUI Maynooth, 2Dublin City University

Biosynthesis of gliotoxin is directed by the multi-gene (gli) cluster in the opportunistic fungal pathogen, Aspergillus fumigatus. Minimal functional cluster annotation is available. The gene gliG, located in the gli cluster, is classified as a glutathione s-transferase by in silico analysis and recombinant GliG exhibits GST and glutathione reductase activity. Two overlapping constructs, each containing part of a marker gene (ptrA) and with homology to gliG flanking regions, were used to disrupt gliG in A. fumigatus (Daku80 and Af293 strains). The generation of a gliG mutant was confirmed using Southern Blot analysis using a digoxigenin-labelled probe specific for an XbaI digested fragment size of 2124 bp in the wild-type and 1668 bp in the gliG mutant. Absence of gliG expression in the mutant was confirmed by Northern analysis. RP-HPLC-DAD and LC-MS analysis of extracts from A. fumigatus wild-type and ΔgliG revealed that gliotoxin (Rt= 14.4 min) was absent from the mutant strains, strongly indicating that gliG is involved in gliotoxin biosynthesis. Interestingly, an additional metabolite (Rt = 12.3 min) was present in mutant culture supernatants which may represent a precursor of gliotoxin (GTP). LC-ToF analysis determined that the metabolic intermediate had a mass of 263 Da and targeted alkylation demonstrated the lack both free thiol residues and an intact disulphide bridge. Reconstitution of gliG into A. fumigatus DgliG restored gliotoxin biosynthesis. Unlike another component of the gli cluster, gliA, it appears that gliG is not involved in the auto-protection of A. fumigatus against exogenous gliotoxin. In conclusion, we confirm a key role for the glutathione s-transferase, GliG, in the biosynthesis of, and not auto-protection against, gliotoxin- which, to our knowledge, is the first time this enzyme has been shown to play a pivotal function in ETP biosynthesis.




Studying the interaction of DON-producers Fusarium graminearum with tolerant or susceptible varieties of Triticum aestivum: a biochemical and molecular approach

CHIARA NOBILI[4] A. Ricelli[1] M. Reverberi[2] S. Gatta[2] V. Scala[3] G. Aureli[3] A.A. Fabbri[2] C. Fanelli[2]

1ICB-CNR, 2Dipartimento di Biologia Vegetale, Università “Sapienza”, Largo Cristina di Svezia 24, 00165, Roma, Italy, 3Unità di Ricerca della Valorizzazione Qualitativa dei Cereali, CRA-QCE, Via Cassia 166, 00191, Roma, Italy, 4ENEA

Under suitable conditions, some fungal species, such as Fusarium, growing on many food commodities, can produce secondary metabolites dangerous for humans and animals.

It has been assessed that one of the main virulence factor involved in the Fusarium head blight (FHB) disease of wheat (leading to a severe reduction of grain yield and quality) is the Fusarium graminearum production of toxins, predominantly deoxynivalenol (DON) that delays germination and growth of wheat plants, inducing hydrogen peroxide (H2O2) production, inhibiting protein synthesis and stimulating cell death in planta.         

Mycotoxigenic fungi contamination is a real issue, especially for cereal industry. Therefore, in order to reduce the diffusion of plant disease and health risks, there is a real need to develop analytical methods able to identify DON-producing fungal variety and to quantify mycotoxins.

In this work, the interaction between two Triticum aestivum varieties, BLASCO (tolerant) and SAGITTARIO (susceptible), inoculated with two F. graminearum strains (Fg126 and Fg8308), was studied.

Recent advances in DNA-based techniques confer to Real Time-PCR (RT-PCR) assays an important role because of the accelerated diagnostic outcome, so that, this method is providing new tools for fungal detection and quantification in complex matrix. Thus, two primer pairs, designed by other authors, on the gene sequences belonging to the thricothecene gene cluster were used to identify high DON producing Fusarium strains through PCR method. Furthermore, a SYBR green Real Time-PCR assay was developed to quantify F. graminearum strains in artificially contaminated soft wheat. These results were correlated with the quantification of ergosterol by HPLC. Moreover, the expression of different genes activated in the interaction environment, was analysed by a relative RT-PCR approach. In the pathogen these genes encode for Fgap1 that is a transcription factor active in the cell defence against oxidative stress, ePG a poligalatturonase involved in cell degradation and tri6, one of the thricotecenes biosynthesis regulator. In T. aestivum, the expression analysis of one glucosyl transferases (gt), one of biochemical mechanisms of resistance to DON is the plant ability to convert DON in a less toxic glucosylated form, and of the pathogenesis-related protein PR1 (PR1) were carried out.

It is known that, among the broad range of defence responses, occurring in planta when Fusarium invasion occurs, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), is one of the earliest events. The activities of three antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) correlated to ROS and of one more enzyme related to the defensive response (lipoxygenase), were monitored.

Finally, the application of HPLC method for the quantitative detection of DON and 15-acetyl DON produced from Fusarium species present on samples, confirmed, also, through ELISA analysis, was described.

In conclusion, as far as fungal diseases are wide diffused, the control of contaminated matrices it’s a priority. Thus, it’s very important to deepen plant-pathogen interaction study, in order to develop control strategies (i.e. quantitative, specie-specific methods) to be applied in diagnostics (i.e. advanced analytical method for mycotoxin detection).





Impact of Grape Berry Resveratrol on the growth of A. carbonarius and on the biosynthesis of

ochratoxin A

Patrizia De Rossi[4] Ricelli, Alessandra[1] Reverberi Massimo[2] Nicoletti Isabella[3] Antonella del Fiore[4] Bello Cristiano[3] De Rossi Antonella[3] Corradini Danilo[3] Fabbri Anna Adele[2] Fanelli Corrado[2]

1CRB-CNR, P.le Aldo Moro 5, 00185, Roma, Italy
2Università degli Studi “Sapienza”, Largo Cristina di Svezia 24 00165 Roma, Italy
3Istituto di Metodologie Chimiche, CNR Area della Ricerca di Roma, C.P. 1000016, MonteRotondo Stazione, Italy

4ENEA C.R.-CASACCIA, Via Anguillarese 301 00123 Roma, Italy

The objective of this research was to investigate whether A. carbonarius contamination induces resveratrol production in grape berry. A possible correlation between OTA production and resveratrol biosynthesis has also been considered. Aspergillus carbonarius is an important ochratoxin A (OTA) producing fungus which is responsible for toxin contamination of grapes and wine. OTA is a secondary metabolite which has been shown to be nephrotoxic, nephrocarcinogenic, teratogenic and immunosuppressive. Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a natural polyphenolic antioxidant found in red wine and grapes has been described as stress metabolite produced by Vitis vinifera in response to biotic and abiotic stress as well as to fungal infection and it has been demonstrated to have a particularly wide spectrum of mycotoxin control. In this study, Vitis vinifera berries were infected, during ripening, by a conidial suspension of A. carbonarius and incubated for 6, 12, 24, 48, 120 hours at 30°C. After incubation, each berry was analyzed, at each time interval considered, for quantifying A. carbonarius, OTA and resveratrol in grapes. Real Time PCR method with specie-specific primers (Acpks), designed on the basis of the OTA-related polyketide synthase sequences, was carried out quantifying the fungal development in grapes. Our results show a correlation between the growth of the fungus and biosynthesis of OTA and resveratrol content into grape berries, leading to hypothesize that some grapevine cultivars are more capable of self-protection against fungal contamination.





Identification of cell wall factor(s) adsorbing Taka-amylase in submerged culture of Aspergillus oryzae

Hiroki Sato, Takahiro Shintani, Katsuya Gomi

 Graduate School of Agricultural Science, Tohoku University, Sendai, Japan

We have observed that Taka-amylase (TAA) activity disappeared in submerged culture of Aspergillus oryzae at the later-stage of cultivation. This disappearance was revealed to be caused by adsorption of TAA on fungal mycelia, but not by degradation by own extracellular proteolytic enzymes. We have also showed that cell wall of A. oryzae prepared from mycelia at the later-stage of cultivation has an adsorption ability for TAA. This suggested that a certain cell wall factor(s) can adsorb TAA, resulting in the disappearance of TAA in liquid medium during cultivation. To identify the adsorption factor(s) in fungal cell wall, we carried out stepwise fractionation of cell wall prepared from mycelia at the later cultivation stage by alkali extraction and cell wall lytic enzymes. The alkali-insoluble fraction of cell wall, CW4, showed high adsorption ability for TAA, but digestion of CW4 with chitinase resulted in a significant decrease in the adsorption ability. These results indicated that the adsorption factor for TAA is chitin, which is one of major polysaccharides in fungal cell wall. However, the cell wall prepared from mycelia at the earlier cultivation stage barely adsorbed TAA, although it contained equivalent amount of chitin to that of later-stage mycelia. Taken together, it is suggested that there exists unidentified factor(s) that could prevent from adsorption of TAA onto the cell wall at the earlier-stage of cultivation and the factor(s) would be removed from or decreased in the cell wall with longer cultivation periods.



Non-ribosomal peptides play an important role in the virulence of the opportunistic pathogen Aspergillus fumigatus

Karen O'Hanlon[1] D. Stack[1] M. Schrettl[2] T. Larsen[3] K. Kavanagh[1] S. Doyle[1]

1National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland, 2Division of Molecular Biology, Innsbruck Medical University, A-6020 Innsbruck, Austria
3Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads 221, 2800 Kgs, Lyngby, Denmark

Aspergillus fumigatus is a ubiquitous filamentous fungus, and a serious opportunistic human pathogen. Availability of the complete genome sequence for A. fumigatus has revealed that there are at least eighteen genes coding for non-ribosomal peptide synthetases (NRPS). NRPS’s are usually large, multi-modular enzymes, comprised of discrete domains, which synthesise bioactive peptides via a thiotemplate mechanism. To date, a wide range of virulence factors have been reported for A. fumigatus, including adhesions, conidial pigments and proteases. Some of the best documented virulence factors for A. fumigatus include Gliotoxin and the iron-chelating Siderophores, which are of NRPS origin. Despite these important findings, there have been few studies relating the majority of A. fumigatus NRPS encoding genes to specific peptide products. This work aims to elucidate the peptide product encoded by a mono-modular NRPS, pesL (Afu6g12050/NRPS11), and to determine a possible role in virulence. A pesL deletion strain was generated, termed ∆pesL. ∆pesL displays severely reduced virulence in the Galleria mellonella model (p < 0.0001). Phenotypic analysis has confirmed increased sensitivity of ∆pesL to H2O2 (> 1 mM) compared to the wild-type (p = 0.05), and severely increased susceptibility towards the antifungal voriconazole (> 0.25 µg/ml) compared to wild-type (p < 0.01). These results indicate a role for pesL in protection against oxidative and antifungal stress within A. fumigatus. Comparative RP-HPLC analysis identified conidial specific material (Rt = 15.9 min; λmax at 220 nm) synthesised by A. fumigatus wild-type. This metabolite was absent from ∆pesL conidia. Increased production of this metabolite was observed in conidial extracts cultured in 2 mM H2O2, indicating up-regulation in response to oxidative stress. This material is currently undergoing further analysis. Furthermore, a recombinant PesL enzyme has been purified for use in an assay to determine the specific PesL amino acid substrate. This will contribute to the currently limited information on fungal NRPS substrate selectivity. Interestingly, another NRPS mutant generated previously, termed ∆pes3 (Afu5g12030/NRPS8) displays increased virulence in the Galleria mellonella model (p < 0.0001). Furthermore, ∆pes3 exhibited severely increased susceptibility towards the antifungal voriconazole (> 0.5 µg/ml) compared to wild-type (p < 0.001). RP-HPLC has not yet revealed a candidate pes3 peptide. However, the search is on-going. This data further highlights the importance that NRPS plays in this serious human pathogen, and may reveal novel drug targets in the future.




Endobacteria affect the metabolic profile of their host Gigaspora margarita, an arbuscular mycorrhizal fungus

Alessandra Salvioli[1] Marco Chiapello[1] Joel Fontaine[2] Anne Grandmougin-Ferjani[2] Luisa Lanfranco[1] Paola Bonfante[1]

1Department of Plant Biology and IPP-CNR, University of Torino, Torino, Italy
2Laboratoire Mycologie/Phytopathologie/Environnement, Université du Littoral, Côte d’Opale, BP 699, 62228 Calais cedex, France

AM fungi are obligate biotrophs of a large spectrum of plants with which they establish a mutualistic symbiosis. The presence of endobacteria living inside the cytoplasm of some AM fungi has long been documented, but the impact of these prokaryotes on fungal biology is still unknown.

The aim of this work was to understand whether the endobacterium Candidatus Glomeribacter gigasporarum has an impact on the biology of its fungal host Gigaspora margarita through the study of the modifications induced on the fungal proteome and lipid profile. The availability of G. margarita cured spores (i.e. spores that do not contain bacteria), represented a crucial tool to enable the comparison between two fungal homogeneous populations in the presence and the absence of the bacterial component. A differential protein expression was detected between wild type and cured spores under different physiological conditions (quiescent, germinating and strigolactone-elicited spores). The results obtained indicate that the fungal primary metabolism does not seem to be affected by the absence of the endosymbiont. By contrast, heat shock proteins are unambiguously upregulated, suggesting that the fungus has to face a stress situation when endobacteria are lacking. Furthermore, the fungal fatty acid profile resulted to be modified both quantitatively and qualitatively in the absence of endobacteria, being fatty acids more abundant in the presence of the endobacterium.

The results not only revealed that endobacteria have important impacts on the host fungal biology, but also offered one of the first contributions to the knowledge of the metabolic features of G. margarita.




Fine-tuning gene expression in filamentous fungi: An inducible and tunable promoter system for Aspergillus niger

Janneke van Gent, Franziska Wanka, Mark Arentshorst, Cees A.M.J.J van den Hondel, Arthur F.J. Ram, Vera Meyer

Leiden University, Institute of Biology Leiden, Department Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands & Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The Netherland

The function of genes is usually inferred from mutants in which the desired gene has been deleted or strongly overexpressed. However, studies at these extreme discrete points give only limited information about the gene functions. Moreover, many overexpression studies make use of metabolism-dependent promoters which often cause pleiotropic effects and thus impose further limitations on their use and significance.
Here we report a promoter system for
Aspergilus niger that can be fine-tuned to user-specified expression levels, that is independent from carbon and nitrogen metabolism, that can be induced within minutes and that shows remarkable reproducibility. The system is based on the tetracycline-dependent promoter and the bacterial rtTA transcriptional activator protein and has been validated under various cultivation conditions. From the data obtained we conclude that the tetracycline-dependent promoter provides rapid and tunable gene control in A. niger. The system should be applicable to other filamentous fungi with only minor modifications.


Purification and cloning of trans-3- and trans-4-proline hydroxylase from the fungus Glarea lozoyensis

Loubna Youssar; Wolfgang Hüttel and Michael Müller

Institut für Pharmazeutische Wissenschaften
Lehrstuhl für Pharmazeutische und Medizinische Chemie
. Freiburg; Germany


Glarea lozoyensis is an anamorph fungus, which was initially assigned Zalerion arboricola based on morphological traits. G. lozoyensis is of pharmaceutical interest since it is producing the antifungal secondary metabolite pneumocandin B0. This cyclic lipopeptide is chemically converted into a water-soluble derivative (caspofungin acetate) that is used against clinically relevant fungi pathogens.


In Pneumocandin B0 trans-4- and trans-3-hydroxyprolines are incorporated which are derived from hydroxylation of L-Proline by proline trans-3-hydroxylase (P3H) and proline trans-4-hydroxylase (P4H),respectively. The P3H activity discovered in G. lozoyensis is unique and specific for pneumocandin B0 biosynthesis.


We are interested in this new selectivity for biocatalysis, but also on the molecular and genetic level. To understand the physiological parameters that influence pneumocandin B0 and C0 production better, we proceed by purification of potential P3H and P4H proteins using gel filtration. The activity is checked in each step by HPLC and LC-MS. In parallel, we are trying to clone these genes by use of some conserved domains known from bacterial proline hydroxylases for designing degenerative primers. In future experiments, we want to express the genes heterologously in E. coli, knock them out in the native strain and study their expression which will allow a deeper understanding of the mechanism of pneumocandin biosynthesis.





Transcriptomic insights into the physiology of Aspergillus niger approaching zero specific growth rate

Thomas R. Jørgensen,1 2 Benjamin M. Nitsche,1 Gerda E. Lamers,1 Mark Arentshorst,1 2 Cees A. van den Hondel1 2 and Arthur F. Ram1 2

1. Sylvius Laboratory, Institute of Biology, Leiden University, Sylviusweg 72, 2333BE Leiden, The Netherlands

2. Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The Netherlands


The physiology of filamentous fungi at growth rates approaching zero has been subject to limited study and exploitation. With the aim of uncoupling product formation from growth, we have revisited and improved the retentostat cultivation method for Aspergillus niger. A new retention device was designed allowing reliable and near complete cell retention even at high flow rates. Transcriptomic analysis was used to explore the potential for product formation at very low specific growth rates. The carbon- and energy-limited retentostat cultures were highly reproducible. While the specific growth rate approached zero (<0.005 h-1), the growth yield stabilized at a minimum (0.20 gDW g-1 maltose). The severe limitation led to asexual differentiation and the supplied substrate was used for spore formation and secondary metabolism. Three physiologically distinct phases of the retentostat cultures were subjected to genome-wide transcriptomic analysis. The severe substrate limitation and sporulation were clearly reflected in the transcriptome. The transition from vegetative to reproductive growth was characterized by down-regulation of genes encoding secreted substrate hydrolases and cell cycle genes, and up-regulation of many genes encoding secreted small cysteine-rich proteins and secondary metabolism genes. Transcription of known secretory pathway genes suggests that A. niger becomes adapted to secretion of small cysteine-rich proteins. The perspectives are that A. niger in approach of zero growth can be used as a cell factory for production of secondary metabolites and cysteine-rich proteins. We propose that the new retentostat method can be used in fundamental studies of differentiation and is applicable to filamentous fungi in general.



The main cell cycle genes in the pathogenic yeast Cryptococcus neoformans

Susumu Kawamoto1, Eric V. Virtudazo1,  Misako Ohkusu1, Tomoko Sonoda2, Satoshi Miura2, Kanji Takeo1

1Medical Mycology Research Center, Chiba University, Chiba, Japan

2 Yokohama City University, Yokohama, Japan


We have been involved in studies towards molecular understanding of cell cycle regulation in the pathogenic yeast Cryptococcus neoformans. Our group has reported the unique cell cycle pattern of C. neoformans, different from that of the model yeast Saccharomyces cerevisiae. In contrast to S. cerevisiae, very little is known about the molecular regulation of C. neoformans cell cycle. To clarify cell regulation at the molecular level, cell cycle control genes in C. neoformans were cloned and analyzed, and further studies are currently being done to confirm their function in C. neoformans cell cycle. The homologues of CDC28/Cdc2 (CnCdk1), the main cell cycle gene which regulates the major processes in eukaryotic cell cycle, and its cyclin counterparts, known to interact with CDC28/Cdc2 and activate it to carry out specific controls throughout different stages of the cell cycle, were isolated and identified from C. neoformans. In addition to CnCdk1, at least three cell-cycle related cyclin homologues were identified in C. neoformans. Analysis of putative amino acid sequences of these cyclin homologues showed that one is a G1 cyclin homologue, named CnCln1. The molecular characterization of the two main cell cycle genes, CnCdk1 and CnCln1, in the pathogenic yeast C. neoformans, will be reported and discussed.






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