A Glutathione S-Transferase, GliG, May Mediate Thiol Incorporation in Gliotoxin Biosynthesis And Is Not Involved in Auto-Protection Against Gliotoxin. Carol Davis1*, Stephen Carberry1, Markus Schrettl1, Dermot Brougham3, Kevin Kavanagh1, John Stephens2 and Sean Doyle1. Department of Biology and National Institute for Cellular Biotechnology, National University of Ireland Maynooth, Co. Kildare, Ireland1. Department of Chemistry, National University of Ireland Maynooth, Co. Kildare, Ireland2. School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland3. *carol.davis@nuim.ie 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 (Δaku80 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 ΔgliG 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.
Engineering intracellular metabolism by altering gene expression of Aspergillus oryzae Junichiro Marui1, Sumiko Ohashi1, Marie Nishimura2, Hideaki Koike1and Masayuki Machida1 1 Natl. Inst. Adv. Indus. Sci. Tech. (AIST), 2 Natl. Inst. Agrobiol. Sci. Aspergillus oryzae is one of the most important organisms in Japanese fermented food industry. Although it hardly produces secondary metabolites, related organisms are producers of diverse metabolites. A major objective of our project is to develop a system using A. oryzae to generate diverse metabolites. The factors affecting expression level of metabolic genes are being studied. To develop the system, novel vectors and host strains of A. oryzae have been constructed. We have replaced promoter regions of some metabolic genes and successfully altered the level of some metabolite productions. Although it is well known that A.oryzae does not produce toxic metabolites, less attention has been paid to its non-toxic secondary metabolites. Genomic analysis revealed that A. oryzae possessed the orthologous gene cluster for penicillin production. The penicillin production was positively regulated by a global gene regulator required for transcriptional expression of the penicillin biosynthetic genes. Overexpression of the biosynthetic genes by a strong promoter yielded a greater than 100-fold increase in penicillin production. Transcriptional repression of a wide range of secondary metabolism genes in A. oryzae is a valuable characteristic for the production of a particular secondary metabolite with higher purity and safety. It appears that genetically engineered A. oryzae should be extremely useful as a cell factory for industrial production of beneficial secondary metabolites.
Non-Ribosomal Peptides play an Important Role in the Virulence of the Opportunistic Pathogen Aspergillus fumigatus K. O’Hanlon1, D. Stack1, M. Schrettl2, T. Larsen3, K. Kavanagh1 and S. Doyle1 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 karen.a.ohanlon@nuim.ie 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
CELL BIOLOGY
The master regulator of the Unfolded Protein Response revisited N.D.S.P. Carvalho, T.R. Jørgensen, M. Arentshorst, C.A.M.J.J van den Hondel and A.F.J. Ram Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands. n.carvalho@biology.leidenuniv.nl In Aspergillus niger, the beta-zip transcription factor HacA plays a central role in the activation of genes involved in the UPR pathway. Activation of HacA is mediated via an unconventional splicing event of 20 nucleotides in its mRNA. In the present study we engineered an A. niger strain that expresses only the activated form of HacA and we show that the removal of the 20 nucleotides results in a constitutive activation of the well established UPR target genes. The wild type strain (HacAWT) and the strain expressing the constitutive active form of HacA (HacACA) were cultured in glucose-limited batch cultures using bioreactors. RNA from the batch cultures was isolated for transcriptomic analysis of the effect of expressing a constitutive activated HacA. Statistical analysis defined 1119 genes as differentially expressed (significance p<0.005) relative to the wt. GO enrichment analysis revealed that the expression of several secretion and protein modification related genes was up-regulated in the HacACA mutant. Biological processes overrepresented in the down-regulated genes include several metabolic pathways, and terms related to transcription and translation. A comprehensive overview of the transcriptional response in the HacACA mutant will be presented.
Mutations in two Golgi apparatus COG proteins affect growth and glycosylation in Aspergillus nidulans. Sara Gremillion1, Darlene Loprete2, and Terry Hill2. 1Department of Biology, Armstrong Atlantic State University, 11935 Abercorn Street, Savannah, GA 31419, USA, 2Departments of Biology and Chemistry, Rhodes College, 2000 N. Parkway, Memphis, TN 38112, USA. Sara.Gremillion@armstrong.edu The swoP1 (swollen cell) and podB1 (polarity defective) mutations in Aspergillus nidulans interfere with establishment and maintenance of polarity. At restrictive temperatures, conidia of both mutants swell to approximately 1.5 times the normal diameter. Conidia of swoP also produce abnormally wide hyphae and establish multiple points of polarity, which grow isotropically before arrest. Genes complementing the mutations of swoP1 and podB1 have strong sequence homology to COG4 (ANID7462.1) and COG2 (ANID8226.1), respectively. In mammals and yeast, COG2 and COG4 are part of a multi-protein structure called the COG (conserved oligomeric Golgi) complex associated with retrograde transport within the Golgi apparatus. A GFP-tagged COG2 displayed a punctuate distribution within fungal hyphae, a pattern consistent with other Golgi protein localization. COG4 was not successfully GFP tagged. Protein overexpression studies provided evidence of intra-complex interactions between COG2 and COG4 as well as between COG2 and COG3. To study the role of these proteins in growth of filamentous fungi, an AlcA promoter replacement strategy was performed. When grown on AlcA-suppressive media, the COG4 AlcA-replaced promoter strain displayed over-swelling and a lack of polarity, while the COG2 AlcA-replaced promoter strain failed to grow. These results link both COG2 and COG4 to normal filamentous growth. A lectin blot using concanavalin A revealed significant differences in protein glycosylation patterns between the swoP1 and podB1 mutants when compared to wild type when grown at restrictive temperatures. The glycosylation patterns of the two mutants were indistinguishable under these conditions.
Signaling through the Aspergillus nidulans orthologue of Pkc mediates septum formation Loretta Jackson-Hayes, Terry W. Hill, Darlene M. Loprete, Britany Chavez, Chassidy Groover, Erinn Ogburn, and Michael Pluta We have shown that the Aspergillus nidulans orthologue of protein kinase C (PkcA) participates in regulating cell wall integrity (CWI) and localizes to sites of cell wall synthesis, including growing hyphal tips and septa. To better understand the mechanisms by which PkcA localizes to tips and septa, we have observed the formation of cortical rings at sites of septation by fluorescently tagged PkcA in hyphae defective in expression of other proteins necessary for septum formation, using either temperature-sensitive mutants or regulation under the AlcA promoter. We have also co-imaged PkcA and other septation proteins bearing complementary tags. Here we report that localization of PkcA to septa lies “downstream” of the functions performed by MobA (Mob1p orthologue), TpmA (tropomyosin), SepA (formin), SepD, SepG, and proteins encoded by two other not-yet-cloned Sep loci. In the absence of function of these proteins, PkcA cortical rings were not observed. PkcA localization lies “upstream” of MyoB (myosin II orthologue), the A. nidulans orthologue of Bud4p (in yeast, a bud site selection marker), and a protein encoded by a third uncloned Sep locus. PkcA cortical rings still form in the absence of function of these proteins, though septa do not develop. SepA, TpmA, MyoB, and MobA all appear to colocalize with PkcA during normal septum formation. While PkcA localizes to the very apex of hyphal tips and to the leading edge of growing septa, the protein phosphatase BimG localizes to sites lateral to the most active sites of growth. Studies with other septation-related proteins are ongoing.
Reduced Expression of SccA Increases Sensitivity to Wall Stress Loprete, D.M., Jackson-Hayes, L., Hill, T.W. Musgrove, J., Ogburn, E. Departments of Biology and Chemistry, Rhodes College, Memphis TN USA Loprete@rhodes.edu Decreased expression of the extragenic suppressor, designated SccA, affects cell wall integrity in the filamentous fungus Aspergillus nidulans. Overexpression of SccA suppresses the phenotype of the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin-binding agent Calcofluor White (CFW). In addition, we have shown that SccA rescues 6 wall-sensitive strains. In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. SccA is predicted to have a single transmembrane domain with 42% of its amino acids residues being serine or threonine, which indicates it is bound to carbohydrates in the cell wall. Homologues exist in the genomes of other filamentous fungi, but not in yeasts or other organisms. A SccA-GFP hybrid localizes to the plasma membrane and septa of vegetative hyphae. When SccA is placed under the control of the regulatable AlcA promoter and grown under low expression conditions (glucose), we observed a sensitivity to CFW, indicating it plays an important role in cell wall integrity. Taking into consideration the protein’s cell surface location and its influence on the function of PkcA, we hypothesize
ColA, a white spore color mutant in Aspergillus niger, identifies the phosphopantetheinyl transferase (PptA) protein which is required for melanin biosynthesis. Joohae Park, Thomas R. Jørgensen, Mark Arentshorst, Patricia A. vanKuyk, Robbert A. Damveld, C.A.M.J.J. van den Hondel, Arthur F.J. Ram Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE, Leiden, The Netherlands A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. In this study, we report the characterization of a color mutant, colA, which was isolated because of a complete loss of pigmentation resulting in white conidia. Pigmentation of the colA mutant was restored by a gene that encodes the A. niger ortholog of the 4’-phosphopantetheinyl transferase protein (PptA). 4’phosphopantetheinyl transferase activity is required for the activation of Polyketide synthases (PKSs) and Non-Ribosomal Peptide Synthases (NRPSs). Complementation analysis showed that the colA mutant is allelic to a previously isolated color mutant, gryA. Sequencing of the colA and gryA loci and the targeted deletion of the pptA gene further confirmed that the colA /gryA mutants are mutated in the pptA gene. Spores from the ∆pptA deletion are paler in color that spores of an A. niger strain disrupted in the pksA gene. PksA encodes the polyketide synthase required for melanin biosynthesis and spores from the pksA disruption strain become fawn colored. Spores from both the ∆pksA and the ∆pptA were hypersensitive to UV-radiation indicating that melanin is required for resistance against UV-radiation. The ∆pksA strain was equally sensitive to hydrogen peroxide as the parental strain, but spores from the ∆pptA strain showed increased sensitivity. The results suggest the involvement of PKS or NRPS-derived metabolites that
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, Miyagi, 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. 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
Essentiality of RNA exosome subunit encoding genes in Aspergillus oryzae. Mizuki Tanaka, Takahiro Shintani, and Katsuya Gomi Grad. Sch. Agric. Sci., Tohoku Univ., Japan The exosome is a multi-subunit 3 f-5 f exonucleolytic complex that is conserved in eukaryotes. The ring-shaped core structure of the exosome is constituted of nine subunits. In yeast, all of nine exosome subunits are essential for viability. On the other hand, in plant, Csl4 is dispensable for growth and development, whereas Rrp41 and Rrp4 are essential for the development of female gametophytes and embryogenesis. Since there has been no report on the exosome itself in filamentous fungi, we attempted to construct the disruptants of genes encoding exosome subunits in Aspergillus oryzae. We have chosen two orthologous genes for csl4 and rrp4 as targets for disruption, and successfully obtained a csl4 disruptant but not an rrp4 disruptant. The disruption of csl4 gene had no apparent defect on growth in A. oryzae. Since the rrp4 disruptant could not been obtained, this gene would be essential for cell viability. Thus, we generated the conditional rrp4 expression strain by using the promoter of nmtA, expression level of which is regulated by riboswitch existed within its 5 f-untranslated region and is repressed considerably in the presence of thiamine. The resultant conditional rrp4 expression strain displayed a remarkable growth defect when thiamine was added to the medium. These results suggested that Rrp4 is essential but Csl4 is not for cell growth in A. oryzae and that function of individual exosome components in A. oryzae is similar to that in plant.
Chitinases of Aspergillus niger upregulated during autolysis. Jolanda van Munster1, Benjamin Nitsche2, Rachel van der Kaaij3, Arthur Ram2, Lubbert Dijkhuizen1, Marc van der Maarel1. 1 Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Groningen University, Haren, The Netherlands. 2 Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands. 3 TNO Quality of Life, Dep. Food and Biotechnology Ingredients, Zeist, 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
COMPARATIVE AND FUNCTIONAL GENOMICS
Tools for Exploration of Aspergillus Gene, Protein, and Sequence Information at the Aspergillus Genome Database (AspGD) Martha B. Arnaud1, Jonathan Binkley1, Marcus C. Chibucos2, Maria C. Costanzo1, Jonathan Crabtree2, Diane O. Inglis1, Joshua Orvis2, Prachi Shah1, Marek S. Skrzypek1, Gail Binkley1, Stuart R. Miyasato1, Jennifer R. Wortman2 and Gavin Sherlock1 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 for Aspergillus genomics and molecular biology, with information curated from the literature and web-based research tools for exploration and analysis of these data. The Sybil Comparative Genomics tool displays alignments of the genomic regions encoding clusters of homologous proteins and syntenic regions from A. nidulans, A. fumigatus, A. flavus, A. oryzae, A. niger, A. clavatus, A. terreus, and Neosartorya fischeri. 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, retrieval, analysis, and download of A. nidulans sequence and curated gene and protein information. 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 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 aspergillus-curator@genome.stanford.edu. All of the data in AspGD are freely available to the public from http://www.aspgd.org/. AspGD is supported by grant RO1 AI077599 from the NIAID at the NIH.
High throughput sequencing of the A. nidulans transcriptome. Mark Caddick, Christopher Sibthorp, Huihai Wu, Neil Hall, Prudence Wong. School of Biological Sciences, The University of Liverpool, Crown Street, Liverpool L69 7ZB, UK. Next generation sequencing is transforming the way in which we can analyse the transcriptome. It potentially provides quantitative information which is superior to microarray data, whilst also providing information about differential use of promoters, splicing and transcript 3’ ends. We are applying this approach to A. nidulans. In the first instance we have specifically investigated transcript start site location, by sequencing mRNA 5’ ends. This has provided genome wide data with over ~3500 transcript start sites being localised to within two nucleotide and over 7000 localised to within four nucleotides. From this we have been able to investigate DNA motifs and features associated with transcription start sites. We will present these data and current work focused on characterising the full transcriptome. Our aim is to provide a community resource that will significantly improve genome annotation and greatly increase our understanding of its flexibility and regulation.
Comparison of the polysaccharide degrading ability of 8 Aspergilli. Isabelle Benoit(1), Pedro Coutinho(2), Hala Al-Bushari(1), Guillermo Aguilar(1), Evy Battaglia(1) Birgit Gruben(1), Blanca Trejo-Aguilar(1), Ad Wiebenga(3), Bernard Henrissat(2), Ronald de Vries(1,3). (1)Microbiology, Utrecht University; (2)AFMB, Univ. Aix-Marseille I & II ; (3)CBS-KNAW Fungal Biodiversity Centre. r.devries@cbs.knaw.nl. Plant polysaccharides are among the most prominent carbon sources for fungi, which degrade these substrates through the production of diverse sets of extracellular enzymes. This topic has been best studied in the Aspergilli, in particular in A. niger and A. oryzae due to the many industrial application of plant polysaccharide degrading enzymes and the good fermentative properties of these fungi. The availability of genome sequences for several Aspergilli has allowed detailed comparisons between these species and enables us to identify differences in the strategies that they employ to release their carbon source from crude plant biomass. CAZy-annotation of the genomes revealed significant differences in the sets of hydrolytic enzymes and growth profiling of these fungi demonstrated strong correlations between genome content and ability to degrade specific polysaccharides. In addition, analysis of the secreted enzymes demonstrated further differences that are likely caused by differences in transcriptional or post-transcriptional regulation. Highlights from these results will be presented. These data will help with the further development of improved enzyme cocktails as they enable a link between the composition of the enzyme set and the efficiency with which different polysaccharides are degraded.
Two GATA factors AREA and AREB negatively regulate arginine catabolism genes of Aspergillus nidulans in response to nitrogen and carbon source. Maria Macios, Piotr Weglenski and Agnieszka Dzikowska Institute of Genetics and Biotechnology, Warsaw University ul. Pawinskiego 5A, 02-106 Warsaw, Poland adzik@ibb.waw.pl In A. nidulans arginine is utilised as a nitrogen and carbon source and it is a good system for investigation the connection between the two global carbon and nitrogen repression regulatory systems. Utilization of arginine depends on the presence and inducibility of two arginine catabolic enzymes arginase and ornithine aminotransferase (OAT) encoded by agaA and otaA genes, respectively. Analysis of different single and double areA and areB mutants have shown that two GATA factors AREA and AREB negatively regulate the expression of arginine catabolism genes under nitrogen repression conditions. AREA and AREB activities depend on carbon source. AREA regulator is necessary for the ammonium repression of arginine catabolism genes under carbon repressing conditions while AREB - under carbon limited, non-repressing conditions. AREA activity was shown to be modulated by a direct protein – protein interaction with NMRA protein which is proposed to bind to the C terminus of AREA and repress its activity in the presence of glutamine (Platt et al., 1996; Andrianopoulos et al., 1998; Lamb 2003 et al., Lamb et al., 2004; Wong et al., 2007). We have shown that these interactions are also important in nitrogen metabolite repression of arginine catabolism genes.
Curation of Aspergillus gene and protein information at the Aspergillus Genome Database (AspGD) Diane Inglis1, Martha Arnaud1, Jon Binkley1, Maria Costanzo1, Marcus Chibucos2, Jonathan Crabtree2, Joshua Orvis2, Prachi Shah1, Marek Skrzypek1, Gail Binkley1, Stuart Miyasato1, Jennifer Wortman2 and Gavin Sherlock1 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 (www.aspgd.org) 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, phenotypes, and Gene Ontology (GO) annotations, as well as tools for exploring these data. The curated information for each gene appears on its Locus Summary page with links to details pages that provide additional information, including phenotype and GO details, sequence and annotation history and a comprehensive list of references. Initially, we have focused on the manual curation of genomic information for Aspergillus nidulans, the best- characterized species of the group. In the future, 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. AspGD is supported by grant RO1 AI077599 from the NIAID at the NIH.
RRMA, an RNA binding protein involved in regulated mRNA degradation. Kinga Krol1, Igor Y. Morozov2, Piotr Weglenski1, Massimo Reverberi3, Mark X. Caddick2, Agnieszka Dzikowska1. 1Institute of Genetics and Biotechnology, University of Warsaw, Poland 2School of Biological Sciences, The University of Liverpool, UK 3Plant Biology, Universita La Sapienza, Roma, Italy kinga@igib.uw.edu.pl RRMA is the RNA binding protein involved in posttranscriptional regulation of gene expression in Aspergillus nidulans. rrmA gene was identified as a suppressor of mutations in arginine/proline catabolic pathway. Independently RRMA protein was shown to bind to the 3’UTR of areA transcript (nitrogen positive regulator). ΔrrmA mutation results in slow growth phenotype and higher sensitivity to oxidative stress. Analysis of main antioxidant enzymes revealed different activity pattern during early development stages in ΔrrmA strain comparing to the control strain. Transcriptional analysis has shown that ΔrrmA mutation results in higher stability of specific transcripts under conditions of oxidative stress and nitrogen starvation. Our results indicate that RRMA plays important role in metabolism of A. nidulans and can be involved in the mechanism of regulated degradation of specific mRNAs in response to specific signals.
Comparative analysis of koji mold's genomes Masayuki Machida1*, Hideaki Koike1*, Ikuya Kikuzato2*, Kazuhiro E. Fujimori1*, Morimi Teruya3*, Masatoshi Tsukahara4*, Yumi Imada4*, Youji Wachi2*, Yuki Satou2*, Yukino Miwa4*, Shuichi Yano4*, Yutaka Kawarabayasi1*, Osamu Yamada5, Koji Jinno6, Hiroshi Horikawa6, Akira Hosoyama6, Takasumi Hattori7, Motoaki Sano8, Koichi Tamano1, Kazurou Fukuda9, Takaomi Yasuhara9, Kenichi Higa7, Shinichi Ohashi5, Kotaro Kirimura4, Masanori Arita10, Kiyoshi Asai10, Keietsu Abe11, Katsuya Gomi11, Shigeaki Mikami5, Takeaki Ishikawa12, Kaoru Nakasone13, Nobuyuki Fujita6, Takashi Hirano1* (1Natl. Inst. Advanced Inst. Sci. Technol., 2Okinawa Sci. Technol. Promotion Center, 3Okinawa Iind. Technol. Center, 4Tropical Technol. Center, 5Natl. Res. Inst. Brewing, 6Natl. Inst. Technol. Eval., 7Waseda U., 8Kanazawa Inst. Technol., 9Asahi Breweries, 10U. Tokyo, 11Tohoku U., 12Brewing Soc. Japan, 13Kinki U., *Okinawa Cutting-edge Genome Project) Koji mold is the traditional name of Aspergillus species that are used for Japanese fermentation industries. Aspergillus oryzae has been widely used in Japanese fermentation industries, Japanese alcohol beverage, soy sauce and so on for longer than a thousand of years. Comparison of A. oryzae genome with those of other Aspergillus species of smaller genome size revealed existence of non-syntenic blocks (NSBs) specific to the A. oryzae genome. Aspergillus awamori is another industrial filamentous fungus, widely used for brewing Japanese traditional spirits, Awamori, in Okinawa prefecture. We have sequenced A. awamori NBRC 4314 (RIB 2604). The total length of non-redundant sequences reached 34.7 Mb consisting of contigs fallen into 44 major linkage groups. High potential of secretory production of proteins has led A. oryzae and A. awamori to extensive use also in modern biotechnology. A. awamori is genetically very close to Aspergillus niger and close to A. oryzae. Like A. niger, A. awamori vigorously produces citric acid, lowering pH of the product. A. awamori is genetically very close to Aspergillus niger. However, mapping of short reads from A. awamori by SOLiD revealed that the species have remarkable difference. Comparison between the genomes of A. awamori and A. oryzae showed higher diversity of genes located on the non-syntenic blocks of the A. oryzae genome. We are currently sequencing various strains of A. awamori by SOLiD. Analysis of the relationship between genetical and phenotypical differences among the strains should provide important information for gene function.
Targeted functional proteomics: A putative translation elongation factor with glutathione s-transferase activity protects Aspergillus fumigatus against oxidative stress. Gráinne O’ Keeffe, Christoph Jöchl, Kevin Kavanagh and Sean Doyle National Institute for Cellular Biotechnology and Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland. Sequencing of the Aspergillus fumigatus genome has led to an increased understanding of the organism; however the functions of many genes remain unknown. A putative translation elongation factor 1Bgamma (EF1Bgamma, termed elfA; 750 bp) is expressed, and exhibits glutathione s-transferase activity, in A. fumigatus [1]. Normally, EF1Bgamma 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 (deltaelfA) was complemented using a hygromycin resistance marker (hph). Southern Blot analysis was used to confirm the generation of deltaelfA and the complemented strain. RT-PCR confirmed the expression of elfA in wild-type and complemented strains, and absence of expression in deltaelfA. The availability of the mutant has facilitated phenotypic analysis of elfA functionality. A. fumigatus wild-type and deltaelfA were grown on AMM plates with the oxidant H2O2 (1 - 5 mM), voriconazole (0.25 - 1 microg/ml), and the thiol-reactive reagent, 4,4’-dithiodipyridine (3 - 7.5 microM). At 37̊C, the elfA mutant was significantly more sensitive (p=0.0003) to H2O2 and (p=0.0056) to 4,4’-dithiodipyridine than wild-type. However, deltaelfA was significantly less sensitive (p=0.0251) to voriconazole 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.
OXYLIPINS EXCHANGE DURING THE INTERACTION BETWEEN A. OCHRACEUS AND TRITICUM DURUM SEEDS Scarpari M.1, Reverberi M.1, Punelli F.1, Zjalic S.1, Ricelli A.2, Fabbri A.A.1, and Fanelli C1. 1Department of Plant Biology, Rome University Sapienza, Roma, Italy; 2 ICB, CNR, Roma, Italy. marzia.scarpari@uniroma1.it The oxylipin metabolism controls mycotoxin biosynthesis, conidiogenesis, sclerotia formation and the interaction with the host in Aspergillus nidulans , A. flavus and A. parasiticus. The subsequent finding of a lox -like gene sequence (AoloxA; DQ087531) in the genome of A. ochraceus indicate that also in this fungus oxylipin metabolsim can play a pivotal role in controlling different pathways. The AoloxA deleted mutant displays a different colony morphology with a remarkable delay in conidia formation and an induction of sclerotia development. AoloxA (-) shows oxylipins biosynthetic pathways switching from 13-HPODE to a prevalent formation of 7,8 and 8,13-DiHODE. A large number of sclerotia, which are considered to be vestigial of the sexual cleistotecia are formed in vitro by AoloxA (-), possibly due to an incretion of diols formation. Further, the reduced oxylipin formation induce a strong reduction of OTA biosynthesis in comparison with the wild type. The seeds of T. durum cv Ciccio contaminated with AoloxA (-) did not accumulate 9-hydroperoxyoctadecadienoic acid and did not express PR1 mRNA whereas WT stimulated both these events. The generalized down-regulation of oxylipins synthesis in AoloxA (-)grown on wheat seeds also confirms the existence of a cross-talk between wheat seeds and A. ochraceus mediated by oxylipins. In conclusion, we suggest that similarly to A. nidulans , A. flavus and A. parasiticus, some of the mechanisms that drive toxin biosynthesis and morphogenesis, which are modulated by fatty acids oxidation by-products, are active in A. ochraceus.
GENE REGULATION
Efficient gene targeting in Aspergillus niger using a transiently disrupted ku70 gene Mark Arentshorst, Arthur F.J. Ram and Vera Meyer Leiden University, IBL, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, The Netherlands. m.arentshorst@biology.leidenuniv.nl Homologous recombination frequencies in filamentous fungi, including Aspergillus niger, are low. Several reports over the last years have shown that mutants defective in the Non-Homologous-End Joining (NHEJ) pathway display increased homologous integration efficiencies up to 80 to 100%. Recently, we described that deletion of the A. niger kusA gene, encoding the orthologue of the Ku70 protein in other eukaryotes, dramatically improved homologous integration efficiency (1). However, deletion of kusA also causes increased sensitivity of A. niger towards UV and X-ray and the consequences of loss of kusA in relation to DNA repair and genome stability are currently unknown. To avoid any potential side effects of a kusA loss-of-function mutation on growth and viability of A. niger, we transiently disrupted kusA. We made use of the counter selectable amdS marker, flanked by 300 bp direct repeats of the kusA gene. Disruption of the kusA gene resulted in similar homologous recombination frequencies compared to the deltakusA strain. After completion of the gene targeting approach, we re-established an intact kusA copy using counter selection on fluoroacetamide, proven by sequencing of the kusA locus. In A. niger, which lacks an sexual reproduction cycle, the transient disruption system is especially important as the NHEJ pathway cannot be restored by performing a sexual cross. References: 1. Meyer, V., Arentshorst, M., El-Ghezal, A., Drews, A.C., Kooistra, R., van den Hondel, C.A.M.J.J., Ram, A.F.J. Journal of Biotechnology, 128 (4), 770-5. 2007.
Significant differences in pentose regulation between two Aspergilli Evy Battaglia1, Anne Leendertse1, Ronald P. de Vries1, 2 1Microbiology, Utrecht University, Utrecht, Netherlands 2CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands e.battaglia@uu.nl D-xylose and L-arabinose are highly abundant components of plant biomass and therefore major carbon sources for many fungi. Fungi produce extracellular enzymes to release these sugars, which are subsequently taken up into the cell and converted through the pentose catabolic pathway. In Aspergillus niger and Aspergillus nidulans, D-xylose release and part of the pentose catabolic pathway are regulated by the transcriptional activator XlnR. In Aspergillus niger, we recently described the transcriptional activator AraR, a homologue of XlnR that controls L-arabinose release and the pentose catabolic pathway. Analysis of A. nidulans AraR showed that it is also involved in pentose (L-arabinose) catabolism in this fungus. However, the growth phenotype of the delta araR from these two species showed significant differences. A. niger delta araR is not able to grow on L-arabinose, whereas for A. nidulans delta araR growth is not affected. The reverse was observed for L-arabitol. We will present data on the molecular difference between the regulation of AraR and the interaction with XlnR in these 2 Aspergilli by analysis of expression of genes of the pentose catabolic pathway. We will demonstrate significant evolutionary differences in regulation of the pentose metabolism, and will hypothesize about the implications hereof for other sugar catabolic pathways.
Implication of FarA transcriptional factor in the expression of lipolytic genes in Aspergillus oryzae Sharon Marie Garrido1, Noriyuki Kitamoto2, Akira Watanabe1, Takahiro Shintani1, and Katsuya Gomi1 1Graduate School of Agricultural Science, Tohoku University, Japan; 2Food Research Center, Aichi Industrial Technology Institute, Japan. FarA is a Zn2Cys6 transcription factor which upregulates genes required for growth on fatty acids in filamentous fungi like Aspergillus nidulans. Homology of all the amino acid sequences and the homology of Zn2Cys6 motifs of FarA between the A. oryzae and the A. nidulans are 83% and 97.5%, respectively. This study determines whether FarA transcriptional factor also works in the regulation of genes responsible for the production of lipolytic enzymes including cutinase particularly in A. oryzae. FarA disruptant was constructed (deltafarA:sC) and confirmed by Southern blotting analysis. The wild-type (WT) and the disruptant strains were grown and induced in minimal liquid medium with 2% of flaxseed oil at different time intervals. Supernatants were analyzed for Western blot and the WT showed the presence of cutinase (CutL1) protein while the disruptant did not. In addition, qRT-PCR and RT-PCR revealed that the expression of cutL1 gene was significantly reduced in the disruptant compared to the WT. Difference of monoacylglycerol lipase gene (mdlB) expression was also found between the WT and the disruptant. These results indicated that the FarA transcriptional factor may be implicated in the gene expression of lipolytic enzymes including cutinase. Acknowledgement: I would like to extend my gratitude to the Federation of European Microbiological Societies and to the ECFG10 Organizing Committee for
RacA is required for actin distribution and affects the localization of the exocytosis machinery in the filamentous fungus Aspergillus niger M. J. Kwon, M. Arentshorst, C. van den Hondel, V. Meyer, and A. Ram Leiden University, IBL, Molecular Microbiology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands Tip growth in filamentous fungus requires coordination of basic cellular processes in the cell such as exocytosis, polarity maintenance, endocytosis and cell wall biosynthesis. The small GTPase RacA, the fungal orthologue of the human Rac1 protein is important for fungal growth. In the absence of RacA, apical dominance of tip growth is lost resulting in apical branching and a hyperbranching phenotype. Actin patches in the racA deletion mutant were found to be hyperpolarized at the extreme apex, while in the wild type strain a smoother gradient of actin patches towards the tip was observed. GFP-RacA localizes to the plasma membrane at the extreme apex of growing hyphae probably, marking the site of exocytosis. To understand the function of RacA in relation to exocytosis, secretory vesicles were visualized by tagging the V-SNARE (SynA) with GFP (GFP-SynA). In wild type cells, GFP-SynA is present on intracellular structures representing secretory and/or endocytotic vesicles. High levels of GFP-SynA are also present in the Spitzenkörper. Finally, GFP-SynA is localized in the plasma membrane. Fluorescence of GFP-SynA at the plasma membrane is highest at the extreme apex (the sites of exocytosis) and decreases in subapical regions. In the racA deletion mutant, SynA localization was similar to the wild type, but the intensity of the signal was less. Whereas the tips of wild type hyphae display a ~10-15 µm gradient of GFP-SynA protein, tips in the ¥ÄracA show a much shorter (< 5 µm) gradient of GFP-SynA. We conclude that the A. niger RacA protein is necessary for precise actin localisation and distribution in hyphal tip cells and that the gradient of GFP-SynA towards the tip is actin dependent.
The RmsA protein – a hub in the protein interaction networks of Aspergillus niger? Vera Meyer, Susann Minkwitz, Tabea Schütze, Cees A.M.J.J van den Hondel, Arthur F.J. Ram Leiden University, Institute of Biology, Department Molecular Microbiology and Biotechnology & Kluyver Centre for Genomics of Industrial Fermentation, The Netherlands Many cells and organisms go through polarized growth phases during their life. Cell polarization is achieved by local accumulation of signaling molecules which guide the cytoskeleton and vesicular trafficking to specific parts of the cell and thus ensure polarity establishment and maintenance. Polarization of signaling molecules is also fundamental for the lifestyle of filamentous fungi such as Aspergillus niger and essential for their morphogenesis, development and survival under environmental stress conditions. Considerable advances in our understanding on the protagonists and processes mediating polarized growth in filamentous fungi has been made over the past years. However, how the interplay of different signaling pathways is coordinated has yet to be determined. We found recently that the A. niger RmsA protein is central for the polarization of actin at the hyphal tip (1). However, we show here that RmsA is also of vital importance for the metabolism, viability and stress resistance of A. niger. This suggests that RmsA could occupy an important position in the global network of pathways that balance growth, morphogenesis and survival of A. niger. 1. Meyer V, Arentshorst M, Flitter SJ, Nitsche BM, Kwon MJ, Reynaga-Pena CG, Bartnicki-Garcia S, van den Hondel CA, Ram AF (2009) Reconstruction of signalling networks regulating fungal morphogenesis by transcriptomics. Eukaryot Cell 8: 1677-1691
Transposon mutagenesis using a resident DNA transposon Crawler in Aspergillus oryzae Hironobu Ogasawara1, Tsutomu Satoh2, Hiroshi Konno2, Yoji Hata3, Saori Takahashi1, and Katsuya Gomi4 1 Akita Res. Inst. Food and Brewing, Akita, Japan. 2 Akita Konno Co. Ltd., Akita, Japan, 3Research Institute, Gekkeikan Sake Co. Ltd., Kyoto, Japan. 4 Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. E-mail:hironobu@arif.pref.akita.jp An active DNA transposon Crawler isolated from the genome of industrially important fungus Aspergillus oryzae transposes under extreme stress conditions. The DNA sequencing surveys revealed that the Crawler element is widely distributed among A. oryzae and A. sojae strains, which are commonly used in Japanese traditional fermentation manufacturing. In the present study, @we analyzed the relationship between various stress stimuli and inhibition of cryptic splicing of the Crawler mRNA by qRT-PCR to enhance the frequency of Crawler-mediated mutagenesis in an A. oryzae industrial strain, AOK139. Under the optimized stress conditions, in which conidiospores were treated in 20mM CuSO4 or 52 @for 6hr, various phenotypic mutants different from the parent strain were isolated. Those exhibited white color in conidiospores, less number of spores formed, shortened aerial hyphae, thin colony mat and so on. DNA sequencing analyses of a white conidia mutant revealed that Crawler was newly inserted within a coding region of wA gene encoding polyketide synthetase, which resulted in wA deficiency. The insertion occurred also at a TA site with duplication according to the manner of Crawler transposition. These results suggested that transposon mutagenesis using active Crawler is potentially valuable to improve characteristics of A. oryzae industrial strains.
Transposon mutagenesis using a resident DNA transposon Crawler in Aspergillus oryzae Hironobu Ogasawara1, Tsutomu Satoh2, Hiroshi Konno2, Yoji Hata3, Saori Takahashi1, and Katsuya Gomi4 1 Akita Res. Inst. Food and Brewing, Akita, Japan. 2 Akita Konno Co. Ltd., Akita, Japan, 3Research Institute, Gekkeikan Sake Co. Ltd., Kyoto, Japan. 4 Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. E-mail:hironobu@arif.pref.akita.jp An active DNA transposon Crawler isolated from the genome of industrially important fungus Aspergillus oryzae transposes under extreme stress conditions. The DNA sequencing surveys revealed that the Crawler element is widely distributed among A. oryzae and A. sojae strains, which are commonly used in Japanese traditional fermentation manufacturing. In the present study, @we analyzed the relationship between various stress stimuli and inhibition of cryptic splicing of the Crawler mRNA by qRT-PCR to enhance the frequency of Crawler-mutagenesis in A. oryzae AOK139 strain. Under the optimized stress conditions, in which conidiospores were treated in 20mM CuSO4 or 52 @for 6hr, various phenotypic mutants different from parent AOK139 were isolated. Those exhibited white color in conidiospore, less number in spore formation, shortened aerial hyphae, thin colony mat and so on. DNA sequencing analyses of a white conidia mutant revealed that Crawler newly inserted within a coding region of wA gene(polyketide synthetase) in reverse direction, which would cause wA deficiency. The insertion occurred also at TA site with duplication according to the manner of Crawler transposition. These results suggested that transposon mutagenesis using active Crawler is potentially valid to improve characteristics of A. oryzae industrial strains.
The transcription activator AtrR regulates gene expression of ABC transporters and contributes to azole drug resistance in Aspergillus fumigatus. Ayumi Ohba1, Kiminori Shimizu2, Takahiro Shintani1, Susumu Kawamoto2, Katsuya Gomi1. 1Graduate School of Agricultural Science, Tohoku university, Japan 2Medical Mycology Research Center, Chiba University, Japan Previously, we demonstrated that a novel transcriptional factor ‘AtrR’ regulated gene expression of these ABC transporters that would function as drug efflux pumps and contributes to the azole resistance in A. oryzae. Orthologous genes of the A. oryzae atrR have been found widely in genomes of Aspergilli, including A. fumigatus, A. nidulans, and A. niger. In the present study, we constructed a disruption mutant of the atrR ortholog in A. fumigatus and investigated the role of the gene in drug resistance. The mutant was similarly hypersensitive to azole drugs, especially susceptible to fluconazole that is not effective to Aspergillus fungi. The mutant also showed reduced expression level of several ABC transporter genes that would function as drug efflux pumps. These results indicate that the transcription factor AtrR also regulates gene expression of ABC transporters and contributes to azole resistance in A. fumigatus. In addition, this suggests that fluconazole resistance of Aspergillus fungi is attributed to the expression of drug efflux ABC transporters regulated by AtrR. This transcriptional factor AtrR provides novel therapeutic targets for the treatment of azole drug resistant fungal
Blue- and red-light photoreceptors regulate the activation by light of conidiation genes in Aspergillus nidulans.Carmen Ruger Herreros, Raúl Fernández-Barranco, María Olmedo,Luis M.Corrochano,David Cánovas.Department of Genetics, University of Seville,Spain The ascomycete A.nidulans is a model organism to study fungal development.Conidiation is controlled by the product of the brlA gene.Many gene products act upstream of brlA,probably allowing the synthesis of chemicals or allowing the transduction of environmental signals to trigger brlA transcription.The A.nidulans genome contains genes for a phytochrome (fphA),two homologs of N.crassa WC-1 and WC-2 (lreA and lreB) and a veA gene. Red and blue light stimulate conidiation in A.nidulans,but mutations in the veA gene allow conidiation in the dark.Recently,it has been shown that the phytocrome FphA interacts with VeA and LreA and LreB.However,the mechanism that the photoreceptors employ to activate conidiation remains unknown.We have found that the expression of several conidiation genes,including brlA,fluG,flbA,flbB and flbC,was regulated by light.The photoactivation of these genes showed a quick reponse with mRNA accumulation increasing after 5 minutes of illumination.brlA mRNA accumulation after illumination increased with time showing maximum values between 30 to 60 minutes and revealed a two-component activation.Deletion of the photoreceptor genes fphA,lreA and lreB reduced the activation by light of the studied genes.None of these genes are essential for gene photoactivation since we observed light-dependent mRNA accumulation in strains with single deletion of photoreceptor genes.On-going experiments provides a model for the light-dependent activation of conidiation.
Dynamic and functional interactions between the components of the trimeric velvet complex in the filamentous fungus Aspergillus nidulans Özlem Sarikaya Bayram1, Özgür Bayram1, Oliver Valerius1, Jennifer Gerke1, Stefan Irniger1, Kap- Hoon Han2, and Gerhard. H Braus1 1 Department of Molecular Microbiology & Genetics, Georg August University, Goettingen, Germany. 2Pharmaceutical Engineering, Woosuk University, Korea. We have recently discovered the trimerinc velvet complex which is comprised of the light-dependent regulator VeA, Velvet-like protein VelB, and master regulator of secondary metabolism LaeA. The velvet complex coordinates development and secondary metabolism upon light signal in Aspergillus nidulans. VeA protein serves as a light-dependent bridging function between VelB and LaeA proteins. We are currently analysing the functional as well as physical relationships between the components of the velvet complex. First data suggest that there might be some subcomplexes regulating development.
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, Department Molecular Microbiology and Biotechnology & Kluyver Centre for Genomics of Industrial Fermentation, The Netherlands 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.
OTHER TOPICS
Impact of Grape Berry Resveratrol on the growth of A. carbonarius and on the biosynthesis of ochratoxin A Patrizia De Rossi1, Alessandra Ricelli2, Massimo Reverberi3, Isabella Nicoletti4, Cristiano Bello4, Antonella De Rossi4, Danilo Corradini4, Anna Adele Fabbri3, and Corrado Fanelli 3 1ENEA, CR-CASACCIA, Roma, Italy 2Universita degli Studi Sapienza, Roma, Italy 3ICB-CNR, Roma, Italy 4Istituto di Metodologie Chimiche, CNR MonteRotondo Stazione, Italy Corresponding author: patrizia.derossi@enea.it 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. 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 30̊C. 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
Development of a novel inducible expression system for the production of heterologous proteins in Aspergillus: successful production of lignolytic enzymes. Sandra de Weert1, Nick van Biezen2, Dennis Lamers2, Jeroen Schouwenberg2, Kees van den Hondel1,2, Arthur Ram1 and Christien Lokman1,2 1 Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands 2 HAN BioCentre, P.O. Box 6960, 6503 GL Nijmegen, the Netherlands For industrial applications and (medical) scientific research efficient(heterologous) production of compounds like, enzymes, antibodies, viral epitopes, chemical compounds and antibiotics is of great importance. For these purposes Aspergillus functions as a suitable production host. Over the years all kinds of commercially available expression systems have been developed. A well established expression system is the one based on the protein Glucoamylase (GlaA). However, in the case of heterologous protein production the efficiency of these systems is still very depending on the protein to be produced. Recently, we identified the inuE gene, encoding for the exo-inulinase protein in A. niger, as the most strongly induced gene in the presence of inulin and sucrose. Characteristics of the system were studied by placing gfp behind the inuE promoter. This reporter strain showed that the inuE gene is highly expressed when grown on inulin and sucrose. No expression was observed when grown on glucose, fructose and xylose indicating a tight control on different carbon sources. This tight control can be a benefit if the heterologous protein to be produced can be a disadvantage for fungal growth.
Gliotoxin- exploiting its anti-fungal effects. Emer Molloy, Stephen Carberry, Markus Schrettl, Gary Jones, Karen O'Hanlon, Kevin Kavanagh and Sean Doyle. Biology, NUI Maynooth, Co. Kildare, Ireland. Aspergillus fumigatus biosynthesizes gliotoxin, yet little is known about the effect of gliotoxin on A. fumigatus. Exposure of A. fumigatus to gliotoxin (14 microg/ml) for 3 hours results in the de novo expression of Cu,Zn SOD, a short chain dehydrogenase and a eukaryotic translation elongation factor (beta1 subunit). Exposure also significantly altered the expression (up and down-regulated > 2x) of 27 other proteins including (i) upregulated: GliT (AFUA_6G09740; reported elsewhere), adenine phosphoribosyl transferase and allergen Asp f3 and (ii) down-regulated: catalase and a peroxiredoxin. Targeted deletion of gliT resulted in hypersensitivity of A. fumigatus deltagliT to gliotoxin (reported elsewhere). Re-introduction of gliT into A. fumigatus deltagliT, using a split marker approach, at a different locus (ctsD;AFUA_4G07040) with selection on gliotoxin-containing media, resulted in deletion of ctsD. Absence of ctsD expression was accompanied by restoration of gliT expression (RT-PCR), and resistance to gliotoxin. Significant (P < 0.001) gliotoxin-mediated growth inhibtion of A. nidulans, A. terreus, A. niger, Cochliobolus heterostrophus and Neurospora crassa was observed at 5 microg/ml gliotoxin. Growth of A. flavus, Fusarium graminarium and A. oryzae was significantly inhibited (P < 0.001) at 10 microg/ml gliotoxin. Potentially, all of these fungal species are suitable for transformation with gliT to facilitate transformant selection on gliotoxin.
Utilization Of Exogenous Heme By Aspergillus niger Angelique C.W. Franken1, B. Christien Lokman2, Arthur F.J. Ram1, Cees A.M.J.J. van den Hondel1,2, Sandra de Weert1 1) Institute of Biology Leiden, Leiden University, Molecular Microbiology & Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands, 2) HAN Biocentre, Laan van Scheut 2, 6525 EM Nijmegen, 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.
Enzymatic Hydrolysis of Lignocelluloses: Identification of Novel Cellulases Genes from Filamentous Fungi Marta Kolasa, Birgitte K. Ahring, Peter S. Lübeck, and Mette Lübeck Copenhagen Institut of Technology, Aalborg University, Denmark Lignocellulosic materials form a huge part of the plant biomass. Cellulose can be degraded to simple sugar components by means of enzymatic hydrolysis. However, due to its complex, crystalline structure it is difficult to break it down and the cooperative action of a variety of cellulolytic enzymes is necessary. Fungi are known to have potential in production of a variety of cellulolytic enzymes. The aim of this work is to discover new thermostable and robust cellulolytic enzymes for improved enzymatic hydrolysis of biomass. For this purpose two screening methods are applied in different fungal strains with high cellulolytic activities: an expression-based method using suppression subtractive hybridization and a targeted genomic screening approach using degenerate PCR. Suppression subtractive hybridization facilitates identification of genes encoding cellulolytic enzymes that are expressed when cultivating a fungal strain in medium with cellulose as the carbon source. By means of degenerate PCR, specific genes, homologous to the genes of previously classified glycoside hydrolases from CAZY database, are searched for in selected strains of Aspergillus sp., Trichoderma sp. and Penicillium sp. Both methods are anticipated to facilitate identification of target genes which subsequently will be cloned and expressed in a relevant fungal host for further characterization of the expressed enzymes. The goal is to introduce new enzymes to industrial processes.
Biochemical producing fungi Lena Nilsson, Birgitte K. Ahring, Mette Lübeck and Peter S. Lübeck Section for Sustainable Biotechnology, Copenhagen Institute of Technology, Aalborg University. We are in the process of developing a biorefinery concept for the use of selected plant biomasses for production of high value biochemicals aiming at replacing chemicals produced from fossil fuels. One aspect will be the engineering of efficient biomass converting fungal strains with the ability to produce high amounts of specific organic acids. Aspergillus niger producing citric acid is a classical example of industrial application of fermentative processes in a filamentous fungal strain. We would like to utilise both the large potential for secretion of hydrolytic enzymes and the organic acid producing machinery of the filamentous fungi for further genetic engineering. We anticipate to initiate the engineering by manipulating central pathways in carbon metabolism i.e. glycolysis to increase the funnelling of sugars to acid production. By initial screening of a large collection of fungal strains isolated from natural habitats we have identified isolates with high production and excretion of organic acids. Among these are several Aspergillus species and one Penicillium sp. with higher excretion of TCA-cycle intermediates, and these strains have been chosen for genetic engineering.
A systems biology approach towards itaconic acid production in Aspergillus An Li, Nicole van Luijk, Martien Caspers, Marloes ter Beek, Jan Jore, Mariet van der Werf, Peter J. Punt TNO Quality of life, Zeist, The Netherlands The black filamentous fungi Aspergillus niger has a long tradition of safe use in the production of enzymes and organic acids, and is widely used in biotechnology as host for the production of food ingredients, pharmaceuticals and industrial enzymes. Besides, Aspergillus niger grows on a wide range of substrates under various environmental conditions. In our research we have addressed the production of one of the commercially interesting building-block organic acids, itaconic acid. To unambiguously identify the itaconic acid biosynthetic pathway several parallel approaches were taken using Aspergillus terreus as parental host strain. Using a combination of controlled fermentation design, reversed genetics and transcriptomics approaches the pathway specific cis-aconitate decarboxylase (CAD) encoding gene was identified. More specifically, data analysis from the transcriptomics study show that the cis-aconitate decarboxylase (CAD) gene and its clustered genes (Class I) are the most highly expressed ones related to itaconate production. Expression of the CAD gene in E.coli proved that this gene encodes cis-aconitate decarboxylase. Further more, expression of the CAD gene in Aspergillus niger resulted in the production of itaconate in the fermentation medium. Further genetic modifications of the itaconic acid metabolic pathway and fermentation medium improvement were initiated to improve itaconate levels.
OXYLIPINS EXCHANGE DURING IN THE INTERACTION BETWEEN A. OCHRACEUS AND TRITICUM DURUM SEEDS Scarpari M.1, Reverberi M.1, Punelli F.1, Zjalic S.1, Ricelli A.2, Fabbri A.A.1, and Fanelli C1. 1Department of Plant Biology, Rome University Sapienza, Roma, Italy; 2 ICB, CNR, Roma, Italy. marzia.scarpari@uniroma1.it The oxylipin metabolism controls mycotoxin biosynthesis, conidiogenesis, sclerotia formation and the interaction with the host in Aspergillus nidulans , A. flavus and A. parasiticus . The subsequent finding of a lox -like gene sequence (AoloxA; DQ087531) in the genome of A. ochraceus indicate that also in this fungus oxylipin metabolsim can paly a pivotal role in controlling different pathways. The AoloxA deleted mutant displays a different colony morphology with a remarkable delay in conidia formation and an induction of sclerotia development. AoloxA (-) shows oxylipins biosynthetic pathways switching from 13-HPODE to a prevalent formation of 7,8 and 8,13-DiHODE. A large number of sclerotia, which are considered to be vestigial of the sexual cleistotecia are formed in vitro by AoloxA (-), possibly due to an incretion of diols formation. Further, the reduced oxylipin formation induce a strong reduction of OTA biosynthesis in comparison with the wild type. The seeds of T. durum cv Ciccio contaminated with AoloxA (-) did not accumulate 9-hydroperoxyoctadecadienoic acid and did not express PR1 mRNA whereas WT stimulated both these events. The generalized down-regulation of oxylipins synthesis in AoloxA (-) grown on wheat seeds also confirms the existence of a cross-talk between wheat seeds and A. ochraceus mediated by oxylipins. In conclusion, we suggest that similarly to A. nidulans , A. flavus and A. parasiticus , some of the mechanisms that drive toxin biosynthesis and morphogenesis, which are modulated by fatty acids oxidation by-products, are active in A. ochraceus .
Investigating the Regulation of GliA Expression in Aspergillus fumigatus. Taylor Schoberle1 and Gregory S May1. 1University of Texas M.D. Anderson Cancer Center, Houston, Texas. Gliotoxin, a member of the epipolythiodioxopiperazine (ETP) class of toxins, is important to virulence in certain host models. Most genes involved in gliotoxin production and transport are located on a gene cluster, which is co-transcribed. Deletion of an essential gene within the gliotoxin biosynthetic pathway, gliP, led to a lack of gliotoxin production within A. fumigatus, as well as a significant reduction in virulence in a steroid treated host. Reduced virulence was a result of the presence of neutrophils within the host, as other labs using neutropenic mouse models did not see this trend. In microarray studies, gliA, the gliotoxin efflux pump, is induced over 30-fold in the presence of neutrophils. Growth of A. fumigatus in medium containing sodium nitrate also significantly induces expression of gliA. Although many studies have been done to elucidate the effects of gliotoxin on host cells, little is known about the expression of the gliotoxin cluster. To identify cis-acting regulatory elements in the gliA promoter, we examined expression from promoter deletion mutants fused to lacZ. Several positive and negative regulatory elements were identified that altered expression in a nitrogen source dependent manner. Using a gliA promoter lacZ fusion reporter construct, we screened for activators of gliA expression and identified candidate plasmids that activate the reporter. We are conducting additional experiments to investigate this regulation. Elucidating the genes that are responsible for the regulation of gliA will lead to a greater understanding of gliotoxin synthesis and transport, which is important to the pathogenesis of A. fumigatus.
Cloning, expression, and characterization of beta-glucosidase genes from a black Aspergillus spp. Annette Sorensen, Peter S. Lubeck, Mette Lubeck, and Birgitte K. Ahring, Section for Sustainable Biotechnology, Copenhagen Institute of Technology, Aalborg University, Denmark, aso@bio.aau.dk Through a broad cellulolytic activity screening of fungi isolated from various locations, a very promising fungus, identified as a black Aspergillus, was selected for further studies. An enzyme extract of this fungus was obtained through solid state fermentation and tested to profile its beta-glucosidase activity. Four beta-glucosidase genes from this fungus have been cloned through pcr with degenerate primers designed from conserved motif regions of known beta- glucosidase genes from Aspergilli followed by genome walking strategies. The goal is to have these genes expressed in Trichoderma reesei. An expression system was constructed with the constitutive ribosomal promoter RP27 for expression of the his-tagged beta-glucosidase genes and with the hygB gene as selection marker. Analysis of the different transformants is currently on-going and subsequently the different beta-glucosidase proteins will be purified for detailed analysis of specific activity, Km, sugar tolerance, thermostability as well as their ability to break down shorter chained oligosaccharides.
Cloning, expression, and characterization of beta-glucosidase genes from a black Aspergillus spp., Annette Sorensen, Peter S. Lubeck, Mette Lubeck, and Birgitte K. Ahring, Section for Sustainable Biotechnology, Copenhagen Institute of Technology, Aalborg University, Denmark, aso@bio.aau.dk Through a broad cellulolytic activity screening of fungi isolated from various locations, a very promising fungus, identified as a black Aspergillus, was selected for further studies. An enzyme extract of this fungus was obtained through solid state fermentation and tested to profile its beta-glucosidase activity. Four beta-glucosidase genes from this fungus have been cloned through pcr with degenerate primers designed from conserved motif regions of known beta- glucosidase genes from Aspergilli followed by genome walking strategies. The goal is to have these genes expressed in Trichoderma reesei. An expression system was constructed with the constitutive ribosomal promoter RP27 for expression of the his-tagged beta-glucosidase genes and with the hygB gene as selection marker. Analysis of the different transformants is currently on-going and subsequently the different beta-glucosidase proteins will be purified for detailed analysis of specific activity, Km, sugar tolerance, thermostability as well as their ability to break down shorter chained oligosaccharides.
POPULATION AND EVOLUTIONARY GENETICS
Examination of species of Aspergillus Section Nigri for fumonisin production and presence of the fumonisin biosynthetic gene fum8. Susca A.1, Proctor R.H.2, Mulè G.1, Stea G.1, Ritieni A.3, Logrieco A.1, and Moretti A.1 1Institute of Sciences of Food Production - CNR, Bari, Italy 2National Center for Agricultural Utilization Research, Peoria, Illinois, USA 3Università degli Studi di Napoli “Federico II”, Italy Fumonisins are mycotoxins associated with cancer and several other serious diseases in humans and animals. Production of the mycotoxins has been reported for over two decades in Fusarium species, but has been reported only recently in strains of Aspergillus niger. In addition, a homologue of the fumonisin biosynthetic gene (FUM) cluster, originally identified in Fusarium verticillioides, has been identified in the genome sequence of A. niger. Here, we examined seven species in Aspergillus Section Nigri that occur on grapes for fumonisin production and presence of the fumonisin biosynthetic gene fum8, which served as a marker for the FUM cluster. Fumonisin B2 (FB2) production was detected in nine of 32 A. niger strains examined, but not in any strains of A. brasiliensis, A. carbonarius, A. foetidus, A. japonicus, A. tubingensis, and A. uvarum that were examined. In addition, PCR and Southern blot analyses provided evidence for the presence of fum8 in 11 A. niger strains but not in strains of the other species examined. These findings indicate that the discontinuity of fumonisin production in grapes isolates of A. niger likely results from absence in some isolates of at least part of the FUM cluster. The results also confirm the taxonomic complexity of A. niger from grapes and provide a possible explanation for previously observed variability in FB2 contamination of grapes and wine.