Biochemistry and Secondary Metabolism

509. Characterization of the genomic region, controlling biosynthesis of host-specific AAL-toxins, on the conditionally dispensable chromosome of the tomato pathotype of Alternaria alternata . Yasunori Akagi¹, Hajime Akamatsu^1,4, Mikihiro Yamamoto², Takashi Tsuge³, Hiroshi Otani¹ and Motoichiro Kodama¹. ¹Plant Pathology Lab, Dept Agriculture, Tottori Univ, Tottori 680-8553, Japan. ²Okayama Univ. ³Nagoya Univ. ⁴Current address: Dept Plant Pathology, WSU, USA.

AAL-toxins are host-specific toxins produced by A. alternata tomato pathotype (A. alternata f. sp. lycopersici), the causal agent of Alternaria stem canker disease of tomato, which cause severe necrosis on susceptible tomato cultivars. AAL-toxins and fumonisins of the maize pathogen Gibberella moniliformis are structurally related to sphinganine and termed sphinganine-analogue mycotoxins. A 120 kb genomic region on a BAC clone that contains the AAL-toxin biosynthetic (ALT) gene cluster in the tomato pathotype was sequenced and compared with corresponding sequences of the fumonisin biosynthetic (FUM) gene cluster in G. moniliformis. The genomic region includes 19 putative ORFs and 12 of these show similarity to the genes in the FUM gene cluster. These genes include fungal Type I PKSs (ALT1), cytochrome P450 monooxygenases, dehydrogenases, aminotransferases, ABC transporters and longevity assurance factors. However, the order of the genes in the ALT gene cluster is different from that in the FUM gene cluster. In addition, the ALT gene cluster resides on a 1.0 Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL-toxin-producing strains of A. alternata and homologues of the genes were not detected in nonpathogenic strains of A. alternata . Genomic sequences of ALT1 and another PKS gene located outside of the ALT gene cluster, both of which reside on the CDCs in the tomato pathotype strains were compared to each other in tomato pathotype strains collected worldwide. This revealed that the sequences of the genes located on the CDCs, from strains with different geographical origins, are identical. Conversely, the sequences of genes located on chromosomes other than CDCs between the same strains are not identical, indicating that the origin of the CDCs might differ from that of the other chromosomes in the tomato pathotype. We propose a hypothesis in which the ability to produce AAL-toxins could be potentially distributed among A. alternata strains by horizontal transfer of the CDCs. This could provide a possible mechanism whereby new pathogens arise in nature.

510. Inhibition of acetyl-CoA carboxylase impairs appressorial melanization and lipid degradation during infection-related morphogenesis in Colletotrichum lagenarium. Makoto Asakura¹, Carine A. J. Masson², Alison M. Hill², Tetsuro Okuno¹, and Yoshitaka Takano¹. ¹Kyoto University, Kyoto, Japan. ²University of Exeter, Exeter, UK.

Soraphen A is known to be a specific inhibitor of acetyl-CoA carboxylase (ACC). We investigated effects of soraphen A on infection mechanism of Colletotrichum lagenarium that causes cucumber anthracnose. Growth of C. lagenarium on nutrient medium was completely inhibited in the presence of 0.5 ug/ml of soraphen A. In contrast, conidia germinated and developed appressoria normally even in the presence of 2.0 ug/ml of soraphen A. However, soraphen A inhibited melanization of appressoria, suggesting that ACC newly synthesizes malonyl-CoA for appressorial melanization. During appressorium formation and maturation, lipid bodies were gradually degraded. Interestingly, non-melanized appressoria treated with soraphen A retained abundant lipid bodies, suggesting inhibition of lipid-body degradation. This phenomenon was not observed in non-melanized appressoria treated with melanin inhibitors, carpropamid and tricyclazole that inhibit conversion of scytalone to vermelon and vermelon to 1.8-DHN, respectively. However, disruption of PKS1 specifically involved in polyketide synthesis for melanin inhibited breakdown of lipid bodies. Thus, inhibition of polyketide synthesis for melanin as well as inhibition of ACC attenuate breakdown of lipid bodies. These suggest that the melanin biosynthesis pathway affects regulation of lipid degradation during infection-related morphogenesis in C. lagenarium.

511. Metabolic Gene Cluster Silencing in Aspergillus nidulans. Jin Woo Bok*, Daan Noordermeer, and Nancy P. Keller. Department of Plant Pathology, University of Wisconsin - Madison, 1630 Linden Drive, Madison, Wisconsin 53706, USA

In contrast to primary metabolism, the genes involved in secondary metabolism are clustered in fungi. Recently a nuclear protein, LaeA, was found to be required for the transcription of several secondary metabolite gene clusters in Aspergillus nidulans (Bok and Keller 2004). Here we show that regulation is confined to secondary metabolite clusters and not other defined metabolic clusters including proline, nitrate and ethanol utilization clusters. One of these clusters contains the positive regulatory (i.e. aflR) and biosynthetic genes required for biosynthesis of sterigmatocystin (ST), a carcinogenic toxin. Analysis of ST cluster expression indicates LaeA regulation of the cluster is location specific as transcription of genes bordering the ST cluster are unaffected in a delta laeA mutant and placement of a primary metabolic gene, argB, in the ST cluster resulted in argB silencing in the delta laeA background. ST cluster gene expression was remediated when an addition copy of aflR was placed outside of the cluster but not when placed in the cluster. Site specific mutation of a S-adenosyl methionine (AdoMet)-binding site in LaeA generated a delta laeA phenotype suggesting the protein to be a methyltransferase. We present a model of LaeA involvement in chromatin regulation of secondary metabolite gene clusters.

512. Characterization of Fusarium verticillioides alternatively spliced and other ESTs. Daren W. Brown, Robert H. Proctor, Robert R.A. Butchko, Foo Cheung*, Christopher Town*, and David F. Kendra. Mycotoxin Research Unit, NCAUR, USDA/ARS, 1815 N. University St., Peoria, IL 61604. *The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850

Fusarium verticillioides is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that have been associated with several animal diseases and cancer in humans. The fumonisin biosynthetic genes are located in a co-regulated 15-member gene cluster spanning 43 kb of genomic sequence. In order to identify genes that regulate fumonisin biosynthesis and that are involved in the F. verticillioides-maize interactions, we generated over 87,000 expressed sequence tags (ESTs) that represent 11,119 unique sequences from nine cDNA libraries prepared from the fungus grown under fumonisin-inducing and non-inducing conditions and in the presence of maize tissues. Of particular interest, we found that 78 of the 700 ESTs that match portions of the 15 fumonisin genes were alternative splice forms (ASFs). Most ASFs are predicted to yield truncated proteins due to stop codons and/or frameshifts in the retained introns or the altered sequence due splice events utilizing an alternate 3' border. We found that the ASFs appeared to be differentially expressed as more were present in libraries derived from older cultures. This, coupled with the high frequency of occurrence of some ASFs suggests they serve a biological role. We have begun to examine the occurrence of ASFs in culture over time using microarrays. The physiological importance of alternative splicing in fungi has not been determined. Understanding their role in fumonisin biosynthesis may open up new avenues to develop strategies to limit fumonisin contamination of agricultural commodities.

513. Isolation, cloning and characterisation of three glutathione-s-transferases in Aspergillus fumigatus. Claire Burns, Rachel Geraghty, Kevin Kavanagh and Sean Doyle National Institute for Cellular Biotechnology, National University of Ireland, Maynooth, Ireland.

Aspergillus fumigatus is a severe fungal pathogen of immunocompromised patients commonly treated with antifungal agents such as amphotericin B. However, treatment is often ineffective and infection frequently leads to death. We postulate that detoxification enzymes such as glutathione-s-transferases (GST) may play a role in survival of the fungus during infection, and would thus provide a novel therapeutic target.

Several putative GSTs were identified from the A. fumigatus genome. Three of these genes, namely gst1, gst2 and gst3, have been cloned and heterologously expressed, yielding proteins of 29-30 kDa. The recombinant proteins exhibited GST activities against CDNB of 0.025 U/mg, 0.006 U/mg and 0.004 U/mg, and glutathione peroxidase activities against cumene hydroperoxide of 0.145 U/mg, 0.025 U/mg and 0.019 U/mg respectively.

 Gst2 and gst3 were basally expressed, and were induced 4-fold and 10-fold in the presence of CDNB, and 3-fold and 5-fold in the presence of hydrogen peroxide. Gst1 was not basally expressed, and was induced by CDNB only. Experiments are ongoing to examine expression in the presence of other xenobiotics. Preliminary results when induced with amphotericin B indicate possible downregulation of gst2 and gst3 and upregulation of gst1.

514. In silico subtraction of Fusarium verticillioides EST libraries to identify potential transcriptional regulators of the FUM gene cluster. Robert A.E. Butchko, Daren W. Brown and Robert H. Proctor. Mycotoxin Research Unit, NCAUR, USDA/ARS, 1815 N. University St., Peoria, IL 61604

Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. These toxins can disrupt sphingolipid metabolism in animals, cause diseases in horses and swine, and have been associated with cancer in laboratory rodents. A cluster of fumonisin biosynthetic genes (FUM) has been described in F. verticillioides. Conspicuously absent from this FUM gene cluster is a transcription regulatory gene. Previous Northern analysis indicated that FUM genes are not expressed at early times points (e.g. 24 hr) in GYAM medium but are highly expressed at later time points (e.g. 96 hr). We have prepared EST libraries from F. verticillioides GYAM cultures, and the presence/absence of FUM gene ESTs in 24- and 96-hr libraries is consistent with the differential expression observed in the Northern analyses. Further comparison of the libraries revealed the presence of a number of EST's with similarities to transcription factors and activators, DNA binding proteins, and zinc finger proteins in the 96-hr library but not in the 24-hr library. We are investigating whether the genes corresponding to these ESTs regulate FUM gene expression. To do this, we have disrupted the putative regulatory genes and developed a rapid screen to assay FUM gene expression. Understanding the transcriptional regulation of FUM genes should provide information that can be used to control fumonisin contamination in maize.

515. Isolation and sequence analysis of polyketide synthase genes from three Fusaria commonly found in corn. Bryce Callighan¹, Keith Johnson¹, Daren W. Brown², ¹ Bradley University, Biology Department, Peoria IL. ² USDA-ARS-NCAUR, Mycotoxin Research Unit, Peoria IL.

Mycotoxins are fungal secondary metabolites that often contaminate grains following plant infestation. Synthesis of many mycotoxins (e.g. fumonisins and fusarins produced by Fusarium verticillioides) is initiated by a member of a family of enzymes called polyketide synthases (PKSs). Degenerate primers were developed that target a highly conserved portion of genomic sequence that encodes the PKS ketosynthase (KS) domain. The first objective of the research was to isolate KS gene fragments from F. verticillioides, F. proliferatum, and F. subglutinans genomic DNA. Based on the genomic sequence of F. verticillioides and F. graminearum, between 10 and 20 PKS genes are expected per genome. To date five novel KS fragments from both F. proliferatum and F. subglutinans have been isolated. We conducted a phylogenetic analysis of the new KS sequences and KS sequences from fungal PKSs with known as well as unknown function. We identified a number of species specific PKS genes as well as PKS genes that appear to be shared by multiple Fusarium. In addition, the unique sequence data has provided us tools to help distinguish between species in mixed cultures.

516. Cloning, characterization and expression of a polyketide synthase gene involved in monacolin k biosynthesis from Monascus sp. Yi-Pei Chen¹², Li-Ling Liaw², Ming-Der Wu¹, Chun-lin Wang¹, Ching-Ping Tseng², Gwo-Fang Yuan¹. ¹Bioresource Collection and Research Center, Food Industry Research and Development Institute. ²Department of Biological Science and Technology, National Chiao Tung University.

Monacolin k, cholesterol serum synthesis inhibitor, is a secondary metabolite synthesized by polyketides from Monascus. In this study, a BAC (Bacterial Artificial Chromosome) clone, mps01, was screened from the mpb01 BAC library constructed with Monascus sp. BCRC 38072 genomic DNA. The putative monacolin k biosynthesis gene cluster was found in mps01 clone, genomic sequencing and Northern blot analysis showed that nine putative genes for monacolin k biosynthesis were located within a 41-kb region and were transcribed when monacolin k was produced. The deduced amino acid sequences encoded by the nine genes, designated mkA¡VmkI, sharing similarities of over 54% with lovastatin gene cluster contained in Aspergillus terreus were assumed to be involved in monacolin k biosynthesis. The mkA gene encoding nonaketide synthase and sfp gene, a phosphopantetheinyl transferase required to convert the expressed apo-PKS to its holo form and obtained from Bacillus subtilis, were coexpressed in Escherichia coli. Novel polyketide compounds produced in the transformant were determined by LC-ESIMS and found at wavelength of 360 nm. Further study on the structure of these polyketides will be presented.

517. Genetic analyses of a peptide synthetase gene from the insect pathogen Metarhizium anisopliae. Yong-Sun Moon (1), Stuart B. Krasnoff (2), John D. Vandenberg (2), Donna M. Gibson (2), and Alice C.L. Churchill (1,3), Boyce Thompson Institute (1), USDA-ARS, Plant Protection Research Unit (2), and Department of Plant Pathology, Cornell University (3), Ithaca, NY, USA

Metarhizium species are at the forefront of efforts to develop entomopathogenic fungi as insect biocontrol agents. Yet we have an incomplete understanding of the biological and genetic factors that make them effective. We have focused our efforts on understanding the roles of toxins as virulence factors in fungal-insect interactions. The principal toxins produced in fermentation by M. anisopliae are the destruxins, a large family of cyclic depsipeptides, which are predicted to be synthesized nonribosomally by a large multifunctional enzyme called a peptide synthetase (PS). We targeted for further study a PS gene fragment (ma267) identified by Freimoser et al. (2003) as an EST that was expressed after 24 hr of fungus growth on insect cuticle-containing medium. We determined that ma267 detects DNA polymorphisms that correlate with relative levels of in vitro destruxins production in several M. anisopliae isolates. Additionally, ma267 gene expression is positively correlated with in vitro destruxins production. We disrupted the ma267 PS gene by Agrobacterium tumefaciens-mediated transformation and homologous recombination and identified several genetically stable knockout (KO) transformants. All KO and ectopic transformants examined exhibited normal growth rates, colony phenotypes, and wild type levels of in vitro destruxins production, suggesting that the ma267 PS gene is not involved in destruxins production in M. anisopliae. Bioassays against beet armyworm are in progress to determine if disruption of the ma267 PS gene affects pathogenicity of the fungus. An understanding of the role of toxins in pathogenicity is essential for enhancing M. anisopliae as a biocontrol agent and to confirm its safety against non-target organisms.

518. Nitrogen source and pH influence aflatoxin production by a previously unrecognized A. flavus morphotype . P. J. Cotty, K.E. Kobbeman, J.E. Mellon, T. Feibelman, K. F. Cardwell, and K.E. Ehrlich. USDA, ARS, Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ

An Aspergillus that produces B aflatoxins and elongate tan sclerotia was isolated from soils collected in North America, Asia, and West Africa. Phylogenetic analysis of the aflJ/aflR A. flavus. All isolates belonged to the same vegetative compatibility group and shared greater than 99% sequence similarity. This morphotype (morphotype P) produced elongate sclerotia, up to 5 mm in length, with a bulbous base and reduced melanin. In contrast to other aflatoxin-producing A. flavus, P morphotype isolates produced 8 to 190 fold more B aflatoxins in a medium with nitrate as the sole nitrogen source than in media with either ammonium or urea as sole nitrogen sources. Other A. flavus isolates and isolates of A. nomius and A. parasiticus produced either similar quantities or more aflatoxins in ammonium-based medium than in nitrate based medium. Low aflatoxin production in ammonium-based medium resulted from sensitivity to low pH created during fermentation on ammonium. Buffering with either succinate or citrate resulted in increased aflatoxin production. Aflatoxin production by both the P morphotype and A. parasiticus (NRRL 2999) was greater in nitrate than in ammonium at pH 2.5. However, at pH 7.5, both fungi initially produced greater quantities of aflatoxins on ammonium medium. Variation in pH sensitivity may cause variation among A. flavus isolates in aflatoxin production. The P morphotype may be useful for studying interrelationships among sclerotial morphogenesis, aflatoxin biosynthesis, and melaninization.

519. Cloning, Expression, and Knockout of Polyketide and Tetramic Acid Synthase Genes from Fungi. Russell J Cox, Thomas J. Simpson FRS, Colin Lazarus, Andy Bailey, Kirstin Eley, Song Zhongshu, Deirdre Hurley, Frank Glod, Thomas P. Nicholson and Ying Zhang. University of Bristol, School of Chemistry, Bristol UK

A rapid cloning procedure has been developed for obtaining polyketide synthase genes from fungi which are associated with the production of particular compounds. The cloning and analysis of genes involved in fungal squalestatin and fusarin biosynthesis will be discussed.

520. Expression analysis of the cross-pathway control genes cpc1 and cpc2 in Acremonium chrysogenum. Jacqueline Dreyer and Ulrich Kück. Algemeine & Molekulare Botanik, Ruhr- University Bochum, D- 44780 Bochum, Germany

The biosynthetic pathway and external factors influencing antibiotic production in Acremonium chrysogenum have already been elucidated. However, the genetic alterations leading to differences in cephalosporin C production between wild type and overproducing strains are still poorly understood. There are several distinct reports indicating that gene regulation at higher level is responsible for the alteration in cephalosporin C production. It has been shown that the gene regulation mediated through glucose in the overproducing strains is clearly different from the one in the wild type strain (Jekosch K, Kück U, 2000 Curr Genet 37: 388-395). Furthermore, several transcription factors have been isolated which play a role in the regulation of gene expression in cephalosporin C biosynthesis (Schmitt EK et al. 2004 In: Molecular Biotechnology of Fungal beta-Lactam Antibiotics and Related Peptide Synthases. Series: Advances in Biochemical Engineering / Biotechnology 88: 1-43).

We have confirmed the presence of two genes cpc1 and cpc2 in Acremonium chrysogenum which are key transcription factors in the amino acid biosynthetic pathway. Preliminary studies on their gene expression by northern analysis and real-time PCR demonstrate that these genes are regulated. The data from wild type and semi- producer strain are compared. A knock-out strain is being generated to investigate the role of cpc1 in secondary metabolism.

521. The aflatoxin biosynthesis cluster gene, hypA is involved in conversion of versicolorin A to sterigmatocystin. Kenneth C. Ehrlich, Beverly Montalbano, Steve Boue, and Deepak Bhatnagar, Southern Regional Research Center/ARS/USDA, PO Box 19687, New Orleans LA 70124

The conversion of polyketide metabolite, versicolorin A, to sterigmatomcystin (ST) probably requires five different enzymatic steps: deoxygenation, Baeyer-Villiger oxidation, lactone cleavage, oxidative decarboxylation, and methylation. The proteins encoded by verA and ver-1 are probably involved in the first two steps. We now report that the aflatoxin biosynthesis cluster gene, hypA, also encodes an enzyme involved in the conversion. Knockout plasmid vector constructs were prepared in pUC18 and used to transform A. parasiticus BN009E niaD-. Five transformants were isolated which accumulated versicolorin A and no longer produced AF. Northern blot and PCR analysis showed that only hypA was disrupted in these transformants and that other genes (verA and ver-1) were expressed at normal levels. Feeding studies confirmed that ST and O-methylST, but not versicolorin A, were efficiently converted to aflatoxins, in the knockout transformants. The enzymatic role of HypA in the conversion has not been established. HypA and its gene have probable membrane-binding and dehydrogenase domains based on a motif search of the GenBank database. We hypothesize that HypA is involved in the necessary lactone cleavage and rearrangement steps which convert the oxidized anthraquinone to the xanthone.

522. Isolation of an Epichloë Endophyte Gene Cluster Involved in Ergovaline Biosynthesis. Damien Fleetwood^1,2, Aiko Tanaka², Barry Scott² and Richard Johnson¹. ¹AgResearch Limited, Palmerston North, New Zealand; ²Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand

Epichloë endophytic fungi are obligate symbionts that colonise the intercellular spaces of temperate pasture grasses of the subfamily Pooideae. This symbiosis is vitally important to agriculture as secondary metabolite alkaloids produced by the endophyte protect the grass host from many herbivores. However some endophyte alkaloids are toxic to grazing animals. One such toxic alkaloid is ergovaline, an ergopeptine that causes toxicosis in cattle and sheep grazing tall fescue and perennial ryegrass pastures in the USA and New Zealand. Here we present the isolation, by degenerate PCR, of a non-ribosomal peptide synthetase gene from Neotyphodium lolii (a common perennial ryegrass endophyte closely related to Epichloë species) that has homology to the Lps2-encoding gene involved in ergopeptine biosynthesis from Claviceps purpurea. Screening of an N. lolii genomic library provided several overlapping clones which have been sequenced. In addition to the putative NRPS gene orthologue (here named lpsB), a second gene has been identified that is also found in the C. purpurea ergopeptine gene cluster, a putative oxidoreductase oxrA. This, along with expression and taxonomic distribution data, provides very strong evidence that we have isolated the ergovaline biosynthetic gene cluster from N. lolii and progress towards the isolation of the full gene cluster is underway. To determine the function of lpsB a targeted gene replacement has been constructed and the mutant introduced into perennial ryegrass to determine the in planta chemical phenotype. Similar experiments are underway with oxrA.

523. Functional analysis of the polyketide synthase genes in Gibberella zeae. Iffa Gaffoor¹, Daren W. Brown², Ron Plattner², Robert Proctor², Weihong Qi¹, and Frances Trail¹. ¹Michigan State University and ²USDA, Peoria IL.

 Type I Polyketide Synthases (PKSs) are multidomain enzymes responsible for synthesizing a gamut of compounds with varied functions. From the genomic sequence of the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum) we identified fifteen putative polyketide synthase genes. We have disrupted each of these genes, along with an additional gene that shares some similarity with PKSs but which we think does not encode a PKS. Individual disruption mutants have been analyzed for traits such as vegetative growth, mycotoxin and pigment production, perithecium production, ascospore discharge and pathogenicity. From these analyses, we have identified PKS genes responsible for zearalenone, fusarin and aurofusarin biosynthesis and biosynthesis of the black perithecial pigment. Expression analysis of these genes under varied culture conditions revealed that they are differentially expressed. While two PKS genes appear to be expressed under most of the conditions tested others are not expressed under any of these conditions. Further analysis of the mutants and identification of the polyketide compounds synthesized by the PKSs will enable us to assign functions to the remaining PKS genes and elucidate their role in the life cycle of G. zeae.

524. Molecular Genetics and Evolutionary Aspects of the Alkaloid Pathway in Claviceps purpurea. T. Haarmann, and P. Tudzynski. Institute of Botany, WWU Muenster, Schlossgarten 3, D-48149 Muenster, Germany

We isolated and cloned genes organized in a cluster of 68.5 kb that could mediate pathway specific steps of the ergot alkaloid biosynthesis. Expression studies showed that all cluster genes are coregulated and that they are only activated under alkaloid producing conditions. The cluster comprises the gene cpd1 which encodes the key enzyme DMATS, four NRPS (cpps1-4), several oxygenases and oxidoreductases and other ORFs not characterized so far. Targeted inactivation of one NRPS (cpps2) led to an ergopeptine-nonproducing mutant which – unlike the parent producer strain – accumulated D-lysergic acid. Cpps2 was shown to encode the monomodular lysergyl-peptidyl-synthetase 2 (LPS2) responsible for the activation of D-lysergic acid. Knock-out experiments with the gene cpP450-1 led also to an ergopeptine-nonproducing mutant which instead accumulated agroclavine, indicating the involvement of the gene product in the formation of the precursors of D-lysergic acid (e.g. elymoclavine). Another aim is to compare different strains of Claviceps particularly with respect to their potential to produce different types of alkaloids (chemical races). Comparison of the cluster sequences of strain P1 (ergotamine producer) with that of strain ECC93 (ergocristine producer) showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of chemical races of C. purpurea is confined to evolution of NRPS module specificity.

525. Crystal structure comparison of two large monofunctional catalases. Wilhelm Hansberg, Adelaida Díaz, Victor-Julián Valdés, Enrique Rudiño-Piñera, Eduardo Horjales. Instituto de Fisiología Celular e Instituto de Biotecnología, Universidad Nacional Autónoma de México, México D. F., México.

Neurospora crassa has two large monofunctional tetrameric catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and is accumulated in conidia. CAT-3 is inducible and is associated with growing cells. Both catalases are modified by singlet oxygen in vitro and in vivo and have similar kinetics and resistance to denaturation. The structure of both enzymes was determined by molecular replacement. Heme group in CAT-1 was a mixture of protoheme IX (heme b) and an oxidized heme (heme d), originated by di-hydroxylation at ring III and subsequent formation of a spirolactone with the propionyl group in C6. The heme d probably is formed by singlet oxygen. In addition, CAT-1 has an unusual covalent bond between the sulfur of a cysteine and the beta carbon of the tyrosine that coordinates the Fe(III) of the heme (1). Different to CAT-1, CAT-3 heme is only heme b and there is no cavalent bond between the glutamine, equivalent to the CAT-1 cysteine, and the essential tyrosine. Other differences between these enzymes are found at the C–terminal domain. Results indicate that both catalases present different mechanisms to contend with molar concentration of hydrogen peroxide.

1) Díaz A, Horjales E, Rudiño-Piñera E, Arreola R, Hansberg W (2004) Unusual Cys-Tyr covalent bond in a large catalase. J Mol Biol 342:971-985.

Financial support: CONACyT C01-40697, DGAPA/UNAM IN228405

526. Molecular analysis of glycolipid production in Ustilago maydis. Sandra Hewald, Beate Teichmann and Michael Bölker. University of Marburg, Dept. of Biology, Karl-von Frisch-Strasse 8, D-35032 Marburg,

Under conditions of nitrogen starvation, Ustilago maydis secretes large amounts of amphipathic glycolipids. These surface active compounds can be grouped into two classes: the ustilagic acids and the ustilipids. Ustilagic acids consist of a cellobiose moiety glycosidically linked to the w-hydroxyl group of 15,16-dihydroxy-hexadecanoic-acid. The ustilipids consist of a 4-O-b-D-mannopyranosyl-D-erythritol which is esterified with palmitoic acid and shorter acyl groups.

We used a reverse genetics approach to identify components involved in glycolipid biosynthesis. Potential glycosyltransferases and cytochrome P450 monooxygenases were identified in the genomic sequence of Ustilago maydis. Deletion of a glycosyltransferase with similarity to macrolide glycosyltransferases of prokaryotic origin resulted in loss of ustilipid production. Mutant, deleted for a cytochrome P450 monooxygenase of the CYP94A family, lost its ability for ustilagic acid production. Both genes are highly expressed under glycolipid producing conditions.

Therefore we used microarray analysis to identify more genes, which are involved in the glycolipid biosynthesis pathway. Potential candidate genes could be identified and are currently tested.

With the glycolipid defective mutants at hand we are now able to determine the biological functions of these amphipathic substances. We could already show that the secreted glycolipids cause a drastic reduction in surface tension of culture medium.

527. The two interacting transcription factors CPCR1 and AcFKH1 controls cephalosporin C biosynthesis and morphogenesis in A. chrysogenum . Birgit Hoff, Danielle Janus, Esther Schmitt and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany

Acremonium chrysogenum is the main industrial producer of the beta-lactam antibiotic cephalosporin C. A complex regulatory network of transcription factors seems to control the expression of at least seven cephalosporin C biosynthesis genes. In this study, we used the RFX transcription factor CPCR1, which is involved in the transcriptional regulation of the cephalosporin C biosynthesis genes, in a yeast two hybrid screen to identify potential protein interaction partners [1]. A cDNA was identified encoding the C-terminal part of a novel forkhead protein, so called AcFKH1, which is the first characterized member of the forkhead gene family in filamentous fungi. AcFKH1 is characterized by two highly conserved domains, the N-terminal forhead associated domain and the C-terminal DNA-binding domain of the winged helix/forkhead type. The observed interaction between CPCR1 and the C-terminus of AcFKH1 in the yeast system was verified in vitro in a GST pulldown assay. Using gel retardation analysis, FKH1 was shown to recognize two forkhead consensus binding sites within the promotor region of the cephalosporin C biosynthesis genes pcbAB/pcbC. Additionally, AcFKH1 is able to bind with high affinity to the SWI5-binding site of the yeast FKH2 protein [2].

In a second approach, we investigated the role of the transcriptions factors CPCR1 and AcFKH1 on fungal morphogenesis. We used cpcR1 and Acfkh1 knockout strains, multicopy strains and retransformants for detailed light and confocal laser microscopic analysis. From the sum of our investigations we concluded that arthrospore formation and cell separation are dependent on the transcription factors controlling cephalosporin C biosynthesis in A. chrysogenum.

[1] Schmitt EK, Bunse A, Janus D, Hoff B, Friedlin E, Kürnsteiner H, Kück U (2004) Eukaryot Cell 3: 121-134

[2] Schmitt EK, Hoff B, Kück U (2004) Gene 342: 269-281.

528. Bimolecular fluorescence complementation (BiFC) – a new tool to visualize protein-protein interactions in living fungal cells. Birgit Hoff and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany

Here we describe a bimolecular fluorescence complementation (BiFC) assay for direct visualization of protein protein interactions in fungal cells. This in vivo approach is based on the formation of a fluorescent complex by two non-fluorescent fragments of the enhanced yellow fluorescent protein (EYFP) brought together by association of interacting proteins fused to these fragments [1]. We have constructed two fungal expression vectors encoding the N- or C-terminus of EFYP with appropriated linker sequences. The application of the expression vectors, and thus of the BiFC technology, was demonstrated by using the two winged helix transcription factors CPCR1 and AcFKH1 from the beta-lactam antibiotic producer Acremonium chrysogenum [2]. Both transcription factors bind to promotor sequences of the pcbAB-pcbC biosynthesis genes. Only when the full length transcription factors were fused to EYFP fragments, yellow fluorescence was observed due to the bimolecular complementation of both chimeric proteins. No fluorescence was observed in strains producing either CPCR1-EYFPC or AcFKH1-EYFPN in combination with the complementing half-EYFP fragment. Under these conditions, the EYFP fragments do not appear in spatial proximity to each other and, consequently, it is impossible to reconstitute a functional yellow fluorescent protein. The nuclear localization of the protein-protein interaction was verified by staining fungal cells with the nucleic acid dye TOTO-3.

Consequently, the BiFC approach should facilitate the visualization of the subcellular sites of protein interactions in the living fungal cell under conditions that closely reflect the normal physiological environment.

[1] Hu CD, Chinenov Y, Kerppola TK (2002) Mol Cell 9:789-798

[2] Hoff B, Kück U (2004) Curr Genet, in press

529. Visualization of Biochemical Pathways at the Saccharomyces Genome Database. Eurie L. Hong¹, Rama Balakrishnan¹, Karen R. Christie¹, Maria C. Costanzo¹, Kara Dolinski², Stacia R. Engel¹, Dianna G. Fisk¹, Jodi E. Hirschman¹, Robert Nash¹, Rose Oughtred², Marek S. Skrzypek¹, Chandra L. Theesfeld¹, Gail Binkley¹, Christopher D. Lane¹, Qing Dong¹, Anand Sethuraman¹, Shuai Weng¹, David Botstein², J. Michael Cherry¹. (1) Dept. of Genetics, Stanford University, 300 Pasteur Drive, Stanford, CA, 94305-5120, USA; (2) Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

 Saccharomyces cerevisiae provides an excellent system for the study of biosynthetic and catabolic pathways and many enzymes involved in these pathways have been identified and characterized. Although there are differences between pathways in S. cerevisiae and other organisms (both prokaryotic and eukaryotic), most summaries of pathway information fail to articulate those differences. Using the Pathway Tools software that is developed and maintained by Peter Karp and his colleagues at SRI International, the Saccharomyces Genome Database (SGD) has released the Yeast Biochemical Pathways tool (http://pathway.yeastgenome.org/biocyc/). This manually curated resource can be searched to view biochemical reactions and pathways as they occur in S. cerevisiae. In addition, pathways can be accessed through hyperlinks on the locus page of enzymes involved in the pathway. This resource also maps data from genome-wide expression analyses onto the pathway overview so that changes in gene expression can be viewed in the context of cellular metabolism.  The Yeast Biochemical Pathways tool can be a powerful resource in the classroom, in the analysis of gene expression data, and as a starting point for further investigation of biochemical pathways in S. cerevisiae or other fungi. SGD can be accessed at http://www.yeastgenome.org/. SGD is funded by the US National Human Genome Research Institute.

530. Comparative Genomics of a Toxin Biosynthetic Gene Cluster in Filamentous Fungi. Donald M. Gardiner *, Anton J. Cozijnsen, David C. Straney #, Barbara J. Howlett. School of Botany, The University of Melbourne, Victoria, 3010. * Current address: Institute for Molecular Bioscience, The University of Queensland, Queensland 4072. # Department of Cell Biology and Molecular Genetics, University of Maryland, College Park MD 20742 USA

Genes responsible for the biosynthesis of secondary metabolites are typically clustered in filamentous fungi. The origin and evolutionary pressures that maintain such clusters are largely unknown. We have cloned a gene cluster encoding enzymes in the biosynthesis of a toxin, sirodesmin, from Leptosphaeria maculans, which causes blackleg disease of canola. Sirodesmin belongs to the epipolythiodioxopiperazine (ETP) class of toxins, which is only produced by fungi and confers toxicity via reduction of a disulfide bond. We are using comparative genomics to determine biosynthetic pathways for ETPs. Putative ETP gene clusters are present in three fungi for which complete genome sequences are available. These are the opportunistic human pathogen Aspergillus fumigatus, the rice blast fungus, Magnaporthe grisea and the wheat head scab fungus, Fusarium graminaerum. A. fumigatus makes the ETP gliotoxin, which causes apoptotic and necrotic cell death, as do the distantly related fungi, Penicillium bilaii and Trichoderma virens. We are attempting to characterise the gliotoxin biosynthetic gene clusters in these fungi. Analysis of the arrangement and sequences of genes in these three clusters may uncover clues about how the clusters evolved.

531. Modelling of Fungal Non-Ribosomal Peptide Synthetases: Prediction of Novel Secondary Metabolites from Fungal Endophytes. Richard Johnson¹, Vic Arcus², T. Verne Lee², Christine Voisey¹ and Greg Bryan¹. ¹ AgResearch Grasslands Research Centre, Palmerston North, New Zealand. ² AgResearch Structural Biology Laboratory, University of Auckland, New Zealand.

Neotyphodium lolii and N. coenophialum are fungal endophytes that live symptomlessly within the intercellular spaces of perennial ryegrass and tall fescue, respectively. These endophytes confer a number of biotic and abiotic advantages to their hosts, many of which are mediated through the production of fungal secondary metabolites. Biosynthetic pathways involved in secondary metabolism are commonly associated with gene clusters in filamentous fungi and the pathways for several key endophyte secondary metabolites (also see abstracts by Young et al. and Fleetwood et al.), have recently been wholly or partially elucidated.

We are particularly interested in biosynthetic pathways containing non-ribosomal peptide synthetases (NRPSs) since these multi-modular enzymes catalyse the formation of small peptides that are highly diverse in structure and activity. We have identified at least 10 novel NRPS genes from N. lolii, and have isolated corresponding BAC clones that may contain associated gene clusters. Predicting the classes of compounds synthesised from these NRPS associated clusters is presently difficult, especially for fungi where the current homology based models, for adenylation domain binding pocket specificity, do not work well for substrate prediction.

Our research aims to refine these homology based models by determining the high-resolution atomic structure of endophyte peptide synthetase adenylation domains with known substrates (for example lpsA, involved in ergovaline biosynthesis). By combining these models with ligand docking software and knowledge-based scoring we hope to be able to predict the likely substrates of fungal NRPSs.

532. Functional characterization of differentially regulated Fusarium verticillioides genes. Nich Jones¹, Keith Johnson¹, Scott E. Baker², and Daren W. Brown³. ¹Bradley University, Biol. Depart., Peoria IL. ²PNNL Chem. Biol. Proc. Dev. Group, Richland, WA. ³USDA-ARS- NCAUR, Mycotoxin Research Unit, Peoria IL.

Fumonisins are secondary metabolites produced by several fungi, including Fusarium verticillioides, and are linked to numerous animal diseases, neural tube defects and cancer in humans. F. verticillioides, normally an endophyte, can cause several diseases of corn. Damaged and fumonisin contaminated corn result in the loss of millions of dollars each year to farmers world-wide. We reasoned that a better understanding of the genetic regulation of fumonisin production as well as the F. verticillioides-plant disease process will enable the development of new strategies to limit fungal damage. Recently, we created nine F. verticillioides cDNA libraries which yielded 87,000 ESTs and compiled into 11,119 unique sequences. The presence of numerous unique ESTs in each library suggested that many of them are differentially expressed. We screened the unique sequences and identified a set that may encode for Zn(II)2 Cys6 or Cys2-His2 Zn(II) transcriptional factors. The pattern of differential expression of many of these genes suggest that some may play a role in regulating fumonisin biosynthesis and/or a role in the fungal-plant disease process. We have begun to examine the role these genes may play by creating disruption vectors. We describes the identification of the target genes based on predicted protein sequence, sequence similarities, differential expression, as well as the development of disruption vectors and our efforts to determine the consequences to F. verticillioides of the loss of these genes.

533. Characterization of the mpkB gene in the model fungus Aspergillus nidulans. Navgeet Kaur and Ana M. Calvo. Department of Biological Sciences, Northern Illinois University, Dekalb, Illinois 60115, U.S.A.

In fungi, environmental signals can be transduced into intracellular responses by the action of MAP kinase cascades, which are similar to the ones found in the mammalian cells. Mitogen-activated protein kinases (MAPK) are a family of serine/threonine protein kinases widely conserved among eukaryotes and are involved in many essential developmental processes such as mating, sporulation and pathogenicity. In this research work we are studying the role of mpkB, an MAP kinase gene found in the model filamentous fungus Aspergillus nidulans. The mpkB gene is a homologue of the FUS3/KSS1 MAP-kinase genes in the yeast Saccharomyces cerevisiae. FUS3 regulates mating in response to pheromones in haploid yeast cells and KSS1 regulates filamentous growth in response to nitrogen limitation in diploid yeast cells. In order to elucidate the role of mpkB in development and secondary metabolism in A. nidulans, we have generated a mpkB disruption strain. Loss of MPKB function results in a blockage in biosynthesis of the mycotoxin sterigmatocystin.

534. Characterization of fungal phosphopantetheinyl transferases involved in primary and secondary metabolism. D. Keszenman-Pereyra¹, N. George², K. Johnsson² and G. Turner¹. ¹Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK; ²Institute of Chemical Sciences and Engineering, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

Primary and secondary metabolic pathways in Aspergillus sp utilize 30-50 phosphopantetheinylated proteins with specialized carrier protein (CP) domains. The conversion of apo-CPs to holo-CPs is catalysed by 4'-phosphopantetheine transferases (PPTases). Blast searches of available filamentous fungal genome sequences revealed that each organism has putative genes encoding Fas2 integrated-, AcpS- (postulated to target mitochondrial Acp1) and NpgA- type PPTases. A. nidulans NpgA seems capable of activating CPs of non-ribosomal peptide synthetases, polyketide synthases and alpha aminoadipate reductase (homologue of yeast Lys2). To analyse the wide substrate specificity of NpgA, the ORF was amplified by PCR from A. nidulans genomic DNA and cloned in frame with N-terminal hexahistidine tag; the PPTase was overexpressed in Escherichia coli and purified by nickel affinity chromatography. The wide substrate specificity of NpgA has been confirmed in vitro by using a range of CP substrates and assays. Moreover, co-expression experiments in E. coli show that NpgA is relatively efficient for the activation of heterologous substrates. NpgA is the first reported example of a eukaryotic PPTase involved in both primary and secondary metabolism. Phylogenetic relationships of fungal PPTases and their substrates will be discussed.

535. Aspergillus nidulans mutants unable to localise glyoxylate cycle enzymes to the peroxisomes are able to utilise acetate. Gillian S. Khew, Sandra L. Murray, Meryl A. Davis and Michael J. Hynes. Department of Genetics, University of Melbourne, Victoria 3010, Australia.

Growth on acetate and fatty acids results in the production of acetyl-CoA which is channeled into the TCA and glyoxylate cycles. The TCA cycle occurs in the mitochondria and the glyoxylate cycle in the peroxisomes. Peroxisomal proteins are directed to the peroxisomes via one of two classes of peroxisomal targeting signals – PTS1 and PTS2. PTS1 targeting occurs via the Pex5 receptor and PTS2 via the Pex7 receptor. These receptors are recycled to the cytosol by Pex6, an ATPase. The A. nidulans genes, acuD (isocitrate lyase) and acuE (malate synthase), encode enzymes specific to the glyoxylate cycle. AcuE possesses a peroxisomal targeting signal 1 (PTS1) whereas AcuD lacks any identifiable PTS1 or PTS2. We are interested in the effects of peroxisomal protein localisation on fatty acid utilisation in A. nidulans. A knockout mutant of the A. nidulans PEX5 orthologue (pexE) mislocalises AcuE to the cytoplasm but is able to target AcuD to the peroxisomes. A mutant in the PEX7 orthologue (pexG), however, mislocalises AcuD but is unaffected in peroxisomal targeting of AcuE. A mutant defective in the PEX6 orthologue (pexF) mislocalises both AcuD and AcuE to the cytoplasm but is nonetheless able to utilise acetate. Most of the peroxisomal mutants studied in our laboratory are sensitive to the presence of fatty acids. The growth of the pexE mutant is particularly inhibited by the presence of all fatty acids tested, including acetate. A pexE/pexG double mutant shows relief of acetate inhibition and is able to grow on acetate.

536. A novel polyketide synthase gene cluster for the biosynthesis of aurofusarin in Gibberella zeae. Jung-Eun Kim¹, Kap-Hoon Han¹, Jianming Jin¹, Hun Kim², Jin-Cheol Kim², Sung-Hwan Yun³, and Yin-Won Lee¹. ¹School of Agricultural Biotechnology, Seoul National University, Seoul 151-921; ²Korea Research Institute of Chemical Technology, Daejon 305-606; and ³Division of Life Sciences, Soonchunhyang University, Asan 336-745, Korea

Mycelia of Gibberella zeae, an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. A screen of insertional mutants of G. zeae, generated using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant to a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains ten additional ORFs, including a putative ORF identified as PKS12 that shares about 40% amino acid identity to type I fungal PKS genes. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. Northern blot analyses revealed that the 10 genes located on the 30-kb region are co-regulated by a putative transcription regulator. This information is the first on the biosynthesis of the red pigment by G. zeae.

537. Endopolygalacturonases from Botrytis cinerea: biochemical properties and interaction with inhibiting proteins. Geja Krooshof¹, Rob Joosten¹, Harry Kester¹, Ilona Kars², Jan van Kan², and Jac Benen¹. ¹Microbiology/Fungal genomics, ² Phytopathology, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.

The phytopathogen Botrytis cinerea harbours at least six endopolygalacturonase-encoding (Bcpg) genes of which Bcpg1 and Bcpg2 are required for full virulence. The endopolygalacturonase (BcPG) enzymes degrade pectin, enabling the fungus to breach the plant cell wall. We expressed five BcPG isozymes in Pichia pastoris, purified them and studied biochemical properties, such as pH optimum, mode of action, and substrate specificity in detail. BcPG3 shows a rather unusual, broad pH optimum and is the only isozyme fully active at pH 3.5. Polygalacturonic acid is a poor substrate for BcPG1 and BcPG4 as compared to BcPG2, BcPG3, and BcPG6. In contrast to BcPG1, 2, and 4, BcPG3 and BcPG6 show extreme processive behaviour on oligogalacturonides longer than four GalpA residues. Only BcPG3 and BcPG6 are able to hydrolyse GalpA dimers.

Since PG activity is important for fungal virulence, the BcPGs are interesting targets for disease control. Therefore, a range of plant extracts was screened to identify potent polygalacturonase-inhibiting proteins (PGIPs). PGIPs from different plant sources have been purified and their interaction with the BcPG isozymes have been investigated using different techniques. Results on the mode of inhibition and binding will be presented.

538. See abstract number 405

539. Recruitment of primary metabolism genes for the insecticidal loline alkaloid gene cluster in grass-endophytes. Brandi L. Kutil, Charles J. Greenwald, & Heather H. Wilkinson. Program for Biology of Filamentous Fungi, Department of Plant Pathology & Microbiology, Texas A&M University, USA

PLP-binding enzymes are ubiquitous ancient biocatalysts. There are five distantly related lineages (defined by both crystal structure and function). Each of these subfamilies within the superfamily has homologs in the archea, eubacteria and eukaryotic superkingdoms. These enzymes function in many primary metabolism pathways. It is expected that diversification into the five subfamilies of PLP-dependent enzymes occurred prior to divergence of the progenitor of these major kingdoms. Subsequently, radiation of the diversity within each of the gene families has involved evolution of different substrate binding specificities and/or tissue specific expression. Discovery of the loline alkaloid secondary metabolite gene cluster in Epichloe and Neotyphodium species has revealed some PLP-dependent enzymes specifically involved in lolines production. LolC is a homocysteine synthase (hcs)-like gene. LolD is an ornithine decarboxylase(odc)-like gene. We investigated the relationship between each of these lol genes, the endophyte primary metabolism genes and publicly available sequences from a variety of eukaryotes, archea and eubacteria. In both gene families the lol genes are most closely related to the primary metabolism paralogs from ascomycetes. Thus, we hypothesize the lol genes were recruited from within the genome of an ancestor to the endophytes. Funded by USDA-NRI.

540. Molecular Cloning and Characterization of the Polyketide Synthetase Gene from Monasucs ruber KCTC6122. Yun Jung Yang, Jae-Yoon Kim¹, In Hyung Lee. Food & Life Science Major, School of Techno Science, Kookmin University, Seoul 136-702, Korea. ¹Biotech Institute, Kookmin University, Seoul 136-702, Korea.

For genetic dissection of biosynthesis of poyketides in Monascus sp., the polyketide synthetase (PKS) gene was cloned and characterized. Monascus sp. produces various polyketides such as pigments, monakolin K, blood pressure-lowering substance, and nephrotoxic citrinin etc. The degenerate PCR primers were designed based on the conserved amino acid sequences of the KS domain of 14 PKS from various fungi. The PCR products showed over 80% sequence identity to other fungal PKS and they were used for screening of the cosmid library of Monascus ruber KCTC6122 and for shotgun cloning. Characterization of the cloned PKS gene is underway and results will be presented.

541. Effects of depleted uranium on the gene expression and germination frequencies of the Arbuscular Mycorrhizal fungus Glomus intraradices. Carlos M. Loya, Marijn de Jong, Andrew Bradford, Manjula Govindarajulu and Peter J. Lammers. Dept. Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA

Arbuscular mycorrhizal (AM) fungi form obligate symbioses with most plant species. The plants benefit from the association via improved mineral uptake and water relations while the fungus receives fixed carbon from the plant. Depleted uranium contamination of the soil presents a significant environmental problem that until now has not been resolved in an ecologically safe manner. It has been shown that AM fungi, such as Glomus intraradices, are able to synthesize metal ligands like citric and oxalic acid that promote depleted uranium (dU) uptake into fungal and plant material. We are attempting to define the genetic and biochemical basis of these processes in order to design a system for dU management. Initial experiments involve monitoring the germination frequency and elongation rates of dU treated germinating spores by microscopic analysis to determine a metal concentration that inhibits germination by 50% (EC50). We next quantified the expression of two fungal genes, Glutathione-S-Transferase (GST) and a putative vacuolar Zn transport protein (Zrc-1), involved in toxic metal responses in three tissues: germinating spores, intraradical and extraradical mycelium. Our results demonstrate significant up-regulation of GST in germinating spores and extraradical mycelium, with minimal changes in Zrc-1. Experiments in progress will follow the expression of these genes after dU addition directly to symbiotic root tissues.

542. Touch Mediated Ca²⁺ signalling in Neurospora crassa in response to mechanical perturbation. Marris, P.I., Hickey, P.C. and Read, N.D. Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH

In the natural envioronment, filamentous fungi respond to contact stimuli (physical surfaces, obstacles and microtopographical features) which elicit various growth and developmental responses. We are investigating the role of Ca²⁺ signalling in response to mechanical perturbation using Neurospora crassa expressing codon-optimized aequorin to measure cytosolic free Ca²⁺ ([Ca²⁺]_c). We have found that mechanical perturbation (microinjection of growth medium) of fungal cultures generates reproducible transient increases in [Ca²⁺]_c in vegetative hyphae (in 18 h-old cultures), germ tubes (3-6 h-old cultures) but not ungerminated conidia. We are presently addressing several questions: (1) Is the Ca²⁺ response to mechanical perturbation dose dependent? (2) Is the [Ca²⁺]_c continuously elevated in N. crassa grown in shake culture or does the Ca²⁺ signalling machinery adapt to continuous mechanical perturbation? (3) Which Ca²⁺-channels, -pumps and/or antiporters are involved in the response to mechanical perturbation? (4) What physiological/growth/developmental responses to mechanical perturbation can be readily measured in Neurospora?

543. Comprehensive functional analysis of all non-ribosomal peptide synthetases in the corn pathogen Cochliobolus heterostrophus. Shinichi Oide and B. Gillian Turgeon. Dept of Plant Pathology, Cornell University, Ithaca NY, 14853

Filamentous fungi have remarkable ability to produce diverse secondary metabolites that are of interest because of their medical, industrial, and agricultural value. Current knowledge of the significance to the producing fungi themselves, however, is limited. We used the corn pathogen, Cochliobolus heterostrophus (Ch) to investigate function of every non-ribosomal peptide synthetase (NRPS). In this study, a set of strains, each carrying a complete deletion of one of the previously identified 11 NPSs (Lee et al, Eukaryotic Cell, in press), was constructed. Double and triple deletion strains were also constructed, by crossing. All strains were examined for their virulence, stress-response, mating, and morphological phenotypes. NPS2, predicted to encode a NRPS involved in siderophore biosynthesis, has a role in sexual development. Deletion of NPS10, which along with NPS6, is conserved in most ascomycetes, causes morphological defects, such as irregular-shaped colonies and reduced asexual sporulation. Deletion of NPS6 leads, concomitantly, to dramatic reduction in virulence on maize and increased sensitivity to oxidative stress. Deletion of NPS6 orthologs from the rice pathogen Cochliobolus miyabeanus, the wheat pathogen, Fusarium graminearum, and Alternaria brassicicola, a pathogen of the model dicot, Arabidopsis thaliana caused the same phenotype as the nps6-deletion strain of Ch. We further examined the functional conservation of NPS6 by introducing the NcNPS6 ortholog from the saprophyte, Neurospora crassa, into the nps6-deletion strain of Ch. NcNPS6 restored virulence of the Chnps6-deletion strain to maize and resistance against oxidative stress, simultaneously. Together, these data demonstrate that NPS6 encodes a NRPS whose product, a presumed small peptide, acts as a defense factor against oxidative stress and has a role in fungal virulence. In summary, these findings suggest that peptides produced by NRPSs play more diverse roles in fungal metabolism than previously thought.

544. The role of peroxisomes in virulence of Candida albicans. K. Piekarska and M. van den Berg, G. Hardy, E. Mol and B. Distel. Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Conclusive evidence has been provided that the ability to switch between yeast and hyphal forms is essential for pathogenesis of C. albicans. Once initiated, the hyphal program not only results in the switch to the hyphal morphology to allow penetration of host tissues and in secretion of proteases and phospholipases to break them down, but it also facilitates adaptation to the new

environmental conditions, such as the hosts macrophages internal environment. Expression analysis has shown (ref.1) that upon internalization of C. albicans by macrophages the yeast upregulates a set of peroxisomal enzymes, notably two glyoxylate cycle enzymes; isocitrate lyase and malate synthase, and enzymes of the beta-oxidation pathway.

Using a PCR-based disruption technique we have constructed pex5delta/pex5delta and pex13delta/pex13delta deletion strains.

Both of these genes are essential for peroxisome formation. To directly test whether peroxisomal beta-oxidation is essential for virulence of C. albicans, both copies of the FOX2 gene encoding the third enzyme of beta-oxidation pathway, were deleted. As expected, none of the constructed double deletion strains was able to grow on oleic acid as a sole carbon source, while the complemented strains showed wild type growth rates on this substrate. Measurements of beta-oxidation activity in these strains revealed that the fox2delta/fox2delta strain is the most severely affected in fatty acid beta-oxidation. All strains are currently tested for virulence in a mice model for candidiasis.

 1. Lorenz, M.C., Bender, J.A. and Fink, G.G.(2004). Euk. Cell, 102, 1076-1087

545. Functional analysis of paxilline biosynthesis genes. Sanjay Saikia¹, Emily Parker² and Barry Scott¹ . ¹Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. ²Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand

Paxilline is an abundant indole-diterpene secondary metabolite produced by the filamentous fungus Penicillium paxilli. The genes involved in paxilline biosynthesis are organized in a cluster containing at least 5 genes including a geranylgeranyl pyrophosphate synthase (paxG), an FAD-dependent monooxygenase (paxM), a prenyl transferase (paxC) and two cytochrome P450 monooxygenases (paxP and paxQ). The aim of this research is to validate the role of P. paxilli genes in paxilline biosynthesis by biochemical and genetic studies. Radiolabeled precursor feeding studies of P. paxilli mutants identified no stable indole-diterpene in paxG, paxM and paxC deletion mutants but identified paspaline and 13-desoxypaxilline in paxP and paxQ deletion mutants, respectively. Further, a strain lacking the core paxilline cluster genes containing a restriction fragment comprising paxG, paxM, paxC and two other putative genes, sec23 and sec25, produced paspaline demonstrating that this set of genes is necessary and sufficient for the formation of the first stable indole-diterpene in P. paxilli. Constructs of wild-type paxP and paxQ complemented corresponding deletion mutants. The deletion mutants of paxP and paxQ accumulate paspaline and 13-desoxypaxilline, respectively. Feeding of paspaline to a mutant lacking the core paxilline cluster genes but containing wild-type paxP resulted in the synthesis of 13-desoxypaxilline, confirming that PaxP catalyzes the conversion of paspaline to 13-desoxypaxilline, reactions that require demethylation and hydroxylation steps. Similar studies with wild-type paxQ showed that PaxQ converts 13-desoxypaxilline to paxilline in a single step hydroxylation.

546. Characterization of the sulfur metabolism network in the phytopathogenic fungi, Magnaporthe grisea . Saint-Macary ME, Beaurepaire A, Gagey MJ, Barbisan C, Beffa R, Lebrun MH, Droux M. Laboratoire Mixte CNRS/BayerCropscience -14-20, rue Pierre Baizet – 69009 LYON

Nutritional needs of pathogenic fungi during interaction with their hosts remain poorly described, although some investigations have been undertaken on nitrogen and carbon assimilation (1). Recent studies suggest that pathogenic fungi need sulfur micronutrient during the infection step (2). The fungal sulfur complex pathway includes both genes corresponding to the described metabolism from plant and from yeast (3,4). Sulfate is assimilated and reduced through the common pathway for all autotrophic organisms. Then, reduced sulfur is incorporated for synthesis of two amino acids, cysteine and methionine. In these final steps, filamentous fungi catalyzed both the interconvertion of cysteine to homocysteine (the sulfur precursor for methionine synthesis) through the direct and the reverse transsulfuration pathways. Methionine synthesis from cysteine involves two enzymes of the direct transsulfuration pathway, cystathionine gamma-synthase and cystathionine beta-lyase followed by the methylation step catalyzed by methionine synthase. Cysteine synthesis proceeds through sulfhydration of activated serine but also from homocysteine through the reverse transsulfuration sequence cystathionine beta-synthase and cystathionine gamma-lyase (5).

Studies on sulfur metabolism in the phytopathogenic fungi Magnaporthe grisea were developed to understand its role during fungal development on planta. The aim of our work is to identify and to highlight the role of the genes involved in the direct and reverse transsulfuration pathway from the available entire genome of M. grisea using genetic and biochemical tools.

1 Solomon P. et al. (2003), Molecular Plant Pathology, 4(3) : 203-210.

2 Pascon R.C. et al. (2004), Microbiology, 150: 3013-3023

3 Droux M. (2004), Photosynthesis research, 79 : 3331-348.

4 Thomas D. and Surdin-Kerjan Y. (1997), Microbiol. Mol. Biol. Rev. , 61 : 503-532

5 Paszewski A. et al. (2000), Sulfur nutrition and sulfur assimilation in higher plants, Brunold C. (ed), pp 93-105

547. Pathway specific regulation of gibberellin biosynthesis in Fusarium fujikuroi – a new type of bZIP transcription factors. Birgit Schönig, Martina Mihlan, Bettina Tudzynski. Institut für Botanik, Westfälische Wilhelms-Universität Münster.

The ascomycete Fusarium fujikuroi produces the economically important phytohormone Gibberellic Acid. One of the genes responsible for GA production is the P450-4 gene encoding the ent-kaurene oxidase. By performing a systematical promotor deletion approach with the P450-4 promotor, a 30 bp region was identified. Its deletion caused a significant decrease in P450-4 transcription. This promotor region was used in a yeast one-hybrid screening to identify proteins interacting with the 30 bp region. A putative transcription factor, GAR (Gibberellic Acid Regulator), has been identified which shows specific binding affinity to the 30 bp region. Its zinc finger domain is most homologous to zinc fingers of "Krüppel like" transcription factors in animals and plants whereas a similar transcription factor had not been described in fungi before. The mutants obtained from a gene replacement experiment show normal mycelium and colony morphology but reduced growth kinetics. Northern blot analysis under GA non-producing and producing conditions revealed a reduced P450-4 transcript level in the mutants. Macroarray experiments will show if GAR affects the transcription of additional genes or if its influence is GA specific. Interestingly, no homologue to GAR could be identified in the genome of the closely related Fusarium graminearum, which also has no GA cluster, whereas in Magnaporthe grisea a GAR homologue and at least two putative GA biosynthetic genes were found.

548. A platform of three gene clusters is required for the biosynthesis of the bioprotective lolitrem alkaloids. Carolyn Young¹, Sanjay Saikia¹, Richard Johnson², German Spangenberg³, Gregory Bryan² and Barry Scott¹. ¹Centre for Functional Genomics, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. ²AgResearch Grasslands Research Centre, Palmerston North, New Zealand. ³Plant Biotechnology Centre, Primary Industries Research Victoria, La Trobe University, Australia

Lolitrems are potent tremorgenic mycotoxins produced by Epichloë and Neotyphodium endophytes in association with their host grass Lolium perenne. These indole-diterpene alkaloids are responsible for the animal syndrome known as ryegrass staggers and have insecticidal properties. Using candidate genes isolated as ESTs from cDNA libraries combined with chromosome walking from a known lolitrem biosynthesis locus (ltm) we have isolated and characterized a ~100 kb platform of three gene clusters required for lolitrem biosynthesis. The clusters are separated by highly repetitive AT-rich sequences that are devoid of open reading frames but contain remnants of retrotransposon sequences. The ltm clusters 1 and 2 contain eight genes, seven of which are orthologues of the characterised P. paxilli paxilline biosynthesis gene cluster (pax). Cluster 3 contains at least two genes, ltmJ, a P450 monooxygenase, and ltmE a gene fusion of a prenyl transferase and a dimethylallyl tryptophan synthase. All 10 ltm genes have similar expression profiles and are highly expressed in planta where the production of lolitrem B is most prevalent. When ltmC and ltmM were placed under the control of a paxM promoter they complemented P. paxilli paxC and paxM deletion mutants, confirming that these two genes are functional orthologues of paxC and paxM. Disruption of the remaining genes will help elucidate the biochemical pathway for lolitrem biosynthesis.

549. A second D-galactose catabolic pathway in Hypocrea jecorina: Involvement of pentose pathway enzymes and implications on cellulase induction. Seiboth B, Gamauf C, Hartl L and Kubicek CP. Institute of Chemical Engineering, TU Vienna, Austria

Hypocrea jecorina is specialized in the degradation of a wide spectrum of plant cell wall polysaccharides. Hemicelluloses are beside cellulose the second most abundant compound in plant cell wall consisting to a major part of sugars such as L-arabinose, D-galactose, and D-xylose. In H. jecorina D-galactose is first phosphorylated by galactokinase, while D-xylose and L-arabinose are metabolized by an interconnected pathway consisting mainly of NADPH-linked reductions and NAD-linked oxidations. Strains defective in galactokinase, convert D-galactose via a second path similar to the pentose catabolic pathways and use some of their enzymes. We have biochemical and genetic evidence that D-galactose is converted to galactitol by a NADPH dependent reduction catalyzed mainly by D-xylose reductase, an enzyme of the D-xylose metabolism. L-arabinitol 4-dehydrogenase, an enzyme of L-arabinose metabolism, catalyzes then the oxidation of galactitol. A third pentose pathway enzyme involved is xylitol dehydrogenase which catalyzes a subsequent step, the oxidation of D-sorbitol to fructose. We also will show that both D-galactose catabolic pathways identified are essential for full cellulase induction on lactose and will discuss the importance of both pathways for inducer formation.

550. Identification and characterization of novel type III polyketide synthases in Aspergillus oryzae. Yasuyo Seshime¹, Praveen Rao Juvvadi¹, Isao Fujii², Katsuhiko Kitamoto¹. ¹Department of Biotechnology, University of Tokyo, Tokyo, Japan. ²Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan

Chalcone synthases (CHSs) are prominent members of type III polyketide synthase (PKS) superfamily, and are known to play a vital role in the biosynthesis of plant phenylpropanoids and flavonoids. By virtue of their agricultural and pharmaceutical values, products of type III PKSs have received significant attention in recent years. Although the distribution of type III PKSs has always been considered to be restricted to either plants or bacteria, the present study reveals the existence of type III PKS-encoding genes in an industrially useful fungus, Aspergillus oryzae. While A. oryzae genome data mining revealed 4 putative CHS-like sequences (csyA, csyB, csyC and csyD), reverse transcription polymerase chain reaction indicated the expression of 3 genes (csyA, csyB and csyD). In addition, we confirmed the presence of type III PKS-encoding genes in other filamentous fungi by using the available fungal genome databases. Phylogenic analysis revealed the distinction of fungal type III PKSs from those of bacteria and plants. Interestingly, the other species of Aspergilli (Aspergillus nidulans, Aspergillus fumigatus) lacked these genes. Significantly, A. oryzae alone contained higher number of putative chalcone synthase homologues in comparison to Neurospora crassa (1), Fusarium graminearum (1), Magnaporthe grisea (2) and Phanerochaete chrysosporium (3). Disruption of these novel PKS genes in A. oryzae is being pursued to elucidate their function in A.oryzae.

551. Asparaginase Genes from Aspergillus nidulans. Ann L. Onton and Patricia M. Shaffer, Department of Chemistry, University of San Diego, San Diego, CA, 92110, USA

L-asparaginase is an amidohydrolase that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia. Asparaginases are classified into two categories, type I and type II, the latter being regulated. Aspergillus nidulans has two asparaginase genes, apnA (on chromosome II) and ahrA (on chromosome VIII). The enzyme expressed by the ahrA gene is categorized as a type II asparaginase [Shaffer et al. (1988) Mol. Gen. Genet. 212, 337-341].

With the help of Cereon Genomics, LLC (subsidiary of Monsanto) and Whitehead Institute (WI) databases we obtained the sequences of both asparaginase genes. From these sequences we prepared PCR primers and used them with genomic DNA [wild type A4(Glasgow)] to produce nucleotide sequences (1214 nucleotides containing the ahrA gene and 706 nucleotides containing the apnA gene), as well as for a mutant of each.

Next we prepared labeled sequences for each gene and probed a UniZap cDNA library. We have the cDNA for the ahrA gene and are in the process of obtaining the cDNA for the apnA gene. The expression of each of these genes is in the planning stages. Since type II asparaginases are used as a cure for childhood acute lymphoblastic leukemia (ALL), this research may have some pharmaceutical significance. We are grateful to Graduate Women in Science for funding and Mark Caddick scientific input.

552. Withdrawn

553. Characterization of metal binding sites in cell wall of Neurospora crassa. T. Naga Sowjanya and P. Maruthi Mohan. Department of Biochemistry, Osmania University, Hyderabad-500 007 (A.P.), INDIA

 Our previous studies showed that significant levels of calcium and magnesium are present on cell wall fraction of N.crassa. Further, the role cell wall calcium in storage function and maintaining the structural integrity was demonstrated. Based on the above work an improved cell wall preparation method to remove SDS and membrane contamination was used to study binding of metal ions. Ca (3 moles 100 mg-1) and Mg (1.5 moles 100 mg-1) binding was distinct between pH 5 - 6 without competitive effects. The bound metals could be desorbed with dilute HCl or EDTA and rebinding could be demonstrated. Co and Cu binding (3 and 4 moles 100 mg-1) displaced 30% of Ca and 50% of Mg respectively. Presence of NaCl (0.5 M) inhibited 30% of Ca binding but Mg binding was not affected. Glutaraldehyde cross-linked walls bound less Ca (30%), while Mg binding was unaffected. Modification of carboxyl groups of cell walls with carbodiimide resulted in 25% loss of Ca binding, while Mg binding was totally abolished. Enzymatic treatment of cell wall (glucanase, trypsin, chitinase and alkaline phosphatase) caused significant loss of proteins and metal binding capacity. Alterations in Co binding to cell walls of cobalt-resistant and sensitive mutants were observed. The physiological significance of distinct Ca and Mg binding sites on cell wall and the influence of toxic metal ions will be discussed.

554. Developmental Expression of Two Forms of Arginase from N. crassa. Gloria E. Turner and R. L. Weiss. University of California, Los Angeles, CA

The physiological role of multiple arginases in N. crassa is not understoood. The two forms are differentially expressed from a single locus (aga) and both proteins are localized to the cytoplasm. The 36-kD protein is expressed under all conditions, whereas the 41-kDa form is detected when N. crassa is grown in the presence of arginine. In this study we determine developmental expression of the two arginase transcripts and proteins by characterizing conidia and conidial germination at 2, 4 and 8 hours. Both major forms of the protein are stored in conidia, however the 1.4-kb transcript is not detected. The RNA's are temporally expressed during early germination. To better understand the role of arginase in conidia and the nature of the temporal expression we examined the effects of related metabolites; arginine, ornithine, proline, glutamate and glutamine on protein storage and temporal expression. These metabolites were used as supplements or sole nitrogen sources. The 1.4-kb transcript was never detected in conidial samples, however the 1.7-kb transcript was detected in conidia under all supplemented conditions. The temporal RNA expression early in germination, found in minimial arginine and ornithine supplementation, is abolished in proline, glutamate or glutamine supplementation as well as nitrogen limitation. In addition to abolishing the temporal expression, proline and glutamine repress aga transcription when they are the only source of nitrogen. Storage of arginase protein was detected in all conidial samples except when glutamate was used as the sole nitrogen source. These results support a novel pathway utilizing arginase for glutamate storage during conidiagenesis.

555. Characterization of four clustered genes associated with the biosynthesis of a red perithecial pigment in Nectria haematococca. Christelle Vasnier, Stéphane Graziani, Marie Dufresne and Marie-Josée Daboussi. Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France

Recently, we have identified a typical polyketide synthase (PKS) required for the synthesis of the red pigment present in the cell wall of perithecia of Nectria haematococca. Clustering between pksN, the gene encoding the PKS and other genes was expected in view of the typical genomic organization of genes involved in secondary metabolism pathways in fungi. Here we report the molecular organization of the genomic region surrounding pksN and the characterization of four novel genes clustered with pksN. The predicted amino acid sequences encoded by these genes designated ppcA, ppcB, ppcC, ppcD are very similar to those of cytochrome P-450 monooxygenase (ppcA), a putative NADH-flavin oxidoreductase (ppcB), an O-methyl transferase (ppcC) and an hypothetical protein displaying a DNA-binding domain typical of a GAL4-like Zn2Cys6 binuclear zinc finger (ppcD).

We performed disruption experiments to determine whether some of these genes are also involved in the red pigment biosynthesis. Disruption of ppcA led to total loss of the pigment suggesting that, as expected, it is part of a new biosynthetic gene cluster designated PP for perithecial pigment. Targeted disruption experiments of other genes are underway. Although sequences identical to the AflR binding sites were foundin the promoter regions of at least two of thefour ppc genes, it is not yet known whether the PP cluster contains an AflR-like regulatory gene.

Further work will allow us to determine the nature of the red pigment and will provide an interesting comparison of the biochemistry and biology between toxins and pigments in fungi.

556. Functional genomics studies for identifying genes involved in aflatoxin formation in Aspergillus flavus. Jiujiang Yu¹, Jeffery R. Wilkinson¹, H. Stanley Kim², William C. Nierman², Gary A. Payne³, Joan W. Bennett⁴, Jong H. Kim⁵, Bruce C. Campbell⁵, Deepak Bhatnagar¹, and Thomas E. Cleveland¹. ¹ USDA/ARS, Southern Regional Research Center, New Orleans, LA, USA. ² The Institute for Genomic Research, Rockville, MA, USA. ³ North Carolina State University, Raleigh, NC, USA. ⁴ Tulane University, New Orleans, LA, USA. ⁵ USDA/ARS, Western Regional Research Center, Albany, CA, USA.

Aflatoxins are secondary metabolites produced mainly by the molds Aspergillus flavus and A. parasiticus. Because these compounds are toxic and extremely carcinogenic to animals, they pose a serious risk to human health. The aflatoxin biosynthetic pathway and its genetic regulation have been studied for decades, revealing a well organized aflatoxin pathway gene cluster consisting of 25 genes within a 70 kb DNA region. In order to better understand the molecular mechanisms that control or regulate aflatoxin production, identification of genes (gene profiling) using A. flavus expressed sequence tags (ESTs) and microarrays is currently being performed. Sequencing and annotation of A. flavus ESTs from a normalized A. flavus cDNA library identified 7,218 unique EST sequences. Genes that are putatively involved in aflatoxin biosynthesis, regulation and signal transduction, fungal virulence or pathogenicity, stress response or antioxidation, and fungal development were identified from these ESTs. Gene profiling using microarrays has thus far identified hundreds of genes that are highly expressed under aflatoxin-producing conditions. Further investigations on the functions of these genes are underway. This research is expected to provide information for developing new strategies for control of aflatoxin contamination of agricultural commodities.