Biochemistry and Secondary Metabolism
458 Identifying Genes Associated with Ochratoxin A Production in Aspergillus carbonarius and Aspergillus niger. Z., Ilic*1, J.I., Pitt2, D.A., Carter1. 1 Dept of Microbiology, University of Sydney, NSW 2006 2 Food Science Australia, North Ryde, NSW 2113
Aspergillus carbonarius and A. niger belong to the family of black Aspergilli. Black Aspergilli are often found in association with certain commodities, such as dried fruit, grapes and wheat. A. carbonarius and to a lesser extent, A. niger, are common producers of a chlorinated cyclic polyketide, ochratoxin A (OA). OA is nephrotoxigenic and is a world-wide problem, only recently found to be caused in some cases by the black Aspergilli. Very little is known about the biosynthesis of this secondary metabolite, and it is important to understand OA biosynthesis in order to develop strategies to control this problem. An initial step in understanding toxin production is to assess the genetic differences between toxigenic and non-toxigenic strains. Random Amplification of Polymorphic DNA (RAPD) analysis was used to investigate the genetic background and diversity of A. niger and A. carbonarius strains of varying toxigenicity. There was no correlation seen between genetic relatedness and degree of to toxigenicity. We have also begun to investigate the genes involved in the OA biosynthetic pathway in A. carbonarius and A. niger, using both degenerate primer PCR and plasmid mutagenesis. The former uses primers based on conserved regions of the gene for polyketide synthase, an important enzyme in the formation of polyketides such as ochratoxin. For plasmid mutagenesis we are optimising a transformation system for A. carbonarius and A. niger to introduce the disruption vector pAN7-1. A simple screen for toxigenicity allows us to identify colonies with insertions into OA biosynthetic genes, thereby allowing the isolation of these genes.
459 Linkage of molecular markers with trichothecene chemotypes in Gibberella zeae. Nancy J. Alexander1, Ronald D. Plattner1, Robert Bowden2, and John F. Leslie2. 1Mycotoxin Research Unit, National Center for Agricultural Utilization Research, USDA/ARS, Peoria, IL, 2Dept. of Plant Pathology, Kansas State University, Manhattan, KS
Gibberella zeae (anamorph Fusarium graminearum) produces mycotoxins, most notably, deoxynivalenol (DON) and nivalenol (NIV). Most of the strains found in North America produce DON, while NIV-producing strains are more common in Asia. It is not known how the biosynthetic pathway of toxin production differs in the two types of producers. Chemically, NIV differs from DON by having an OH group at the C4 position. In an attempt to distinquish genetic control of toxin formation, we crossed a high DON-producer with a low NIV-producer and analyzed 99 progeny. Progeny were scored for type of toxin produced as well as relative amount of toxin. Southern hybridizations and polymerase chain reaction (pcr) were used to detect polymorphism at the TRI5 (trichodiene synthase) locus. Results suggest that the type of toxin produced, either NIV or DON, was controlled by a single gene which was completely linked to TRI5. The amount of toxin produced, either high or low, was controlled by another single gene which was unlinked to TRI5 or the toxin type locus. These results should help in our investigations of the ecology of the organism as well as a better understanding of toxin biosynthetic pathways.
460 Characterization of a Locus Required for Mycotoxin Biosynthesis in Aspergillus spp. Yongqiang Zhang, Nancy Keller. Department of Plant Pathology and Microbiology Texas A&M University, College Station, TX 77843-2132
Sterigmatocystin (ST) is a carcinogenic polyketide produced in several Aspergillus species including A. nidulans. In A. nidulans, the ST biosynthesis genes (stc genes) are clustered on chromosome IV. Mutagenesis and subsequent screening have identified 23 mutants that were defective in ST biosynthesis and unlinked to the stc gene cluster. Diploid analysis demonstrates that five of these mutants likely comprise one locus. Furthermore, a genetic segregation study shows that this locus is linked to yA and biA on chromosome I. Two cosmids of the A. nidulans trpC library complement the mutated locus. Subcloning and transformation studies identify a 17 kb subclone of these cosmids which can complement the locus. Sequencing data reveals that this 17 kb DNA fragment contains a fatty acid transporter homolog, a translation elongation factor homolog as well as other less defined ORFs. Further investigation to identify the gene required for ST/AF biosynthesis is under way.
461 Cloning and characterization of a polyketide synthase gene (pksN1) required for synthesis of a perithecial red pigment in Nectria haematococca. Stephane Graziani. Marie-Josťe Daboussi. Universitť Paris Sud, IGM, Orsay France
Most studies of pigment biosynthesis in fungi have focused on dark brown pigments, melanins, shown to be important pathogenic factors for both plant- and animal- pathogens. In contrast to this biosynthesis, little is known about pigment biosynthesis in reddish fungi. In Nectria haematococca, mutants affecting the coloration of mycelia and perithecia (hyperproduction of red pigments or albinos) have been obtained through mutagenesis. The analysis of these mutants showed that the lack of pigmentation of the perithecium wall and that of mycelia result from the operation of two distinct biochemical pathways. Some of the pigments formed in the vegetative phase are naphtoquinones. The composition of the pigment found in the wall of perithecium is unknown. Here we reported on the cloning of the gene which is required for the red coloration of perithecia by complementation of the I4 mutant (white perithecia) by a cosmid library. Subcloning identified a 5 kb DNA fragment able to restore the mutant phenotype. DNA sequencing revealed high degree of homology with polyketide synthase genes. On the basis of the presence of at least the beta-ketoacyl synthase and acyl transferase domains usually present in polyketide synthases and fatty acid synthases, we propose that the I4 gene encodes the polyketide synthase which initiates red pigment biosynthesis. In addition, we initiated studies on the expression of this gene in order to determine if it is developmentally regulated.
462 Loline biosynthesis in Neotyphodium uncinatum: identifying biosynthetic genes with the use of subtractive hybridization. Martin J. Spiering1, Jimmy D. Blankenship1, Heather H. Wilkinson2, and Christopher L. Schardl1. 1Department of Plant Pathology, University of Kentucky, Lexington, Kentucky and 2Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, U.S.A.
The insecticidal 1-aminopyrrolizidine alkaloids (e.g. N-formyl and N- acetyl loline) are present in the grass meadow fescue (Lolium pratense) when infected by the fungal endophyte Neotyphodium uncinatum, significantly strengthening the plant∆∆s resistance to insect attack. For isolating key genes involved in loline biosynthesis in N. uncinatum we have adjusted culture growth conditions to induction of loline production in mycelium at high levels and to suppression of production, respectively. RNAs isolated from mycelium grown under these conditions were used as śśtester∆∆ (producing) and śśdriver∆∆ (suppressed) in subtractive hybridization. Northern analysis showed significant expression of subtracted transcripts only under loline-producing conditions. By using the subtracted cDNAs as probe, several hybridizing (śś+∆∆) clones have been identified from a cDNA library of loline- producing mycelium. Sequence analysis of a śś+∆∆ clone indicated a highly significant match with an aspartate kinase, an enzyme involved in the biosynthesis of methionine and threonine. In addition, we have cloned a DNA fragment closely linked to a locus controlling loline production in the endophyte Epichlo festucae. Analyses indicated a highly significant similarity to O-acetyl-homoserinesulfhydrylase (OAH), an enzyme likewise involved in the biosynthesis of methionine. The OAH-like gene is highly expressed during loline production in N. uncinatum, and was detected only in loline-producing endophytes. These findings suggest a significant role in loline biosynthesis of enzymes normally functioning in methionine biosynthesis, possibly by directing methionine to production of polyamines (e.g. spermidine), which are intermediates in the biosynthesis of related plant pyrrolizidine alkaloids. This work was supported by the U.S. National Science Foundation Integrative Plant Biology Program 9808554.
463 Antimicrobial and antichagasic activities produced by Talaromyces flavus. Tatiana Pereira da Silva de Freitas, Jairo Kenupp Bastos, Sťrgio de Albuquerque, Suraia Said. Faculdade de CiÍncias FarmacÍuticas de Ribeir„o Preto, Universidade de S„o Paulo, Ribeir„o Preto, Brasil.
Chagas' disease, which is caused by Trypanosoma cruzi, attacks about 116 million people in the world and the lack of efficient and nontoxic medicine is a constant concern in tropical countries. In addition, microbial resistance to antibiotics is increasing and many antibiotics are no longer effective. Thus, the search for new antichagasic and antimicrobial drugs is desirable. In the present study T. flavus (CCT3138) was inoculated in a two-step culture, the final broth was partiotioned with chloroform and butanol and the extracts were analyzed. The best antimicrobial activity was detected in the chloroform extract obtained from cultures after 192 hours of incubation. The extract from Czapeck medium had the best activity against Candida albicans (76% more active than the standard miconazole 4 mg/ml) and against Micrococcus luteus (88% more active than the standard penicillin G 0.6075 U/ml). The extract from Takeuchi medium showed the best activity against Staphylococcus aureus (200% more active than the standard used, i.e. penicillin G 1.215 U/ml). The chloroform extracts from the Takeuchi and Czapeck media had the same activity against Escherichia coli as the standard used (streptomicin 77.7 U/ml). The activity contained in the water extract from the culture broth of Czapeck medium, which was incubated for 96 hours, was responsible for the lysis of 97.58% of trypomastigote forms of T. cruzi (red blood cells remained normal). The antichagasic activity was also detected in other extracts but with a lower effect. The standard used to compare the efficiency of the extracts was Gentian violet, which killed 100% of the trypomastigote forms of the parasite. Supported by FAPESP (Proc. n . 00/06847-5).
464 Comparative sequence analysis of trichothecene biosynthesis gene clusters from DON/NIV chemotypes of Gibberella zeae. Theresa Lee, Daewoong Oh, Hye-Seon Kim, Jeong-Kwan Lee, Sung-Hwan Yun1, and Yin-Won Lee. School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea, 1Division of Life Sciences, Soonchunhyang University, Asan 336-745, Korea
G. zeae, one of major causative fungi of cereal scab, is chemotaxonomically divided into two groups based on trichothecene production; deoxynivalenol(DON) chemotype and nivalenol(NIV) chemotype. To study genetic diversity of trichothecene production by these two chemotypes frequently found in Korea, the gene cluster required for trichothecene biosynthesis in each chemotype was cloned and sequenced. H-11 was a representative of DON chemotype isolated from corn and 88-1 was a NIV producer from barley. Sequencing analysis revealed a 23kb and a 26kb gene cluster along with unlinked Tri101 genes from H-11 and 88-1, respectively. Each gene cluster consisted of 8 Tri genes and 2 other ORFs (probably Tri9 and Tri10) with the same order and transcriptional directions as that of Fusarium sporotrichioides. Comparative sequence analysis showed that all the genes except Tri7 were very conserved among H-11 and 88-1, ranging from 87.8 to 95.9% at nucleotide level and from 81.8 to 97.3% at amino acid level. However, Tri7 was strikingly different showing 78.8% similarity at nucleotide and 53.9% at amino acid level. Sequence analysis of Tri7 from other DON and NIV producers revealed that all the Tri7 ORFs from DON chemotypes tested carried several mutations and an intron containing different numbers of a 11bp-tandem repeat while none of the NIV chemotype tested had the repeat or mutations. This result indicates that Tri7 may be a key enzyme for differentiation of DON and NIV in the trichothecene biosynthetic pathway. Further study to determine a function of Tri7 in DON/NIV chemotypes is in progress.
465 Genetic manipulation of Gibberella zeae for increasing outcross Jeong-Kwan Lee, Sung-Hwan Yun1, Theresa Lee, and Yin-Won Lee. School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea, 1Division of Life Sciences, Soonchunhyang University, Asan 336-745, Korea
G. zeae, a self-fertile (homothallic) ascomycete is an important plant pathogen causing head blight of wheat and barley, and stalk or ear rot of maize. In addition, the fungus produces mycotoxins including trichothecenes and zearalenone on diseased crops so it has been a potential threat to human and animal health. However, homothallism of this fungus has been an obstacle to genetic analysis of pathogenicity as well as mycotoxin production of G. zeae. To circumvent this barrier, several G. zeae isolates producing either deoxynivalenol (DON) or nivalenol (NIV) were genetically manipulated to be used as parents of sexual crosses in this study. First, each representative of two chemotypes was transformed with either of two drug resistance genes (HygBR for resistance to hygromycin B and GenR for geneticin), creating transformants with either DON;HygBR or NIV;GenR. A significant number of sexual progeny showing resistance to both drugs were easily obtained from a cross between two different transformants and the progeny segregated independently for the mycotoxin chemotype and drug resistance. Second, a mating type gene (MAT-2) of a DON producer, which carries two opposite MAT genes (MAT-1 and MAT-2) in a single nucleus, was deleted and replaced with a vector carrying a green fluorescent protein gene. The resulting MAT-2-deleted strain was self-sterile, suggesting that both MAT genes are required for homothallism of G. zeae. A cross between this MAT-2- deleted DON producer and a homothallic NIV producer produced fertile recombinant perithecia on the mating plate although the fertility was lower than that of self of wild type. Ascospores from those perithecia segregated independently for green fluorescence and mycotoxin chemotype, suggesting normal meiosis occurred in this cross. Thus, use of drug resistance genes and MAT gene manipulation provided simple and efficient ways to obtain sexual recombinants of G. zeae, which allows genetic analysis of this fungus.
466 Enhancement of antimicrobial activity produced by Aspergillus fumigatus. Niege AraÁari Jacometti Cardoso Furtado, Suraia Said, Izabel Yoko Ito, Jairo Kenupp Bastos. Faculdade de CiÍncias FarmacÍuticas de Ribeir„o Preto, Universidade de S„o Paulo, Ribeir„o Preto, Brasil.
In the course of a screening program for new antibiotic producers, a strain of Aspergillus fumigatus was isolated from Brazilian soil samples (Pantanal, MS). This fungus was first grown on seed medium to achieve biomass and after 24 hours it was transferred to a production medium. A pool of living or autoclaved microorganisms was added or not to the fungus culture on the second day in the production medium at the ratio of 1:6 to increase antibiotic production. After different times the culture broths were separated from mycelium and extracted with chloroform and butanol. Extracts were analyzed by HPLC and submitted to antimicrobial activity evaluation by agar diffusion assay and bioautography. There was a 55% increase in activity against Staphylococcus aureus, a 63% increase against Candida albicans and more than 100% against Micrococcus luteus in chloroform extracts from the broth of cultures in which the pool of living or autoclaved microorganisms was present. The chromatographic profiles of chloroform extracts from the two culture conditions were different. By bioautography two additional active compounds were detected in chloroform extract from the broth of cultures grown in the presence of pooled microorganisms and 1,3,5-trimethoxybenzene and 2,5-dimethoxyphenol were identified in this extract. Supported by FAPESP (Proc. n . 99/09850-8).
467 Localisation of ACV synthetase of Aspergillus nidulans. William Vousden and Geoffrey Turner. Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, Western Bank, Sheffield, UK
The biosynthetic pathway for penicillin is now well understood from extensive studies in both Penicillium chrysogenum and Aspergillus nidulans. There is also evidence for compartmentalisation of the pathway, since it has been shown that acyl transferase, the enzyme responsible for the final step in biosynthesis, is located in microbodies. Localisation of L-aminoadipyl-L-cysteinyl-D-valine synthetase (ACVS), the peptide synthetase which carries out the first step in the pathway, proved more difficult using available antibodies, though there had been reports that it might be membrane associated, possible within the vacuole. We have used a GFP fusion construct in an attempt to resolve this problem. GFP was fused to the 5' end of the ACVS, since previous work had indicated that loss of some of the N-terminal amino acids did not prevent penicillin biosynthesis. This fusion gene was placed under the control of the strong, regulatable alcA promoter, and introduced into A. nidulans using a gene replacement technique. A single copy transformant was analysed for penicillin production and GFP localisation within the mycelium. The strain was still able to produce penicillin from the GFP-ACVS fusion, though at a lower yield than wild-type, indicating partial functioning of the gene fusion. Fluorescence microscopy revealed that the GFP was found throughout the mycelium, but appeared to be excluded from the vacuoles. It appears that vacuolar localisation of ACVS is not required for penicillin biosynthesis.
468 Functional analysis and expression studies of the gibberellin biosynthesis genes in Gibberella fujikuroi. Bettina Tudzynski, Martina Mihlan and Jessica Schulte. Westfšlische Wilhelms-Universitšt MŁnster, Institut fŁr Botanik, Schlossgarten 3, 48149 MŁnster, Germany
Gibberella fujikuroi produces large amounts of gibberellins under conditions of nitrogen limitation. Recent studies have shown that at least 6 genes of the gibberellin (GA)-biosynthetic pathway are clustered in chromosome 4 in the G. fujikuroi genome; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (cps/ks), a GA-specific geranylgeranyl diphosphate synthase (ggs2) and four cytochrome P450 monooxygenases. (Tudzynski et al. 1998, Tudzynski and HŲlter,1998). To study the function of several genes of this cluster, we used a gene replacement approach followed by GC-MS and HPLC analysis of the mutant culture fluids as well as feeding experiments. Since the biosynthesis of fungal gibberellins includes at least 13 enzymatic steps, we expected some of the biosynthesis genes to have a multifunctional character. To find out the pathway steps catalyzed by each of the P450 monooxygenases, we transformed the single genes into a G. fujikuroi GA-defective mutant which lacks the entire GA gene cluster due to a large deletion on chromosome 4 (LinnemannstŲns et al. 1999). The single genes are highly expressed in this mutant strain, clearly demonstrating that the regulation factors needed for their expression are not located within the gene cluster. Using this procedure we have identified the multifunctional character of both P450-1 and P450-4. Because the gibberellin biosynthesis is regulated by ammonium, we cloned several genes which are involved in nitrogen regulation, such as the general nitrogen regulators, areA and nmr, as well as gs and gdh1 encoding a glutamine synthase and a NAD-dependent gluatamate dehydrogenase. Gene replacement of areA led to a significant reduction of gibberellin formation by repressing the expression of the GA-pathway genes. Complementation of the areA-deficient mutant with the areA wild-type copy completely restored the gibberellin production, which suggests that the positive acting regulatory protein AREA directly controls the transcription of the pathway (Tudzynski et al 1999). References B. Tudzynski, H. Kawaide, and Y. Kamiya (1998). Curr. Genetics, 34: 234-240. B. Tudzynski and K. HŲlter (1998). Fungal Genetics and Biology, 25: 157-170. B. Tudzynski, V. Homann, B. Feng, and G.A. Marzluf (1999). MGG, 261: 106-114. P. LinnemannstŲns, T. Vo , P. Hedden, P. Gaskin and B. Tudzynski (1999). Appl. Env. Microbiol. 65: 2558-2564.
469 Isolation and characterization of a second gene in the pyridoxine synthesis pathway of fungi, plants, archaebacteria and a subset of eubacteria. Marilyn Ehrenshaft and Margaret E. Daub, Dept. of Plant Pathology, NCSU, Raleigh, NC, 27695
Recent work in our laboratory uncovered definitive evidence that the majority of the biological world uses a pathway for pyridoxine (vitamin B6) synthesis that diverges from that of the well-characterized Escherichia coli pathway. The Cercospora nicotianae PDX1 gene was originally isolated as part of a project to genetically characterize C. nicotianae resistance to its own endogenous photosensitizer, cercosporin, and to other photosensitizers of diverse structure and solubility. PDX1 homologues belong to one of the most highly conserved protein families yet identified, and are found in fungi, plants, and archaebacteria, as well as in many eubacteria that do not encode homologues to the E. coli pyridoxine genes. Homologues to the gene we are now calling PDX2 were first noted because they were consistently found in the same subset of organisms that encoded PDX1 homologues. In some organisms they were found in the same operon (Bacillus subtilis and Haemophilus influenzae) or within 500 bp of each other (yeast). We have isolated the C. nicotianae PDX2 gene using a degenerate primer strategy. We provide direct evidence this gene is involved in pyridoxine synthesis via complementation of pyridoxine auxotrophs and also by our production of a pyridoxine auxotroph via gene disruption of pdx2. We show that PDX2 proteins are less well conserved than their PDX1 counterparts but contain several conserved protein motifs. We also present evidence to support the hypothesis that the enzymatic step catalyzed by the PDX1 protein precedes that of the step catalyzed by the PDX2 protein.
470 New mutants of the carotenoid pathway of Neurospora crassa. Javier Avalos1, Loubna Youssar1, Nabil Arrach1, and Tom J. Schmidhauser2. 1Departamento de Genetica, Universidad de Sevilla, Spain 2Department of Biology, University of Louisiana at Lafayette, LA, USA
Three novel kinds of carotenoid mutants of N. crassa have been identified. Two of them were isolated taking advantage of the accumulation of a higher proportion of final products of the carotenoid pathway upon photoinduction at low temperature. Using illumination at 6 C as screening conditions, two types of pale reddish mutants have been isolated. Two of them lack neurosporaxanthin and accumulate a mixture enriched in at least one polar carotenoid. Both mutants are complemented by al-2 and their mutant al-2 alelles have been sequenced. They are presumably affected in the cyclase activity of Al-2, as deduced from the phenotype and the location of the mutations in the cyclase domain of similar genes in other fungi. The chemical nature of the carotenoids accumulated by these mutants is under investigation. Two mutants of a second type have a different reddish pigmentation, lack neurosporaxanthin and are not complemented by al-2. Experiments to determine their biochemical phenotype are under way. Extensive UV mutagenesis was carried out in an attempt to isolate mutants pigmented in the dark. No mutants could be identified out of 500,000 survivors from a wild type strain, but two orange mutants were isolated out of 20,000 from an ovc mutant. The ovcphenotype is characterized by an increased carotenoid biosynthesis in the light. The two mutants, called lic (light independent carotenogenesis) accumulate carotenoids in the dark and show colonial growth and enhanced conidiation. Phenotypic characterization of these mutants is in progress.
471 Isolation and characterization of two key regulatory enzyme genes involved in isoprenoid metabolic biosynthesis of Aspergillus nidulans. Guangyi Wang and Jay D. Keasling. Chemical Engineering, U. C. Berkeley, Berkeley, CA
Isoprenoids are a fascinating family of compounds derived from mevalonate. Fungal isoprenoids comprise a structurally diverse group of primary and secondary metabolites, which range from fungal plant hormones, to mycotoxin, to antimicrobial agents, to pharmaceutically important antibiotics. In many cases, the low production level of the isoprenoid of interest in the native source is a major limiting factor for practical extract and purification before use. 3-hydroxy-3-methylgutaryl-CoA reductase (HMGR) and isopentenyl pyrophosphate (IPP) isomerase are two key regulatory enzymes in the isoprenoid metabolic pathway. HMGR catalyzes the reduction of HMG-CoA to mevalonate and IPP isomerase converts IPP to dimethylallyl diphosphate (DMADP), a reversible reaction. Over-expression of the two enzymes has been shown to dramatically increase carotenoid production of interest in several systems. In order to engineer metabolic biosynthesis of isoprenoids in Aspergillus nidulans, genomic fragments of HMGR and IPP isomerase have been isolated from A. nidulans by homology. Sequence analysis revealed high homology of HMGR and IPP isomerase gene fragments from A. nidulans with the HMGR and IPP isomerase genes from other organisms. The genomic DNA fragments have been used as probes to isolate full-length genes from a cosmid genomic library of A. nidulans.
472 Mapping a locus involved in loline alkaloid expression in the fungal endophyte Epichlo festucae. Gang, Liu1, Mayfield, Coreline T.1, Schardl, Christopher L2. And Wilkinson, Heather H1. 1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas and 2Department of Plant Pathology, University of Kentucky, Lexington, Kentucky
Loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) are fungal secondary metabolites often present in grasses symbiotic with endophytes in the genera Epichlo and Neotyphodium. Endophytes producing loline alkaloids provide grass hosts with enhanced protection from herbivory, drought and pathogens. In an earlier study, segregation analysis supported a single locus contributing to a naturally occurring lolines expression polymorphism in Epichlo festucae (Lol+ vs. Lol-). Identification and cloning of an AFLP marker tightly linked to the Lol+ phenotype has permitted map based cloning of at least a portion of that locus. A 6,144 clone bacterial artificial chromosome (BAC) genomic library of a Lol+ E. festucae parent was constructed. Current analysis centers around one 115 kb BAC clone containing the AFLP marker. End sequencing of this and overlapping BACs provided additional markers (0.2 to 4.5 kb in length). Southern analysis showed that markers identified at one end of the BAC are present in both the Lol+ and the Lol- parent. Markers located closer to the opposite end of the BAC, from 85 to 115 kb away from the shared region, only hybridize to DNA from the Lol+ parent. Preliminary sequence analysis indicated genes in this unique region related to those for ornithine decarboxylase (ODC) and an O-acetylhomoserinesulfhydrylase/thio-lyase (OAH). ODC catalyzes putrescine synthesis and OAH can serve in a methionine synthesis pathway. Both putrescine and methionine are precursors of spermidine, so it is of interest that, based on their structure, loline alkaloids have previously been suggested to be spermidine derivatives. Our data support a relationship between lolines and polyamines.
473 Analysis of an ergovaline-deficient Neotyphodium endophyte resulting from a peptide synthetase gene knockout. R. D. Johnson1, P. Damrongkool1, J. Wang2, C. L. Schardl2 and D. G. Panaccione1. 1 West Virginia University, Morgantown; 2 University of Kentucky, Lexington.
Toxic ergopeptines are produced by several fungi, including the ergot fungus Claviceps purpurea and the tall fescue endophyte Neotyphodium coenophialum. Ingestion of ergopeptines, particularly ergovaline, by livestock results in poor performance and toxicoses. The biosynthetic pathways involved in the production of these compounds has begun to be elucidated and appears to require the activity of two peptide synthetases. Several lines of evidence, from C. purpurea, indicate that a gene named Cp605 encodes one of these peptide synthetases and we have shown that this gene has homologues among ergopeptine producing endophytes of the Clavicipitaceae. We have knocked out a Cp605 homologue (LPS1) in Neotyphodium sp. strain Lp1 by gene replacement and have analysed its effect on ergovaline production during the association with perennial ryegrass. HPLC analysis showed that grass associations containing the knockout strain of Lp1 failed to produce ergovaline. However, competitive PCR (using primers to an internal fragment of LPS1) on grass associations containing the gene knockout transformant, ectopic transformants and wild type Lp1, established that fungal biomass was not effected in planta. Interestingly, absence of ergovaline production appears to be associated with the presence of a novel HPLC peak of grass origin.
Supported by the CSREES, USDA, under agreement number 98-35303-6663.
474 Genetic analysis of four genes in the paxilline biosynthesis gene cluster from Penicillium paxilli. Lisa McMillan, Carolyn Young and Barry Scott. Institute of Molecular BioSciences, College of Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Indole-diterpenes are a family of potent tremorgenic mammalian mycotoxins and known inhibitors of maxi-K ion channels. While the chemical complexity of indole-diterpenes are well documented, very little is known about the nature of their biosynthesis or the biochemical intermediates. We recently cloned a gene cluster for the biosynthesis of paxilline, an indole diterpene synthesized by Penicillium paxilli and a proposed intermediate in the biosynthesis of other indole-diterpenes (Young et al. 2001). The cluster is predicted to lie within a 50 kb region on chromosome Va and to contain 17 genes, including a geranylgeranyl pyrophosphate (GGPP) synthase (paxG), two FAD-dependent monooxygenases (paxM and paxN), and two cytochrome P450 monooxygenases (paxP and paxQ) (Young et al. 1998, 2001). The gene structures of four of these genes, paxG, paxM, paxP and paxQ, were further characterised by RT-PCR and RACE. Gene replacements using homologous recombination were performed and the chemical phenotypes determined by TLC and HPLC. The four gene replacements were all paxilline negative. Deletion of paxG and paxM have no detectable intermediates suggesting an early role for the enzymes encoded by these genes, while paxP and paxQ gene replacements accumulate the intermediate paspaline and 13-desoxypaxilline respectively. This is the first indole-diterpene gene cluster to be cloned and characterised.
475 Molecular cloning of indole-diterpene biosynthetic genes from Neotyphodium lolii.Carolyn A. Young and Barry Scott. Massey University, IMBS, Palmerston Nth, New Zealand
Lolitrem B is well documented as the cause of ryegrass staggers in animals grazing on Neotyphodium lolii-infected pastures. Although much is known about how lolitrem B is produced, the genes and proteins responsible have not been isolated. Working with Penicillium paxilliwe have recently identified a biosynthetic gene cluster for producing paxilline, a proposed intermediate of lolitrem B. This cluster is comprised of 17 genes, including paxG,a geranylgeranyl pyrophosphate synthase (GGPPS), paxMand N, two monooxygenases, and paxPand Q,two P450 monooxygenases. Two independent copies of GGPPS are present in the P. paxilligenome where paxGis specifically responsible for paxilline production and the second, ggs1,is required for primary isoprenoid biosynthesis.
To target the N. loliipaxilline homologues, degenerate PCR was used with primers designed to paxG,the most highly conserved gene in the paxilline pathway. Two 220 bp fragments, CY28 and CY29, were isolated and shown to encode two distinct GGPPS genes. Southern analysis of Epichlo typhinaand three N. loliistrains showed CY29 sequences are present in all species, while CY28 sequences are seen only in N. loliistrains indicating the former is a homologue of ggs1and the latter is a homologue of paxG.Sequence analysis of a lambda clone containing CY28 revealed three genes, lolG, encoding a GGPPS, lolM, a monooxygenase and lolP, a P450 monooxygenase. All three genes share strong similarity to those found in the paxilline gene cluster. Further analysis will confirm the role of these genes in lolitrem B production.
476 Independent expression of AGK and AGPR in Neurospora crassa. Mazen Karaman and Richard L. Weiss. Department of Chemistry and Biochemistry University of California, Los Angeles
The complex arg-6 locus in Neurospora crassa encodes a polyprotein precursor for two early mitochondrial arginine biosynthetic enzymes, acetylglutamate kinase (AGK) and acetylglutamyl phosphate reductase (AGPR). This polyprotein is processed into two mature proteins as it is translocated into the mitochondria. Processing involves cleavage of the polyprotein at three different sites. Cleavage of the first site, upstream of the proximal AGK, removes the mitochondrial targeting sequence. The two other sites are upstream of the distal AGPR and cleavage removes a 20-amino acid region connecting both enzymes. The presence of a multi-protein coding gene is rare in eukaryotes; however, arg-6 homologues have persisted in polyprotein form in N. crassa and three other fungi. This work examines the biological significance of this unusual gene/protein structure by investigating the effects of encoding AGK and AGPR on separate genes. Genes expressing AGK and AGPR independently of each other have been introduced into an arg-6 null strain. The independently expressed proteins can rescue the auxotrophic phenotype of the null mutant with little effect on growth rate or mitochondrial targeting of the proteins. AGK, AGPR, and arginine levels, however, appear to be compromised in these transformants. The results indicate that the polyprotein structure is important for normal expression of AGK and AGPR which, in part, is needed to maintain normal levels of mycelial arginine.
477 Effect of the nap mutation on growth, carotenogenesis, and respiratory chain in Neurospora crassa. Tatiana A. Belozerskaya1, Yuri V. Ershov2, Natalia N. Gessler2, Elena P Isakova3, Eugene I Shurubor3, Ludmila V. Gorpenko3, and Renata A Zvyagilskaya3. 1A.N.Bach Inst. Biochem., Russian Acad. Sci., Lab. of Evol. Biochem., Moscow, Russia. 2A.N.Bach Inst.Biochem., Lab.vitamins, Moscow Russia. 3A.N.Bach Inst.Biochem., Lab.oxid.phosph., Moscow Russia
Down-regulation of metabolism due to reduced solute flux in the transport mutant nap brought about the slowed growth without sacrificing general homeostasis (carotenoid levels and biosynthetic rate). The mutant strain was found to have an elevated level of carotenoids both in dark and in the light, and higher intracellular ATP content. A comparative study has been made of mitochondria isolated from wild-type Neurospora and from nap. Oxidation of succinate and exogenous NADH by mitochondria from wild-type cells, but not by mutant cells, at the stationary growth phase was accompanied by cyanide-resistant respiration (16 to 32% of the total respiration). By contrast, in nap cells, the electron flux invariantly (at all growth stages examined) was mediated by cytochrome oxidase with the minor contribution of the nonphosphorylating alternate oxidase. An essential difference between mitochondria from wild-type and nap was also revealed during respiration with pyruvate (plus malate). Thus, higher intracellular ATP content in the transport-defective mutant might serve to regulate carotenoid pathway in nap. Further characterization of the antioxidant systems in the two strains will be presented. The work was supported by the Russian Foundation for Basic Research grant # 98-04-48382.
478 ABC transporters in Fusarium proliferatum. Cees Waalwijk, Thamara Hesselink and Gert Kema. Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Among the toxigenic species of the genus Fusarium, F.proliferatum is known to produce the largest spectrum of mycotoxins, including beauvericin, fumonisin, fusaproliferin and moniliformin. These secondary metabolites are produced within the cytoplasm and must be exported to the exterior to excert their toxic effects. ABC transporters are likely candidates for the secretion of these compounds. In Saccharomyces cerevisiae, analyses of the entire genome sequence has revealed the presence of 29 ABC-transporter genes, that have been grouped into 6 classes based on the topology of the deduced proteins. Degenerate primers were designed that are based on the sequences from the genes in the two largest classes, and these primers were used to amplify PCR fragments from several toxigenic Fusarium spp. Sequence analyses revealed that these fragments show homology to different genes. Subsequently, a BAC library of F. proliferatum (constructed in pBeloBAC 11 and consisting of 10 genome equivalents with 50kb-100kb inserts was screened by PCR. Several positive clones were identified and sequence analysis revealed complete genomic copies of two ABC transporters, FpABC1 and FpABC2, with strong homologies to the so-called PDR- and MDR-like ABC transporters, respectively. Northern blot analysis was performed to study the expression of ABC1and ABC2 under different conditions including those inducive for mycotoxin production as well as under conditions of fungicide stress. Several ABC-like gene homologs derived from published EST sequences were also included in these experiments.
479 The Antifungal Protein from Aspergillus giganteus causes permeabilization of fungal membranes. T. Theis, M. Wedde, U. Stahl. Department of Microbiology and Genetics, University of Technology Berlin, 13355 Berlin, Germany
Aspergillus giganteus is known to secrete two small basic proteins, the ribonuclease alpha-Sarcin and the Antifungal Protein (AFP). AFP inhibits the growth of several filamentous fungi mainly from the genera Aspergillus and Fusarium . The minimal inhibitory concentration (MIC) ranges from 0.1 microgram/ml for Fusarium oxysporum up to 200 microgram/ml for Aspergillus nidulans (1). The protein has no effect on the growth of yeast or bacteria. AFP shares some structural features with other antimicrobial proteins whose inhibitory effect is often due to an interaction with membranes. A similar mode of action was proposed for the AFP. Previous studies revealed that after treating AFP-sensitive fungi with AFP the protein is localized at the hyphal membrane (1). A more detailed analysis of the mode of action was carried out by using an assay based on the uptake of SYTOX-Green (2). Current data from this assay indicates that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization, measured as SYTOX-Green uptake. The concentration of the protein and time span of incubation are also important factors for AFP- induced permeabilization. Membrane permeabilization could readily be detected after 5 min. of incubation. The results from the uptake assay correspond very well to those of the localization experiments. It is therefore reasonable to conclude that the growth inhibitory effect of AFP is due to permeabilization of fungal membranes.
(1): T. Theis et al. (1999) ECFG5 Abstract Book: IV-35 (2): K. Thevissen et al. (1999) Appl. Environ. Microbiol. 65: 5451-5458
480 Interaction of propionate, acetate and glucose metabolism in Aspergillus nidulans. Matthias Brock and Wolfgang Buckel. Philipps-Universitat Marburg, Karl-von-Frisch-Strasse, 35032 Marburg, Germany
Filamentous fungi are able to grow on propionate as sole carbon and energy source via the methylcitrate cycle. However, propionate inhibits growth of Aspergillus nidulans on glucose but not on acetate. The growth is inhibited even stronger in a methylcitrate synthase deletion strain. Methylcitrate synthase is a key enzyme of the methylcitrate cycle and catalyses the condensation reaction of propionyl-CoA and oxaloacetate to form methylcitrate. During growth on glucose/propionate medium high amounts of propionyl-CoA are accumulated in the mutant strain. Under these conditions succinyl-CoA synthetase is inhibited strongly. At 0.2 mM acetyl-CoA and 0.4 mM propionyl-CoA the in vitro activity of succinyl-CoA synthetase is 95% inhibited, whereas citrate synthase and 2-oxoglutarate dehydrogenase are not affected. A second target for inhibition of glucose metabolism is the pyruvate dehydrogenase complex. Growth of wildtype and mutant A. nidulans strains on glucose/propionate show an excretion of pyruvate in a millimolar range, probably due to the inhibition of pyruvate dehydrogenase. Addition of acetate to glucose/propionate medium showed a restoring effect on growth, implicating a shunt of building blocks from acetate into the impaired citrate cycle. Despite the fact that acetate metabolism is CreA regulated, significant amounts of acetate are taken up during growth on glucose/propionate/acetate medium. The uptake of acetate is not observed in A. nidulans wildtype strains during growth on glucose/acetate medium. These observations implicate that cometabolism of propionate interferes with normal metabolic regulation, probably by simulating starvation through inhibition of enzymes from other metabolic pathways.
481 Characterization of progesterone receptors and identifiaction of some of their agonist/antagonists in Rhizopus nigricans. Helena Lenasi, University of Ljubljana, Faculty of Medicine, Institute of Biochemistry, Ljubljana, Slovenia.
It is known that steroid hydroxylating enzyme system containing cytochrome P450 as a terminal oxydase could be induced in filamentous fungus Rhizopus nigricans. Enzymes could be induced by progesterone and different steroidal inducers, most probably with the purpose of rendering fungitoxic substrates into less toxic hydroxylated products. During our investigation of the mechanism of the induction process by progesterone we revealed specific progesterone binding molecules in the cytosol and in the plasma membrane. The structure/affinity relationship of cytosolic binding sites and various ligands was examined by cross-displacement studies using different labeled and nonlabeled steroids. Binding components exhibited the highest affinity for progesterone, followed by R5020 and testosterone, whereas dexamethasone and estradiol-17beta showed no binding ability. Mammalian progesterone antagonists onapristone and mifepristone were not able to displace labeled progesterone indicating an important structural difference between progesterone receptors in mammals and lower eucaryotic microorganism. In search for the possible other antagonists of progesterone we examined the action of alpha-naphtoflavone which was not able to induce hydroxylating enzymes in R. nigricans. The displacement of labeled progesterone by alpha-naphtoflavone was as efficient as by nonlabeled progesterone. Moreover, alpha-naphtoflavone prevented in a dose-dependent manner the induction of enzymes by progesterone. These results strongly suggest the antagonistic action of alpha-naphtoflavone as well as the involvement of soluble progesterone receptors in the enzyme-induction by progesterone in R. nigricans. Further potential inducers/ligands originating from natural fungal growing environment are under investigation.
482 Dimethylallyltryptophan synthase genes associated with endophyte ergot alkaloids. Christopher L. Schardl and Jinghong Wang. University of Kentucky, Plant Pathology, Lexington, Kentucky, USA
Ergot alkaloids produced by some mutualistic, seed-borne fungal symbionts of grasses (the Neotyphodium spp. endophytes) are associated with livestock toxicosis. A potential solution involves disrupting the gene for the first commitment step in ergot alkaloid biosynthesis. We cloned the dimethylallyltryptophan synthase (DMAT synthase) gene (dmaW) from Claviceps fusiformis, C. purpurea and Balansia obtecta, and confirmed identities of the gene products. We then compared predicted amino acid sequences, derived degenerate primer sets for PCR, and cloned four dmaW homologs from three Neotyphodium spp. The deduced amino acid sequences shared 60-74% identity with DMAT synthases from Claviceps spp. and B. obtecta. Transcripts of dmaW were detected from cDNA libraries of tall fescue symbiotic with N. coenophialum, but not from N. coenophialum grown on potato dextrose agar nor from non-symbiotic tall fescue. Since N. coenophialum produces alkaloids in planta but not in uninduced culture, this result is consistent with dmaW encoding the determinant step in the pathway. The presence or absence of dmaW homology in 19 endophyte species was analyzed by Southern blot and PCR. All ergot alkaloid producers investigated contained dmaW homology and most ergot alkaloid non-producers lacked such homology, further indicating that dmaW is dedicated to biosynthesis of ergot alkaloids. Therefore, dmaW genes of grass endophytes are appropriate targets for knock-outs to generate ergot alkaloid non-producing endophytes to be incorporated into forage grass breeding lines and cultivars. USDA-NRI 98-35303- 6663
483 A genomic and biochemical approach to trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum. Daren W. Brown, Susan P. McCormick, Nancy J. Alexander, Robert H. Proctor, and Anne E. Desjardins. Mycotoxin Research Unit, USDA/ARS, National Center for Agricultural Utilization Research, Peoria, IL 61604
The trichothecenes T-2 toxin and deoxynivalenol (DON) are fungal natural products that are toxic to both animals and plants. Their importance in the pathogenicity of Fusarium spp. on crop plants has inspired efforts to understand the genetic and biochemical mechanisms leading to trichothecene synthesis. In order to better understand T-2 toxin biosynthesis by Fusarium sporotrichioides and DON biosynthesis by F. graminearum, we compared the nucleotide sequence of the 23-kb core trichothecene gene cluster from each organism. This comparative genomics allowed us to predict proteins encoded by two trichothecene genes, TRI9 and TRI10, that had not previously been described from either Fusarium species. Differences in gene structure were also correlated with differences in the types of trichothecenes the two species produce. Gene disruption experiments showed that F. sporotrichioides TRI7 (FsTRI7) is required for acetylation of the oxygen on C-4 of T-2 toxin. Sequence analysis indicated that F. graminearum TRI7 (FgTRI7) is non-functional. This is consistent with the fact that the FgTRI7 product is not required for DON synthesis in F. graminearum because C-4 is not oxygenated.
484 Influence of Corn Silk Extracts and Flavonoids on Differentiation and Mycotoxin Synthesis by Aspergillus nidulans. Carlos Bucio, Hugo A. Luna and Doralinda Guzman-de-Pena. Unidad Irapuato. CINVESTAV-IPN, Micotoxin Laboratory, Irapuato, Guanajuato, Mexico
Several reports have indicated that plant substances can modifyd mycotoxin synthesis on Aspergilli suggesting a chemical communication between plant and fungus. However, information about the effect of specific plant regulators and flavonoids present in silk corn, on the differentiation process of the fungi and its capacity to produce aflatoxins, has not been evaluated. In this work we study the effect of these compounds on differentiation and synthesis of Sterigmatocystin by Aspergillus nidulans. One (naphthalene-acetic acid) out of eight growth regulators showed an inhibitory effect on both processes. The ethanolic silk extract showed a similar effect. Silk tissues on the contrary showed an increase on sporulation and mycotoxin synthesis.Studies on the effect of these substances on the expression of genes involved in sporulation (flbA and brlA1) and mycotoxin synthesis (aflR) are in progress.
485 Nonribosomal peptide synthetases of Cochliobolus heterostrophus. Bee-Na Lee, Olen C. Yoder and B. Gillian Turgeon. Novartis Agricultural Discovery Institute, San Diego, California, U.S.A.
Small molecules produced by fungi or bacteria are key candidates as effectors of the plant microbe interaction. Members of one class of these are peptides synthesized nonribosomally by multifunctional enzymes called nonribosomal peptide synthetases (NRPSs). The genes encoding these enzymes are large, ranging from 7 to 50 kb. All NRPSs exhibit a similar modular organization. Each module contains six core signature sequences in separate domains. One domain (5 core signatures) is responsible for amino acid activation (adenylation) and the other (core 6) is responsible for polymerization of amino acid residues. Recently, a conserved peptide synthetase (CPS1) was characterized from Cochliobolus heterostrophus. Deletion of CPS1 reduces virulence on corn. To further explore the role of NRPSs in fungal virulence, we are analyzing all NRPS-encoding genes in the C. heterostrophus genome. For functional analysis, primers are designed to generate constructs that delete the conserved core sequences (core 1-core 6) of at least one module. Deletion constructs are linearized and used for fungal transformation. Each transformant carrying a targeted deletion is verified by PCR and then screened on plants for changes in virulence compared to wild type.
486 Identification of differentially expressed genes during aflatoxin biosynthesis in Aspergillus flavus. Greg R. OBrian and Gary A. Payne. Department of Plant Pathology, North Carolina State University, Raleigh NC 27695-7616.
Aflatoxins are polyketide derived secondary metabolites whose biosynthesis can be induced by simple sugars. We are interested in identifying those genes differentially expressed during aflatoxin biosynthesis and profiling their expression during aflatoxin biosynthesis. Candidate genes for study were identified by hybridizing an array of 100,000 Aspergillus flavus cDNA clones with probes made from mRNA collected from cultures of the fungus in the log phase of aflatoxin production. A subset of clones expressed primarily during aflatoxin biosynthesis was chosen for sequence analysis. Quality EST sequences (100 base pairs or greater with at least phred 20) were obtained for over 2200 clones. Sequence analysis revealed the presence of over 600 unique clones. Many of the unique ESTs showed no homology to sequences in the public databases. Among ESTs with homology to sequences with known functions are those coding for signal transduction pathways, secondary metabolism, glucose regulation, cell wall biosynthesis, and cell cycle control. We have developed a microarray containing replicates of our unique clone set and are in the process of further analyzing these clones to establish a gene expression profile during aflatoxin biosynthesis.
487 Production and use of expanded DNA microarrays for characterization of trichothecene production. Andrew G. Tag1, Andrew W. Peplow1, Tzung-Fu Hsieh2, Terry L. Thomas2, and Marian N. Beremand1. 1Texas A&M University, Plant Pathology, College Station, TX, USA. 2Texas A&M University, Biology, College Station, TX, USA.
We have been developing DNA microarrays to investigate global gene expression and the regulatory networks involved in the control of primary and secondary metabolic pathways associated with trichothecene biosynthesis in Fusarium sporotrichioides. Based on our previously generated EST database and results from our early phase DNA microarrays, we have recently constructed an expanded array which contains 300 defined genes. A series of genes which are both positively and negatively coordinately regulated in association with trichothecene biosynthesis have been identified. Detailed information about the expanded arrays and the transcriptional profile data obtained from using them in conjunction with a selected group of toxin mutants will be presented.
488 Cloning polyketide pigment biosynthetic genes from the chestnut blight fungus Cryphonectria parasitica and chemical analyses of pigment mutants. Alice C. L. Churchill1, Heather McLane1, Sagar S. Mungekar1, Tara Sirvent2, Stuart Krasnoff2, Anna Herforth2, and Donna Gibson2. Boyce Thompson Institute1 and USDA, ARS, Plant, Soil, and Nutrition Laboratory2, Ithaca, NY.
The orange and yellow pigments of Cryphonectria parasitica consist of a family of aromatic polyketides of anthraquinone derivation. Emodin and the other pigments of C. parasitica exhibit numerous and diverse biological activities; however, their roles in the biology of the fungus are unknown. We are using insertional mutagenesis (REMI) and PCR amplification of conserved domains of polyketide synthases (PKSs) to clone genes involved in polyketide pigment production in C. parasitica. Using REMI, we have isolated thirteen mitotically stable pigment mutants, several of which were isolated as fast- growing sectors from primary transformants. Altered pigment production in many of the REMI mutants generally correlates with significantly reduced asexual sporulation and impaired fertility in sexual crosses. Five of ten pigment mutants failed to produce perithecia in crosses; each of the remaining crosses produced only a few perithecia in comparison with control transformants, which produced numbers of perithecia and levels of pigment comparable to wild type strains. Chemical profiles of several pigment mutants, analyzed via HPLC, show significant alterations in amounts and ratios of some of the known pigments; several unknown compounds are being further characterized by HPLC-MS. We have also used PCR amplification, with primers designed to conserved domains of PKSs, to clone putative pigment biosynthetic genes. We have isolated fragments of seven unique genes from C. parasitica that show significant sequence similarity to other fungal and bacterial PKS and fatty acid synthase genes. We are conducting complementation analyses of pigment mutants with hybridizing cosmid clones to assess their roles in pigment biosynthesis.
489 Role of the eukaryotic domain of acetylglutamate kinase in Neurospora crassa. Catherine McKinstry, Richard L. Weiss. UCLA, Chemistry and Biochemistr, Los Angeles, California, USA
Arginine biosynthesis in Neurospora crassa is regulated primarily by feedback inhibition of the first two enzymes of the pathway, acetylglutamate synthase (AGS) and acetylglutamate kinase (AGK). Previous genetic studies suggested a coordinate mechanism of inhibition mediated by interaction between AGS and AGK. Mutations in the gene for AGK (arg-6) have been identified which affect the activity or feedback sensitivity of AGK and/or AGS. The yeast-two-hybrid system has been used to demonstrate direct interaction between these two enzymes and to define the interaction domain of AGK as a unique C-terminal region found only in the enzyme from eukaryotic organisms. This domain has been termed the eukaryotic domain. N. crassa arg-6 mutant strains have been transformed with a plasmid encoding AGK lacking the eukaryotic domain. The behavior of the transformants supports an important role for the eukaryotic domain in protein-protein interaction and coordinated feedback inhibition.
490 Molecular cloning of a gene encoding nucleoside diphosphate kinase and its biochemical properties in Aspergillus nidulans. Nak-Jung Kwon, Hye-Jin Kang1, Nam-Shik Lee, Seung-Hyen Ka, Kong-Joo Lee1, and Suhn-Kee Chae. Research Center for Biomedicinal Resources and Division of Life Science, Paichai University, Taejon, Korea, and 1Center for Cell Signaling Research and College of Pharmacy, Ewha Womans University, Seoul Korea
Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of the terminal phosphate group of a nucleoside triphosphate to a nucleoside diphosphate. Human NDPK-A (nm23-H1) was first isolated as a tumor metastasis suppressor and coexisted as hetero-hexamers with Nm23-H2, also known as the c-myc transcription factor PuF. Autophosphorylation and serine/threonine specific protein phosphotransferase activities were reported for Nm23. However, relevant functions of Nm23 on the various cellular processes are largely unknown. In Aspergillus nidulans, we have been isolated a Nm23 homolog gene (ndkA) by PCR with a degenerated primer set. Aspergillus NDPK consists of 154 amino acids with 65% sequence identity to human Nm23. The NDPK was coded from an ORF of 462bp, interrupted by four introns located on chromosome II. The 1.2Kb transcript was detected in northern analysis and the amount of transcript was decreased during asexual development. Over-expression of the sense and the antisense RNA did not affect growth and differentiation. No evidence on the existence of other isotypes was obtained from non-stringent Southern and western analysis. Biochemical properties of recombinant as well as native NDPK purified using ATP-Sepharose affinity chromatography were examined. Aspergillus NDPK was existed as a homo-tetramer (78KDa) judged from gel filtration chromatography. NDPK and autophosphorylation activities were demonstrated and both enzymatic activities were more thermostable than human NDPK. Supported by a grant #1998-001-F00771 from KRF, Korea.
491 Four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides. Seo, J.-A., Proctor R. H., and Plattner, R.D. USDA ARS NCAUR, Mycotoxin Research, Peoria, Illinois, USA
Fumonisins are mycotoxins that cause several fatal animal diseases, including cancer in rats and mice. These toxins are produced by the maize pathogen Fusarium verticillioides (syn. F. moniliforme) and can accumulate in maize infected with this fungus. We have identified four F. verticillioides genes (FUM6, FUM7, FUM8, and FUM9) immediately downstream of FUM5, a previously identified polyketide synthase gene that is required for fumonisin biosynthesis. Gene disruption analysis revealed that FUM6 is required for fumonisin production and Northern blot analysis revealed that expression of the four new genes is correlated with fumonisin production. In contrast, the expression of five genes located upstream of FUM5 are not correlated with fumonisin production. Nucleotide sequence analysis indicated that the predicted FUM6 translation product is most similar to cytochrome P450 monooxygenase/reductase fusion proteins, while the predicted products of FUM7, FUM8 and FUM9 are most similar to type III alcohol dehydrogenases, class-II a-aminotransferases and dioxygenases, respectively. These four groups of enzymes catalyze the same general types of reactions expected to be necessary for fumonisin biosynthesis given the chemical structures of the toxins. Together, these data indicate that FUM5, FUM6, FUM7, FUM8, and FUM9 form part of a fumonisin biosynthetic gene cluster in F. verticillioides.
492 Characterization of a gene cluster with a putative role in toxin production in the ascomycete Leptosphaeria maculans. Alexander Idnurm1, Janet L. Taylor2, and Barbara J. Howlett1. 1School of Botany, The University of Melbourne, Parkville, Victoria 3010, Australia. 2Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada.
Leptosphaeria maculans is a loculoascomycete that causes blackleg disease of canola (Brassica napus) and other Brassica species. During a screen of L. maculans Expressed Sequence Tags (ESTs), a cDNA was identified with low sequence similarity to aflR, the zinc finger regulatory gene for toxins such as aflatoxin and sterigmatocystin production in Aspergillus species. Aflatoxins and related compounds have not been reported in L. maculans. To investigate the role of this putative regulatory gene in metabolite production in the fungus, a cosmid (34 kb) containing this gene was obtained and the flanking regions of the gene sequenced to seek the presence of other genes that may have a role in biosynthesis of a secondary metabolite. So far, a peptide synthetase gene, similar to those that produce cyclic antibiotics in bacteria, and a transport protein gene have been found. These genes are being mutated in the fungus by targeted gene disruption. Resultant mutants will be screened for the presence/absence of known L. maculans metabolites and inoculated onto canola plants to see if these genes play a role in plant disease.