Apparent genetic redundancy facilitates ecological plasticity for nitrate transport.

Aspergillus nidulans possesses two high-affinity nitrate transporters, encoded by the nrtA and the nrtB genes. Mutants expressing either gene grew normally on 1-10 mM nitrate as sole nitrogen source, whereas the double mutant failed to grow on nitrate concentrations up to 200 mM. These genes appear to be regulated coordinately in all growth conditions, growth stages and regulatory genetic backgrounds studied. Flux analysis of single gene mutants using 13NO3(-) revealed that K(m) values for the NrtA and NrtB transporters were approximately 100 and approximately 10 microM, respectively, while V(max) values, though variable according to age, were approximately 600 and approximately 100 nmol/mg dry weight/h, respectively, in young mycelia. This kinetic differentiation may provide the necessary physiological and ecological plasticity to acquire sufficient nitrate despite highly variable external concentrations. Our results suggest that genes involved in nitrate assimilation may be induced by extracellular sensing of ambient nitrate without obligatory entry into the cell.

Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans.

The Aspergillus nidulans cnxE gene, required for molybdenum cofactor biosynthesis, was isolated by functional complementation of an Escherichia coli mogA mutant strain. The deduced CnxE polypeptide consists of two domains which display similarity to the E. coli proteins MoeA and MogA, respectively, separated by a putative hinge region of around 58 amino acid residues which is notably histidine rich. A deletion mutant lacking the entire cnxE gene, including both MoeA-like and MogA-like domains, was identified. Compared to the wild type, a small increase in the intermediate precursor Z was observed in the deletion strain but was significant only under conditions in which the molybdoenzyme nitrate reductase was induced. Elevated levels of the pathway intermediate molybdopterin were found both under nitrate reductase-inducing and non-inducing conditions in the deletion mutant compared to the wild type. This increase is in contrast to previous results for cnxABC, cnxF, cnxG, and cnxH mutants, in which the levels of molybdopterin were substantially reduced, and therefore supports previously published classical genetic and biochemical studies that indicated that the CnxE protein is likely to be involved in the final stages of molybdenum cofactor biosynthesis. We have found no evidence during our chemical analysis for any involvement of this protein in the intermediate section of the molybdenum cofactor biosynthetic pathway (i.e. in the synthesis of molybdopterin from precursor Z), as has been suggested previously for E. coli MoeA. The 2.5-kb cnxE transcript is not abundant and appears to be expressed constitutively.

Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.

An heterologous transformation system for entomopathogenic fungi B. bassiana and M. anisopliae was developed based on the use of A. nidulans nitrate reductase gene (niaD). B. bassiana and M. anisopliae niaD stable mutants were selected by treatment of protoplast with ethane methane sulphonate (EMS) and regenerated on chlorate medium. The cloned gene was capable of transforming B. bassiana and M. anisopliae at a frequency of 5.8 to 20 transformants per microg of DNA. Most of them were mitotically stable.

The Aspergillus nidulans panB gene encodes ketopantoate hydroxymethyltransferase, required for biosynthesis of pantothenate and Coenzyme A.

Ketopantoate hydroxymethyltransferase, which is encoded by the panB gene in the lower eukaryote Aspergillus nidulans, is essential for the biosynthesis of coenzyme A, while the pathway intermediate 4'-phosphopantetheine is required for penicillin production. Ketopantoate hydroxymethyltransferase could also serve as a target for anti-fungal drugs, since it is not present in mammals. Clones of panB were identified by complementation of the corresponding mutant, and the DNA sequence of the gene was determined. The fungal panB gene encodes a predicted protein of molecular mass 37.7 kDa, containing two short sequence motifs, LeuValGlyAspSer and GlyIleGlyAlaGly, that are completely conserved between prokaryotic and eukaryotic homologues. The mutation panB100 was found to result in deletion of Gly-168, the last glycine within the latter conserved motif. Analysis by gel filtration suggests that the fungal PanB protein can be expressed in Escherichia coli as an active octameric enzyme. The panB transcript is present in low abundance and, most probably, a small increase in transcript levels occurs in the absence of exogenous pantothenate.

Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants.

We describe the primary structure of eukaryotic molybdopterin synthase small and large subunits and compare the sequences of the lower eukaryote, Aspergillus nidulans, and a higher eukaryote, Homo sapiens. Mutants in the A. nidulans cnxG (encoding small subunit) and cnxH (large subunit) genes have been analyzed at the biochemical and molecular level. Chlorate-sensitive mutants, all the result of amino acid substitutions, were shown to produce low levels of molybdopterin, and growth tests suggest that they have low levels of molybdoenzymes. In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations. Thus on the basis of chlorate toxicity, it is possible to distinguish between amino acid substitutions that permit a low level of molybdopterin production and those mutations that completely abolish molybdopterin synthesis, most likely reflecting molybdopterin synthase activity per se. Residues have been identified that are essential for function including the C-terminal Gly of the small subunit (CnxG), which is thought to be crucial for the sulfur transfer process during the formation of molybdopterin. Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity suggesting that this residue resides in a region important for correct folding of the fungal protein. Many years ago it was proposed, from data showing that temperature-sensitive cnxH mutants had thermolabile nitrate reductase, that CnxH is an integral part of the molybdoenzyme nitrate reductase (MacDonald, D. W., and Cove, D. J. (1974) Eur. J. Biochem. 47, 107-110). Studies of temperature-sensitive cnxH mutants isolated in the course of this study do not support this hypothesis. Homologues of both molybdopterin synthase subunits are evident in diverse eukaryotic sources such as worm, rat, mouse, rice, and fruit fly as well as humans as discussed in this article. In contrast, molybdopterin synthase homologues are absent in the yeast Saccharomyces cerevisiae. Precursor Z and molybdopterin are undetectable in this organism nor do there appear to be homologues of molybdoenzymes.

The two subunits of human molybdopterin synthase: evidence for a bicistronic messenger RNA with overlapping reading frames.

Molybdoenzymes are ubiquitous and require a prosthetic group called the molybdenum cofactor for activity. We provide evidence here that the two heteromeric subunits (MOCO1-A and MOCO1-B) of human molybdopterin synthase, which is involved in the conversion of precursor Z to molybdopterin in the molybdenum cofactor biosynthetic pathway, are spe-cified by a single bicistronic mRNA with overlapping reading frames. The transcript is in low abundance and shows variable tissue distribution. We propose that leaky scanning of the first translational initiation codon for MOCO1-A by 40S ribosomal subunits occurs, allowing recognition of the AUG for the downstream MOCO1-B reading frame. Such a genetic arrangement may result in a constant ratio and close proximity of lowly expressed enzyme subunits which should, a priori, be especially advantageous for assembly in complex mammalian cells. The MOCO1 locus resides on human chromosome 5.

Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) from Aspergillus nidulans.

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.

The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis. Molecular characterization and analysis of in vivo generated mutants.

The product of the Aspergillus nidulans cnxF gene was found by biochemical analysis of cnxF mutants to be involved in the conversion of precursor Z to molybdopterin. Mutants cnxF1242 and cnxF8 accumulate precursor Z, while the level of molybdopterin is undetectable. The DNA sequence of the cnxF gene was determined, and the inferred protein of 560 amino acids was found to contain a central region (residues around 157 to 396) similar in sequence to the prokaryotic proteins MoeB, which is thought to encode molybdopterin synthase sulfurylase, ThiF, required for thiamine biosynthesis, and HesA, involved in heterocyst formation, as well as eukaryotic ubiquitin-activating protein E1. Based on these similarities, a possible mechanism of action is discussed. Sequence comparisons indicate the presence of one and possibly two nucleotide binding motifs, Gly-X-Gly-X-X-Gly, as well as two metal binding Cys-X-X-Cys motifs in this central region of the CnxF protein. Seven in vivo generated A. nidulans cnxF mutants were found to have amino acid substitutions of conserved residues within this central region of similarity to molybdopterin synthase sulfurylase, indicating that these seven amino acids are essential and that this domain is crucial for function. Of these seven, the cnxF1285 mutation results in the replacement of Gly-178, the last glycine residue of the N-proximal Gly-X-Gly-X-X-Gly motif, indicating that this motif is essential. Mutation of the conserved Arg-208, also probably involved in nucleotide binding, leads to a loss-of-function phenotype in cnxF200. Alteration of Cys-263, the only conserved Cys residue (apart from the metal binding motifs), in cnxF472 suggests this residue as a candidate for thioester formation between molybdopterin synthase and the sulfurylase. Substitution of Gly-160 in two independently isolated mutants, cnxF21 and cnxF24, results in temperature-sensitive phenotypes and indicates that this residue is important in protein conformation. The C-terminal CnxF stretch (residues 397-560) shows substantial sequence conservation to a yeast hypothetical protein, Yhr1, such conservation between species suggesting that this region has function. Not inconsistent with this proposition is the observation that mutant cnxF8 results from loss of the 34 C-terminal residues of CnxF. There is no obvious similarity of the CnxF C-terminal region with other proteins of known function. Two cnxF transcripts are found in low abundance and similar levels were observed in nitrate- or ammonium-grown cells.

The tigA gene is a transcriptional fusion of glycolytic genes encoding triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in oomycota.

Genes encoding triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are fused and form a single transcriptional unit (tigA) in Phytophthora species, members of the order Pythiales in the phylum Oomycota. This is the first demonstration of glycolytic gene fusion in eukaryotes and the first case of a TPI-GAPDH fusion in any organism. The tigA gene from Phytophthora infestans has a typical Oomycota transcriptional start point consensus sequence and, in common with most Phytophthora genes, has no introns. Furthermore, Southern and PCR analyses suggest that the same organization exists in other closely related genera, such as Pythium, from the same order (Oomycota), as well as more distantly related genera, Saprolegnia and Achlya, in the order Saprolegniales. Evidence is provided that in P. infestans, there is at least one other discrete copy of a GAPDH-encoding gene but not of a TPI-encoding gene. Finally, a phylogenetic analysis of TPI does not place Phytophthora within the assemblage of crown eukaryotes and suggests TPI may not be particularly useful for resolving relationships among major eukaryotic groups.

The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis.

The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3-4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function.

3-Hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterization.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first specific enzyme of the isoprenoid pathway, which leads to several classes of primary and secondary metabolites such as sterols, quinones, carotenoids and gibberellins. The structural gene of HMG-CoA reductase was isolated from the ascomycetous fungus Gibberella fujikuroi. Additionally, the most conserved region of this gene was also isolated from another plant pathogenic fungus, Sphaceloma manihoticola. Both ascomycetous fungi use the plant hormone gibberellin to induce an elongation of infected host plants, and in the case of S. manihoticola of plant tumors. Sequence analysis revealed a high degree of similarity between the deduced amino-acid sequences in the C-terminal catalytic domains of all known HMG-CoA reductases, but the highest degree was found between the sequences of both analysed ascomycetes. In contrast to Saccharomyces cerevisiae, Ustilago maydis and plants, G. fujikuroi and S. manihoticola possess only a single copy of this gene, although the product of HMGR (mevalonate) is the precursor for essential sterol and quinone biosynthesis and secondary metabolites such as gibberellins. RNA-blot and hybridization experiments showed that gene expression is not influenced by either glucose or ammonium excess.

Secondary metabolite production in filamentous fungi displayed.

We report the potential of differential display technology for the isolation of genes of biotechnological interest. We have assessed the usefulness of the technique for the cloning of genes involved in the production of secondary metabolites, many of which are of industrial use or interest. We have used the complex pathway for the biosynthesis of gibberellins, as well as bikaverin and carotenoids, present in the filamentous fungus Gibberella fujikuroi as a test system. From a total display of approximately 16000 PCR products for each RNA sample, 100 were derived from the derepressed but not the repressed condition. These products were analysed by Northern blotting and a subset of 16 such PCR products showed differential expression at the transcript level. A number of different mRNA species were observed on this basis which varied in their size. Hence, this approach appears suitable for the isolation of genes involved in the complex pathways often required for the synthesis of secondary metabolites in organisms which are genetically intractable. Moreover, the method has the advantage that it is quick, differential displays being obtained after 2 days and DNA clones in 6 days.

Expression and antisense inhibition of transgenes in Phytophthora infestans is modulated by choice of promoter and position effects.

Procedures were identified for manipulating the expression of genes in the oomycete fungus, Phytophthora infestans. The activities of five putative promoter sequences, derived from the 5' regions of oomycete genes, were measured in transient assays performed in protoplasts and in stable transformants. The sequences tested were from the ham34 and hsp70 genes of Bremia lactucae, the actin-encoding genes of P. infestans and P. megasperma, and a polyubiquitin-encoding gene of P. infestans. Experiments using the GUS reporter gene (encoding beta-glucuronidase) demonstrated that each 5' fragment had promoter activity, but that their activities varied over a greater than tenfold range. Major variation was revealed in the level of transgene expression in individual transformants containing the same promoter::GUS or promoter::lacZ fusion. The level of expression was not simply related to the number of genes present, suggesting that position effects were also influencing expression. Fusions between the ham34 promoter, and full-length and partial GUS genes in the antisense orientation blocked the expression of GUS in protoplasts and in stable transformants.

Zinc fingerprinting for Phytophthora species: ZIF markers.

We have assessed the potential of using zinc finger markers for identification of fungal species using Phytophthora as a model organism, since it is particularly difficult to classify. The results show that such markers are suitable for species identification of Phytophthora but do not appear to aid strain identification within species.

Transformation of Gibberella fujikuroi: effect of the Aspergillus nidulans AMA1 sequence on frequency and integration.

A stable and reproducible transformation selection system for Gibberella fujikuroi protoplasts based on the Aspergillus nidulans arg B gene, encoding ornithine transcarbamylase, has been developed. Inclusion into the vector of the A. nidulans DNA fragment (AMA1), which permits plasmid autonomous replication in A. nidulans, A. niger and A. oryzae, appeared to permit autonomous replication of G. fujikuroi although the transformation frequency was increased by only two-fold. Transformation was also achieved using the bacterial hygromycin B resistance gene under the control of G. pulicaris and A. nidulans promoters.

The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons.

The Aspergillus niger niaD gene has been sequenced and the inferred nitrate reductase (NR) protein found to consist of 867 amino acid residues (97 kDa). The gene is interrupted by six small introns, as deduced by comparison with the niaD gene of Aspergillus nidulans. The positions of these putative introns are conserved between the two fungi, although the sequences are dissimilar. The niiA gene, encoding nitrite reductase, the second reductive step in the nitrate assimilation pathway, is tightly linked to niaD and divergently transcribed in A. niger, similar to the general organisation in the related fungi, Aspergillus oryzae and A. nidulans. The nucleotide (nt) sequences of the intergenic region between niiA and niaD (excluding the ATG translation start codon) of A. niger (1668 nt) and A. oryzae (1575 nt) were determined and compared with the previously determined A. nidulans (1262 nt) sequence. No striking extended nt regions of homology are observed in spite of the fact that transgenic strains with fungal niaD or the two control genes required for induction and repression show virtually normal regulation. Fungal NR shows considerable aa homology with higher plant NR, particularly within the co-factor domains for flavin adenoside dinucleotide, heme and molybdopterin cofactor.

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway.

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.

Actin in the oomycetous fungus Phytophthora infestans is the product of several genes.

Actin (ACT) in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and other filamentous fungi where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. The complete nucleotide sequence of both genes has been determined. Unlike the actin-encoding genes (act) of other filamentous fungi, no introns are obvious in the coding region, a feature shared with the act genes of certain protists. Northern blotting and primer extension studies of the mRNA show that actA and actB are actively transcribed in mycelium, sporangia and germinating cysts but only at a low level in the case of actB. Both genes display bias in their codon usage. This is more extreme in actA. The deduced ACTB protein is strikingly similar to that of the Phytophthora megasperma actin and is more diverged from other actins than ACTA.