UPR-independent dithiothreitol stress-induced genes in Aspergillus niger.

A subtraction library was prepared from cultures of Aspergillus niger that had or had not been exposed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn2+-containing superoxide dismutase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control conditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stresses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined.

The transposable element Tan1 of Aspergillus niger var. awamori, a new member of the Fot1 family.

Aspergillus niger var. awamori has transposable elements that we refer to as Vader and Tan1 (transposon A. niger). Vader was identified by screening unstable nitrate reductase (niaD) mutants for insertions. Four of the isolated niaD mutants were shown to contain a small insertion element. This 437 bp insertion element, Vader, is flanked by 44 bp inverted repeats (IR) and is present in approximately 15 copies in the genomes of two A. niger strains examined. A synthetic 44 bp oligomer of the inverted repeat of Vader has now been used to clone, via the polymerase chain reaction, a 2.3 kb Tan1 element. The Tan1 element has also been isolated from a partial genomic library. Tan1 is present as a single copy in A. niger var. awamori. The Tan1 element has a unique organization: IR-ORF-IR-IR-Vader-IR. The single open reading frame (ORF) (1668 bp) encodes a putative transposase homologous to Fusarium oxysporum Fot1 and Magnaporthe grisea Pot2. Immediately 3' to the second inverted repeat, which bounds the transposase, is a copy of the AT-rich Vader element. We hypothesize that at some stage the independent Vader element, although inactive by itself, arose from Tan1, resulting in current strains with only one copy of Tan1 providing transposase activity and numerous mobile copies of Vader dispersed in the genome.

Strain improvement of chymosin-producing strains of Aspergillus niger var. awamori using parasexual recombination.

Parasexual recombination was used to obtain improved chymosin-producing strains and to perform genetic analysis on existing strains. Chlorate resistance was used to select for a variety of spontaneous nitrate assimilation pathway mutations in strains previously improved for chymosin production using classical strain improvement methods including mutation and screening, and selection for 2-deoxyglucose resistance (dgr). Diploids of these improved strains were generated via parasexual recombination and were isolated on selective media by complementation of nitrate assimilation mutations. A preliminary genetic analysis of diploid and haploid segregants indicated that the dgr trait, resulting in overexpression of chymosin, was recessive. Also, mutations in two different dgr genes resulted in an increased level of chymosin production. When these mutations were combined via parasexual recombination, the resulting haploid segregants produced about 15% more chymosin than either parental strain. CHEF gel electrophoresis was used to determine the chromosomal location of the integrated chymosin DNA sequences, and to verify diploidy in one case where the chromosome composition of two haploid parents differed.

Chromosomal and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes.

An electrophoretic karyotype has been established for Trichoderma reesei strain QM6a, and several of its derivatives, by pulsed-field gel electrophoresis. All strains examined appear to have seven chromosomes with a total genome size of approximately 33 megabases (Mb). The sizes of the chromosomal bands in strain QM6a are approximately 6.2, 6.0, 5.1, 4.2 (doublet), 3.6 and 3.2 Mb. Genes encoding the cellulase complex and xylanases of T. reesei have been mapped, as have several other genes.

Commercial levels of chymosin production by Aspergillus.

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa.

A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.

Biochemical characterization of the molybdenum cofactor mutants of Neurospora crassa: in vivo and in vitro reconstitution of NADPH-nitrate reductase activity.

Molybdenum cofactor (MoCo) mutants of Neurospora crassa lack both NADPH-nitrate reductase and xanthine dehydrogenase activity. In vivo and in vitro studies to further characterize these mutants are now reported. The MoCo mutants nit-9A and nit-9B are capable of growing, albeit poorly, with nitrate as the sole nitrogen source, provided high levels of molybdate are present. The MoCo mutants nit-9A, nit-9B and nit-9C, but not nit-1, nit-7 or nit-8, have significant levels of NADPH-nitrate reductase when grown in nitrate medium containing 30 mM molybdate. In vitro reconstitution experiments using cell free extracts of the N. crassa MoCo mutants and E. coli HB101 as a source of wild-type MoCo were performed. MoCo from E. coli was capable of reconstituting NADPH-nitrate reductase activity to nit-1, nit-7 and nit-8. Molybdate is required for the in vitro reconstitution of NADPH-nitrate reductase activity. It was not possible to in vitro reconstitute NADPH-nitrate reductase activity in the MoCo mutants nit-9A, nit-9B or nit-9C.

Effect of the gln-1b mutation on nitrogen metabolite repression in Neurospora crassa.

In Neurospora crassa, synthesis of the enzymes of nitrate assimilation, nitrate reductase and nitrite reductase, was repressed by the presence of ammonium, glutamate, or glutamine. This phenomenon was a manifestation of the regulatory process termed nitrogen metabolite repression whereby alternative pathways of nitrogen acquisition are not expressed in cells enjoying nitrogen sufficiency. However, the glutamine synthetase mutant gln-1b had derepressed levels of the nitrate assimilation enzymes. The inability of glutamine to achieve nitrogen metabolite repression in this mutant militated against its potential role as the direct effector of this regulation.

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

The regulation of nitrate assimilation in Neurospora crassa: the isolation and genetic analysis of nmr-1 mutants.

Four mutants of Neurospora crassa have been isolated which have altered regulation of nitrate reductase. They each carry a mutation which results in derepressed synthesis of nitrate reductase even in the presence of glutamine. They map to a single locus which has been designated nmr-1 and which is located between am and gln on linkage group VR. The mutations appear to affect only nitrate assimilation. The nit-2, nit-3 and nit-4/5 mutations are epistatic to nmr-1 since the double mutants have the single nit mutant phenotype. For nitrate reductase synthesis, the nmr-1 mutation is epistatic to am such that the double mutant is derepressed even in the presence of glutamate or glutamine. In all other respects however, the double mutant exhibits the am phenotype. We suggest therefore that the nmr-1 mutations do not directly affect the regulation of nitrate reductase at the level of transcription but instead act post-transcriptionally.

The regulation of nitrate assimilation in Neurospora crassa: biochemical analysis of the nmr-1 mutants.

Neurospora crassa nmr-1 mutants, selected on the basis of their sensitivity to chlorate in the presence of glutamine, have elevated levels of the nitrate assimilation enzymes, NADPH-nitrate reductase and NAD(P)H-nitrite reductase. Immunoelectrophoretic determinations show that the higher nitrate reductase activities in nmr-1 mutants are due to greater enzyme concentrations. The half-life of nitrate reductase in these mutants is unaltered. As in wild-type, expression of nitrate assimilation in nmr-1 mutants is dependent on induction by nitrate. Reduced nitrogen metabolites like ammonium and glutamine still repress this expression in nmr-1 mutants, but not as effectively as in wild-type. Enzymatic activity measurements in double mutant strains confirm that the nit regulatory loci, nit-2 and nit-4/5, are epistatic to nmr-1, but nmr-1 is epistatic to nit-3, the nitrate reductase structural gene. The results imply that nmr-1 is involved in post-transcriptional control of nitrate assimilation.

The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa.

Growth of Neurospora crassa on media containing NH4+ leads to the repression of a variety of permeases and alternative pathways which would generate NH4+, so called "ammonium repression." The mutant am2 which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-1a has derepressed levels of the aforementioned systems unless grown with glutamine. The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am2 and gln-1a when they are ammonium derepressed. Th mechanism of NH4+ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH4+ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa: effect of the gln-1a locus.

Nicotinamide adenine dinucleotide phosphate (reduced form)-nitrate reductase was freed from ammonium repression in a Neurospora crassa mutant having drastically lowered glutamine synthetase activity, gln-1a. The general phenomenon of nitrogen metabolite repression required glutamine or some aspect of glutamine metabolism.

The regulation of hexokinase and phosphoglucomutase activity in Aspergillus nidulans.

The levels of glucose-6-phosphate and 6-phosphogluconate dehydrogenase in wildtype cells of Aspergillus nidulans varied with the carbon and nitrogen source. In general, hexokinase activity did not vary with carbon or nitrogen source. The ammonium derepressed mutant amrA1 had only 50% of the wildtype level of hexokinase. Phosphoglucomutase activity was low in wildtype cells grown with nitrate, but high in cells grown with ammonium when glucose was the carbon source. A non-inducible mutant, nirA-1, in the regulatory gene for nitrate reductase, had high phosphoglucomutase activity when grown with nitrate or ammonium. A constitutive mutant nirAc1, in the regulatory gene for nitrate reductase had low phosphoglucomutase activity when grown with nitrate or ammonium. The mutants nir-1 and nirAc1 are recessive and semi-dominant respectively for abnormal phosphoglucomutase activity.

In vivo and in vitro studies of nitrate reductase regulation in Asperillus nidulans.

Induced wildtype cells of A. nidulans rapidly lost NADPH--linked nitrate reductase activity when subjected to carbon and or nitrogen starvation. A constitutive mutant at the regulatory gene for nitrate reductase, nir Ac 1, rapidly lost nitrate reductase activity upon carbon starvation. This loss of activity is thought to be due to a decrease in the NADPH concentration in the cells. Cell free extracts from wildtype cells grown in the presence of nitrate, rapidly lost their nitrate reductase activity when incubated at 25 degrees C. NADPH prevented this loss of activity. Wildtype cells grown in the presence of nitrate and urea have a higher initial NADPH:NADP+ ratio and cell free extracts from such cells lost their nitrate reductase activity slower than extracts of cells grown with nitrate alone. The Pentose Phosphate Pathway mutant, pppB-1, had a lower NADPH concentration compared with the wildtype grown under the same conditions and cell free extracts lost their nitrate reductase activity more rapidly than the wildtype. Cell free extracts of nirAc-1 and a non-inducible mutant for nitrate reductase, nirA- -14, upon incubation lost little of their nitrate reductase activity.