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.

Identification of a novel gene hbrB required for polarised growth in Aspergillus nidulans.

We have cloned a novel gene, hbrB, by complementation of a temperature sensitive hyperbranching (hbr) mutant of Aspergillus nidulans. The mutant, hbrB3, exhibits hyperseptation and shows a marked increase in hyphal branching at the restrictive temperature. A genomic library incorporating the AMA1 sequence, which confers autonomous replication on the plasmid, was used to clone the gene. Co-ordinate loss of the complementing plasmid and wild type phenotype was shown. The 847 amino acid predicted product of the hbrB gene shows significant identities with Neurospora crassa and Aspergillus fumigatus hypothetical proteins but no other obvious relative outside the filamentous fungi. The gene was placed under control of the alcA promoter and downregulation resulted in a loss of polarity phenotype. These results suggest that A. nidulans hbrB is specific to filamentous fungi and is essential for hyphal growth and polarity. Index descriptors: Aspergillus nidulans; branching; hbrB; hyphal growth and polarity

Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei.

Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5' ends of 21888 cDNA clones were sequenced from which a unique set of over 5000 were also 3' sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.

On the safety of Aspergillus niger--a review.

Aspergillus niger is one of the most important microorganisms used in biotechnology. It has been in use already for many decades to produce extracellular (food) enzymes and citric acid. In fact, citric acid and many A. niger enzymes are considered GRAS by the United States Food and Drug Administration. In addition, A. niger is used for biotransformations and waste treatment. In the last two decades, A. niger has been developed as an important transformation host to over-express food enzymes. Being pre-dated by older names, the name A. niger has been conserved for economical and information retrieval reasons and there is a taxonomical consensus based on molecular data that the only other common species closely related to A. niger in the Aspergillus series Nigri is A. tubingensis. A. niger, like other filamentous fungi, should be treated carefully to avoid the formation of spore dust. However, compared with other filamentous fungi, it does not stand out as a particular problem concerning allergy or mycopathology. A few medical cases, e.g. lung infections, have been reported, but always in severely immunocompromised patients. In tropical areas, ear infections (otomycosis) do occur due to A. niger invasion of the outer ear canal but this may be caused by mechanical damage of the skin barrier. A. niger strains produce a series of secondary metabolites, but it is only ochratoxin A that can be regarded as a mycotoxin in the strict sense of the word. Only 3-10% of the strains examined for ochratoxin A production have tested positive under favourable conditions. New and unknown isolates should be checked for ochratoxin A production before they are developed as production organisms. It is concluded, with these restrictions, that A. niger is a safe production organism.

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.

Identification and cloning of a mobile transposon from Aspergillus niger var. awamori.

Aspergillus niger var. awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of the niaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in both A. niger var. awamori and A. niger. A single copy of Vader was detected from only one of the two laboratory strains of A. nidulans which were also examined. Insertion of the Vader element into the niaD gene of A. niger var. awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements at niaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. Mutants niaD392 and niaD436 reverted at a frequency of 9x10(-3) and 4x10(-2), respectively. Two of the mutants, niaD587 and niaD410, reverted at a lower frequency of 6x10(-4).

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.

Cloning and preliminary characterization of a molybdenum cofactor gene of Neurospora crassa.

A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli ch1D molybdenum cofactor mutants (ch1D, ch1D::Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band of N. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants (ch1A, ch1B, ch1C, ch1D, ch1E, ch1G and ch1M), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the ch1D mutant. In vitro reconstitution experiments using wild-type, ch1D and ch1D; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. crassa MoCo mutants nit-1, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from ch1D; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original ch1D mutant.

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.