The regulatory protein NIT4 that mediates nitrate induction inNeurospora crassa contains a complex tripartite activation domain with a novel leucine-rich,acidic motif

Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys₆/Zn₂ zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.     

NIT2, the nitrogen regulatory protein of Neurospora crassa,binds upstream of nia,the tomato nitrate reductase gene,in vitro

Summary The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.     

Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa,alter promoter element recognition

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.     

Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa

Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.     

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

Summary 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.     

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function

Summary This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.     

Identification and cloning of a mobile transposon fromAspergillus 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 theniaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in bothA. niger var.awamori andA. niger. A single copy of Vader was detected from only one of the two laboratory strains ofA. nidulans which were also examined. Insertion of the Vader element into theniaD gene ofA. 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 atniaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. MutantsniaD392 andniaD436 reverted at a frequency of 9x10^-3 and 4x10^-2, respectively. Two of the mutants,niaD587 andniaD410, reverted at a lower frequency of 6x10^-4.     

NiaA,the structural nitrate reductase gene of Phytophthora infestans: isolation,characterization and expression analysis in Aspergillus nidulans

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene and its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia ^- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia ^- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli -glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.     

Genetic analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardii

Summary Six mutants (305, 301, 203, 307, 104 and 102) of Chlamydomonas reinhardii, all defective in nitrate reductase (NR) activity, have been genetically analyzed. All except 102 carry single Mendelian mutations.Mutant 305, defective in diaphorase activity and mutant 301, defective in terminal enzyme activity, did not give rise to wild-type recombinants when crossed to each other or with the nit-1 mutant isolated from strain 137c (which is actually a double mutant nit-1 nit-2). Nit-1 was shown to lack both diaphorase and terminal activities. Whether the mutated sites in 305 and 301 are located in a unique cistron (nit-1) or in two adjacent cistrons (nit-1a and nit-1b) coding for a diaphorase subunit and a terminal subunit of NR is discussed in the light of previous biochemical findings.The 203 mutation affecting a regulatory gene did not recombine with nit-2, the other mutated locus present in strain 137c.Mutants 307, 104 and 102, all lacking molybdenum cofactor for both NR and xanthine dehydrogenase, where shown to be affected in different loci. The genes mutated in 307 and 104 have been designated nit-3 and nit-4, respectively. The 102 strain is mutated in two non-linked loci, nit-5 and nit-6, with both mutations required to confer the mutant phenotype. One of these cryptic mutations is present in the wild strain 21gr.The results indicate that at least six or seven loci are involved in the production of an active NR enzyme: one (nit-1) or two (nit-1a and nit-1b) cistrons to produce the NR apoproteins responsible for the partial activities diaphorase and terminal, one locus (nit-2) for the regulation of NR synthesis, and four loci (nit-3, nit-4, nit-5 and nit-6) to produce the molybdenum cofactor. The loci nit-1a and nit-2 seem to correspond to the nit-A and nit-B loci described by Nichols and Syrett (J Gen Microbiol 108:71–77, 1978).Abbreviations NR nitrate reductase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - MoCo molybdenum-containing cofactor - PD parental ditype - NPD non-parental ditype - TT tetratype - WT wild type - BVH reduced benzyl viologen     

NRE,the major nitrogen regulatory protein of Penicillium chrysogenum,binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters

NRE, the nitrogen regulatory protein of Penicillium chrysogenum, contains a single Cys2/Cys2-type zinc-finger motif followed immediately by a highly basic region. The zinc-finger domain was expressed to Escherichia coli as a fusion protein with -galactosidase. In order to test the putative DNA-binding ability of NRE, the intergenic promoter region of the nitrate reductase/nitrite reductase gene cluster (niiA-niaD) of Penicillium was sequenced. Our results show that NRE is a DNA-binding protein and binds to the intergenic promoter regions of the P. chrysogenum niiA-niaD and acvA-pcbC gene cluster, encoding the first two enzymes in penicillin biosynthesis. Three of the four high-affinity NRE-binding sites contained two GATA core elements. In one of the recognition sites for NRE, one GATA motif was replaced by GATT. The two GATA elements showed all possible orientations, head-to-head, head-to-tail and tail-to-tail, and were separated by between 4 and 27 bp. Missing-contact analysis showed that all three purines in both of the GATA core sequences and the single adenine residue in each of the complementary TATC sequences were involved in the binding of NRE. Moreover, loss of purines in the flanking regions of the GATA elements also affect binding of NRE, as their loss causes reduced affinity.     

Isolation and characterization of xyl mutants in a xylose-utilizing yeast,Pichia stipitis

Summary Mutants of the xylose-utilizing yeast, Pichia stipitis, unable to grow on xylose as the sole carbon source were isolated and characterized. The mutants were deficient in either xylose reductase or xylitol dehydrogenase. By immunological means and upon analysis of revertants, both mutant types could be attributed to the structural genes XYL1 and XYL2, which code for xylose reductase and xylitol dehydrogenase, respectively. These data support previous assumptions that both NADH- and NADPH-dependent xylose reductase activity are due to one protein or gene, respectively. Revertant analysis of xyl1 mutants has revealed the existence of a second xylose reductase gene in P. stipitis. This gene is very likely cryptic. Its corresponding xylose reductase activity is NADPH-dependent.     

Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides

Two overlapping cosmid clones (Cos1-1 and Cos9-1) carrying the Tox5 gene were isolated from a library of F. sporotrichioides strain NRRL 3299 genomic DNA. These cosmids were used to transform three T-2 toxin-deficient mutants that are blocked at different steps in the trichothecene pathway. Both cosmids restored T-2 toxin production to Tox3-1 ^– or Tox4-1 ^– mutants but neither restored T-2 toxin production to a Tox1–2 ^– mutant. The production of T-2 toxin by the complemented Tox3-1 ^– and Tox4-1 ^– mutants, as well as the production of diacetoxyscirpenol by the cosmid-transformed Tox1-2 ^– mutant, were 2- to 10- fold higher than in strain NRRL 3299. In addition, those transformants carrying Cos9-1 produced significantly higher levels of trichothecenes than transformants carrying Cos1-1. Two different DNA fragments (FSC13-9 and FSC14-5), representing the region of overlap between the cosmid clones, were isolated. These fragments specifically complemented either the Tox3-1 ^– mutant (FSC14-5) or the Tox4-1 ^– mutant (FSC13-9). The trichothecene-production phenotype of these transformants was similar to NRRL 3299. These results suggest that two or more genes involved in the biosynthesis of trichothecenes are closely linked to Tox5.     

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli -glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.     

Molybdate repair of molybdopterin deficient mutants from Chlamydomonas reinhardtii

Summary The phenotypically wild strain I₃ of Chlamydomonas reinhardtii, carrying a cryptic mutation at the nit-6 locus, was distinguished from strains 21gr (cryptic mutant at nit-5) and 6145c (wild type) because of the ability of I₃ to grow on nitrate media containing 2mM tungstate.Molybdopterin-cofactor (MoCo) mutants 102 (double mutant at nit-5 and nit-6) and 104 (mutant at nit-4) grew on nitrate media supplemented with high concentrations of molybdate, although final cell densities were 40–60% lower and generation times 3.5 to six fold longer than for wild type. Under these conditions, nitrate reductase (NR) activity of the mutants, when measured either in situ or in vitro, was practically undetectable. The MoCo defective mutant 307 (nit-3) was not molybdate repairable. Although NR activity was not restored in vitro by molybdate in any of the MoCo^– mutant strains, their extracts had free NR-diaphorase subunits together with NR-subunits assembled into high molecular weight species.Our results indicate that: a) nit-4, nit-5 and nit-6 loci are responsible for molybdate processing in the cell; b) nit-3 may encode a component of the pterin moiety biosynthetic route; c) NR subunits can assemble in the presence of an inactive MoCo; d) high concentrations of molybdate can replace partially in vivo but not in vitro the function of nit-4 and the combined function(s) of the nit-5 and nit-6 gene products.     

DNA-mediated transformation of the secondarily homothallic basidiomycete Coprinus bilanatus

Summary The Coprinus cinereus trp-2 gene which encodes a trifunctional protein of the tryptophan biosynthetic pathway was used to transform a trp-2 mutant of the secondarily homothallic species Coprinus bilanatus. Southern blot analysis confirmed the presence of transforming DNA in the genome of transformants and indicated the presence of tandemly duplicated copies of the plasmid in some of these. Although these two species of Coprinus are regarded as closely related, no cross-hybridisation between the homologous trp-2 genes was detected.     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

Two family G xylanase genes from Chaetomium gracile and their expression in Aspergillus nidulans

With oligonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into Aspergillus nidulans and found to be expressed with their own promoters.