Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.     

Mutational analysis of the C-terminal region of AREA,the transcription factor mediating nitrogen metabolite repression inAspergillus nidulans

InAspergillus nidulans the positive-acting, wide domain regulatory geneareA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of theareA product (AREA) are inessential for at least partial expression of most genes subject to regulation byareA. Paradoxically,areA ^r 2, a ?1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for theareA ^r 2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterisedareA ^r 2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutantareA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. TheareA ^r 2 product retains some function with respect to the expression ofuaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with theareA ^r 2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5 flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5 regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea, is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^– mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^– transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^– transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Cloning and molecular characterisation of the amdR controlled gatA gene of Aspergillus nidulans

Summary The gamma-amino-n-butyrate transaminase gene (gatA) of Aspergillus nidulans is one of several genes under positive control by the regulatory gene amdR (also called intA). The gatA gene has been cloned from a cosmid library by complementation of a gatA mutation. The sequence of a 2.6 kb genomic fragment containing gatA has been determined. An open reading frame of 1497 bp within this sequences is interrupted by three putative introns and predicts a protein of 55 kDa. Northern analysis confirms control of gatA RNA levels by amdR and also indicates that gatA is not strongly regulated by areA-mediated nitrogen metabolite repression. A. nidulans transformants containing multiple copies of a plasmid carrying an 88 bp fragment from the 5 untranscribed region of gatA grew poorly on substrates whose utilisation is dependent on genes controlled by amdR. This indicated titration of limiting amounts of the amdR gene product by this 88 bp fragment. Comparison of this sequence with the 5 region of the coregulated gene, amdS, reveals probable sites of action for the amdR protein.     

Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces.

Summary A 7.2 kbBglII restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and -galactosidase, was cloned inStreptomyces lividans from the DNA ofS. griseus ATCC 10137. This gene (namedsaf) showed a positive gene dosage effect on production of extracellular enzymes. When thesaf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores inS. lividans and also retarded actinorhodin production inStreptomyces coelicolor. Thesaf gene hybridized with specific bands in the DNA of severalStreptomyces strains tested. A 1 kb fragment containing thesaf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of M_r 10 500. This ORF is contained within a fragment of 432 by which retained activity inStreptomyces. A fragment with promoter activity is present upstream of thesaf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea,is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^? mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^? transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^? transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5′ regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.     

Expression of fungal genes involved in penicllin biosynthesis

Carbon catabolite repression and pH regulation are regulatory circuits with a wide domain of action in the Plectomycetes. Penicillin biosynthesis is one of the pathways which are under their control. The conclusions obtained so far, which are based on studies of the genetic and molecular regulation of the penicillin pathway of Aspergillus nidulans, would have been much harder to produce using an organism such as Penicillium chrysogenum (the industrial penicillin producer). However, A. nidulans and P. chrysogenum are close in terms of their phylogeny and one can reasonably predict that the conclusions about A. nidulans, which are summarized in this review and which are of unquestionable biotechnological relevance, will be extrapolable to the industrial organism.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.     

Ribosomal RNA genes in species of theCynareae tribe (Compositae). II

Summary The structure of the ribosomal DNA has been analyzed in three species of theCynareae tribe using Southern blot hybridization.Silybum marianum possesses two types of ribosomal genes 12.3 and 13.4 kb long;Cirsium vulgare has at least four types of rDNA repeats 10.6, 10.5, 11.7 and 11.9 kb long;Carlina acaulis only one type of ribosomal genes 9.7 kb long. The rRNA genes of the three species studied showed an identical restriction mapping in the 18 S and 25 S regions. However species differentiation in length and/or nucleotide sequences are present in the external spacer and very probably in the internal transcribed spacer.By cytophotometric studies and byin vitro rRNA/DNA filter hybridization, the DNA amount/4 C nucleus and the rDNA percentage were calculated in nine species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Cynara cardunculus subsp.cardunculus (wild artichoke),Onopordon acanthium, O. illyricum, Galactites tomentosa, Carduus nutans, Silybum marianum, Cirsium vulgare andCarlina acaulis. The DNA amount/4 C nucleus in eight species are similar, ranging from 4.24 pg inGalactites tomentosa to 5.96 pg inCirsium vulgare, whileCarlina acaulis has a DNA amount/ 4C nucleus of 11.8 pg. The rDNA percentages range from 0.192% inOnopordon acanthium to 1.022% inSilybum marianum, whileCarlina acaulis has 0.038% of rDNA. This latter species is clearly distinct within theCynareae tribe.     

A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.