Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

Summary The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5 non-coding regions show significant homology with UAS _GAL and UAS _QA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5 sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.     

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.     

Gene order in the qa gene cluster of Neurospora crassa

Summary Four different types of crosses have been used to establish the order of the four genes in the qa gene cluster of Neurospora crassa, which encode the following proteins involved in the inducible catabolism of quinic acid: a regulatory (activator) protein (qa-1), catabolic dehydroquinase (qa-2), quinate dehydrogenase (qa-3), and dehydroshikimate dehydrase (qa-4). The four crosses involved (1) the ordering of the four qa genes relative to the closely-linked me-7 locus; (2) the ordering of the three other qa genes relative to a qa-1 ^S mutant; (3) the use of a three factor cross-qa-3xqa-4 qa-2 and (4) the use of four factor crosses-qa-1 ^S xqa-3 qa-4 qa-2. The results of all four types of crosses agree in establishing an apparently definitive proximal to distal order, within the right arm of linkage group VII, i.e., qa-1 qa-3 qa-4 qa-2 me-7.The significance of a definitive establisment of the gene order within the qa cluster for an understanding of the organization and mechanism of genetic regulation in this cluster is discussed.     

In vivo overproduction of the pentafunctional arom polypeptide in Aspergillus nidulans affects metabolic flux in the quinate pathway

Summary The shikimate pathway and the quinic acid utilisation (QUT) pathway of Aspergillus nidulans and other fungi share the two common metabolic intermediates, 3-dehydroquinic acid (DHQ) and dehydroshikimic acid (DHS), which are interconverted by two isoenzymes, catabolic 3-dehydroquinase, (cDHQase) and biosynthetic dehydroquinase (bDHQase). bDHQase is one of five consecutive enzymatic activities associated with the pentafunctional arom protein encoded by the complex AROM locus, whereas cDHQase is encoded by the single-function QUTE gene, one of seven genes comprising the QUT gene cluster in A. nidulans, which is required for the catabolism of quinate to protocatechuate. We addressed the question of how much (if any) leakage there is of the two common substrates between the two pathways, by increasing the concentration of the arom protein in vivo by means of recombinant DNA technology. We demonstrated that constitutive overproduction of the arom protein by 12-fold in the presence of quinate inhibits germination of conidiospores, but showed that 12-fold quinate-inducible overproduction of arom protein does not have this effect. In addition we showed that a qutE mutant (lacking cDHQase) can grow with quinic acid as sole carbon source when the arom protein is overproduced fivefold. The data are most simply interpreted as simple competition for common substrates by the enzymes of the two pathways and demonstrate that any channelling function of the arom protein in vivo is relatively leaky.     

Effect of mutations in the qa gene cluster of Neurospora crassa on the enzyme catabolic dehydroquinase.

Catabolic dehydroquinase, which functions in the inducible quinic acid catabolic pathway of Neurospora crassa, has been purified from wild type (74-A) and three mutants in the qa gene cluster. The mutant strains were: 105c, a temperature-sensitive constitutive mutant in the qa-1 regulatory locus; M-16, a qa-3 mutant deficient in quinate dehydrogenase activity; and 237, a leaky qa-2 mutant which possess very low levels of catabolic dehydroquinase activity. The enzymes purified from strains 74-A, 105c, and M-16 are identical with respect to behavior during purification, specific activity, electrophoretic behavior, stability, molecular weight, subunit structure, immunological cross-reactivity, and amino acid content. The mutant enzyme from strain 237 is 1,500-fold less active and appears to have a slightly different amino acid content. It is identical by a number of the other criteria listed above and is presumed to be a mutant at or near the enzyme active site. These data demonstrate that the qa-1 gene product is not involved in the posttranslational expression of enzyme activity. The biochemical identity of catabolic dehydroquinase isolated from strains 105c and M-16 with that from wild type also demonstrates that neither the inducer, quinic acid, nor other enzymes encoded in the qa gene cluster are necessary for the expression of activity. Therefore the combined genetic and biochemical data on the qa system continue to support the hypothesis that the qa-1 regulatory protein acts as a positive initiator of qa enzyme synthesis.     

Genetical and Molecular Analyses of qa-2 Transformants in NEUROSPORA CRASSA

Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants.     

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.     

Genetical and biochemical aspects of quinate breakdown in the filamentous fungus Aspergillus nidulans

In the ascomycetous fungus Aspergillus nidulans, the expression of two inducible, contiguous or closely linked genes (qutB and qutC) which encode enzymes for quinate breakdown to protocatechuate, appears to be controlled by the product of a tightly linked third gene (qutA). The qut gene cluster locates on chromosome VIII. The catalytic steps required for this conversion are dehydrogenase, dehydroquinase, and dehydratase, and these activities are induced by the presence of quinate in a similar manner. The dehydroquinase enzyme has been purified and shown to be multimeric, consisting of 20–22 identical subunits of approximately 10,000 MW. The enzyme has a pI value of 5.84, a K _m of 5×10^–4 m, and an amino acid composition that lacks tryptophan and cysteine. The enzyme also cross-reacts with rabbit antibodies raised against Neurospora crassa catabolic dehydroquinase.This work was supported in part by European Molecular Biology Organisation grants to J.R.K. and A.R.H. and by National Institutes of Health Grant GM23051 to N.H.G.     

Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

Summary A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 Kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.Standard Abbreviations Used SSC Standard saline citrate (3 M Sodium Chloride, 0.15 M Sodium citrate) - EDTA Ethylenediaminetetra acetic acid - DTT Dithiothreitol - PMSF Phenyl methyl sulphonyl fluoride - TEMED N N N N, Tetramethylethylenediamine - Md Megadaltons - Kd Kilodaltons     

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.     

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.     

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping

Summary TheAspergillus nidulans gene coding for acetamidase (amdS) was introduced intoA. niger by transformation. Twelve Amd⁺ transformants were analysed genetically. TheamdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed)A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for theamdS insert have the Amd⁺ phenotype. We exploited the opportunity for two-way selection inA. niger: transformants can be isolated based on the Amd⁺ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologousamdS gene inA. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI betweenpabA1 andcnxA1, providing evidence for a single transformational insert. The results also show that theamdS transformants ofA. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use ofamdS transformants for mitotic gene mapping in other fungi.     

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

Summary When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.     

Organization and expression inPseudomonas putida of the gene cluster involved in cephalosporin biosynthesis fromLysobacter lactamgenus YK90

TheLysobacter lactamgenus YK90pcbAB gene encoding -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is located immediately upstream of thepcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. ThepcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411 593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within thepcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. The domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthatase 2 ofBacillus brevis. ThepcbAB gene was expressed under the control of thelac promoter inPseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis inP. putida led to the accumulation of -lactam antibiotics. Deletion analysis of an open-reading frame located between thecefE andcefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis inL. lactamgenus appear to be clustered in the orderpcb AB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.