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.     

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.     

Cloning of a sequence of Aquaspirillum magnetotacticum that complements the aroD gene of Escherichia coli

A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.     

Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

Catabolic dehydroquinase which functions in the inducible quinic acid catabolic pathway in Neurospora crassa has been purified 8000-fold. The enzyme was purified by two methods. One used heat denaturation of contaminating proteins; the other used antibody affinity chromatography. The preparations obtained by these two methods were identical by all criteria. The purified enzyme is extremely resistant to thermal denaturation as well as denaturation 0y urea and guanidine hydrochloride at 25 degrees. It is irreversibly inactivated, although not efficiently dissociated, by sodium dodecyl sulfate and guanidine hydrochloride at 55 degrees. At pH 3.0, the enzyme is reversibly dissociated into inactive subunits. At high concentrations catabolic dehydroquinase aggregates into an inactive, high molecular weight complex. The native enzyme, which has a very high specific activity, has a molecular weight of approximately 220,000 and is composed of identical subunits of 8,000 to 12,000 molecular weight each. The native enzyme and the subunit are both asymmetric.     

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.     

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa

Summary Theqa-2 gene ofNeurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. TheQUTE gene ofAspergillus nidulans corresponds to theqa-2 gene ofN. crassa. The plasmid pEH1 containing theQUTE gene fromA. nidulans was used to transform aqa-2 ^– strain ofN. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to theQUTE gene probe. Northern blot analyses indicated thatQUTE mRNA was produced in the transformants. The functional integrity of theQUTE gene inN. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-typeN. crassa. The evidence that theQUTE structural gene ofA. nidulans is inducible when introduced into theN. crassa genome implies that theN. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5 to theQUTE gene.     

Organization of Enzymes in the Common Aromatic Synthetic Pathway: Evidence for Aggregation in Fungi

Centrifugation in sucrose density gradients of partially purified extracts from six species of fungi, i.e., Rhizopus stolonifer, Phycomyces nitens, Absidia glauca (Phycomycetes), Aspergillus nidulans (Ascomycetes), Coprinus lagopus, and Ustilago maydis (Basidiomycetes), indicate that the five enzymes catalyzing steps two to six in the prechorismic acid part of the polyaromatic synthetic pathway sediment together. The sedimentation coefficients for these enzymes are very similar in the six species and are comparable to those previously observed for the multienzyme complexes (arom aggregates) of Neurospora crassa and Saccharomyces cerevisiae. These results are interpreted as indicating the presence in each of these fungi of arom aggregates, presumably encoded by arom gene clusters similar to those in N. crassa and S. cerevisiae. Evidence has also been obtained for the presence in two species (A. nidulans and U. maydis) and the absence in the other four species of a second dehydroquinase isozyme which is distinguishable from the synthetic activity on the basis of both thermostability tests and S values. This second dehydroquinase, which is apparently involved in the catabolism of quinic acid via a pathway similar to that in N. crassa, is inducible in A. nidulans (as it is in N. crassa), but constitutive in U. maydis. These comparative findings are discussed in relation to the organization, evolution, and possible functional relationships of synthetic and catabolic aromatic pathways in fungi.     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Characterization of qa-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques.

Genetic and complementation mapping studies using 20 qa-2 mutants defective for catabolic dehydroquinase indicate that the qa-2 gene encodes a single polypeptide chain and is the structural gene for catabolic dehydroquinase, a 220,000-molecular-weight protein composed of identical 10,000-molecular-weight subunits. Many qa-2 mutants are capable of reversion, but no evidence has yet been obtained for nonsense mutations in this gene. The biochemical consequences of the mutations in two complementing qa-2 strains (M239 and M204) have been determined. Both mutants have extremely low levels of catalytic activity and form a heterocaryon with about 4% of the wild-type activity. As assayed by immunological cross-reactivity, mutant M239 and the heterocaryon have nearly wild-type levels of native-molecular-weight catabolic dehydroquinase protein, whereas M204 has no detectable amount of this protein. Thus it is concluded that M239 has a mutation at or near the catalytic site which reduces the activity 10,000-fold but has little or no influence on the formation of the native multimeric structure. In contrast, M204 apparently has a mutation that severely inhibits aggregation and may have only a minor effect on the inherent potential for catalytic conversion at the reactive site. The heterocaryon would appear to form a mixed multimer with the monomeric subunits from M239 providing the aggregated structure and those from M204, the catalytically active moiety.     

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: an alternate cycloisomerase motif.

3-Carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.     

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa.

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.     

Mutagenicity of neocarzinostatin in Neurospora crassa

Neocarzinostatin (NCS) is an acidic, single-chain polypeptide of 109 amino acids that has shown some antitumor activity in clinical trials. NCS is mutagenic in recA+ strains of Escherichia coli, but not in recA strains; on the other hand, a defect in the nucleotide-excision-repair pathway has no effect on the mutagenicity of NCS in E. coli. Similar results are seen in mammalian cells. Excision-repair-deficient xeroderma pigmentosum (XP) cells repair NCS-induced DNA damage at the same rate as repair-proficient XP heterozygotes, and X-ray-sensitive ataxia telangiectasia fibroblasts are also sensitive to NCS. I have investigated the mutagenicity of NCS in the ad-3 forward-mutation test in nucleotide excision-repair-sufficient and -deficient heterokaryons of Neurospora crassa. Resting conidia from a repair-sufficient strain, H-12, and a nucleotide-excision-repair-deficient strain (uvs-2) H-59, were exposed to NCS. These conidia were assayed for survival and ad-3 forward mutation. The results show that H-59 is more sensitive to the killing and mutagenic activities of NCS than is H-12. These data indicate, in contrast to E. coli and mammalian cells, that the nucleotide-excision-repair pathway of N. crassa does repair NCS-induced lesions. In other experiments, ad-3 mutants induced by NCS in H-59 were characterized to determine the spectrum of NCS-induced mutation. The results show that NCS induces both intracistronic mutations and multilocus deletions in H-59.     

Ribosomal RNA genes of Neurospora crassa: multiple copies and specificities

Ribosomal RNA genes were isolated from the germinated conidial and mycelial cells of N. crassa by repeated cycles of 3H-DNA:rRNA reactions followed by hydroxyapatite chromatography. Specificity of multiple copies of those rDNAs with respect to N. crassa cell types was studied. The fraction of N. crassa germinated conidial in vitro labelled 3H-DNA recovered in the presence of rRNA isolated from the same cell type was about 2.2%, when compared with approximately 1.2% rDNAs obtained in mycelial cells. These isolated rDNAs reacted specifically to 26S and 17S rRNAs of eukaryotic (N. crassa) organisms and did not react with 4S tRNAs. rRNA:rDNA reassociation kinetics studies indicate that 90% of the rRNA genes were homogeneous and not identical with the other 10% rRNA genes isolated from N. crassa mycelia. These studies suggest that the possible heterogeneity of rDNA sequences of N. crassa cannot be attributed to inclusion of any tDNA sequences as has been shown in the heterogeneity of rDNA sequences of the bacterium Escherichia coli. The heterogeneity of multiple copies of N. crassa rDNAs could be due to differences in internal or external spacer regions of N. crassa rRNA genes.     

Circadian and light-induced expression of luciferase in Neurospora crassa

We have constructed a plasmid vector for expressing firefly luciferase in Neurospora crassa under control of the light- and clock-regulated ccg-2 (eas) promoter. The sequence of the luciferase gene in the vector has been modified to reflect the N. crassa codon bias. Both light-induced activity and circadian activity are demonstrated. Expression of luciferase in strains carrying mutant frequency alleles shows appropriate period length alterations. These data demonstrate that luciferase is a sensitive reporter of gene expression in N. crassa. Our results also show that the modified luciferase is expressed in Aspergillus nidulans.     

An analysis of gene-finding programs for Neurospora crassa.

MOTIVATION: Computational gene identification plays an important role in genome projects. The approaches used in gene identification programs are often tuned to one particular organism, and accuracy for one organism or class of organism does not necessarily translate to accurate predictions for other organisms. In this paper we evaluate five computer programs on their ability to locate coding regions and to predict gene structure in Neurospora crassa. One of these programs (FFG) was designed specifically for gene-finding in N.crassa, but the model parameters have not yet been fully 'tuned', and the program should thus be viewed as an initial prototype. The other four programs were neither designed nor tuned for N.crassa. RESULTS: We describe the data sets on which the experiments were performed, the approaches employed by the five algorithms: GenScan, HMMGene, GeneMark, Pombe and FFG, the methodology of our evaluation, and the results of the experiments. Our results show that, while none of the programs consistently performs well, overall the GenScan program has the best performance on sensitivity and Missing Exons (ME) while the HMMGene and FFG programs have good performance in locating the exons roughly. Additional work motivated by this study includes the creation of a tool for the automated evaluation of gene-finding programs, the collection of larger and more reliable data sets for N.crassa, parameterization of the model used in FFG to produce a more accurate gene-finding program for this species, and a more in-depth evaluation of the reasons that existing programs generally fail for N.crassa. AVAILABILITY: Data sets, the FFG program source code, and links to the other programs analyzed are available at http://jerry.cs.uga.edu/~wang/genefind.html. CONTACT: eileen@cs.uga.edu.     

Molecular comparison of the negative-acting nitrogen control gene,nmr, inNeurospora crassa and otherNeurospora and fungal species

In Neurospora crassa, the expression of unlinked structural genes which encode nitrogen catabolic enzymes is subject to genetic and metabolic regulation. The negative-acting nmr regulatory gene appears to play a role in nitrogen catabolite repression. Using the N. crassa nmr gene as a probe, homologous sequences were identified in a variety of other filamentous fungi. The polymerase chain reaction was used to isolate the nmr-like gene from the exotic Mauriceville strain of N. crassa and from the two related species, N. intermedia and N. sitophila. Sequence comparisons were carried out with a 1.7-kb DNA segment which includes the entire coding region of nmr plus 5' and 3' noncoding sequences. The size of the nmr coding region was identical in all three Neurospora species. Approximately 30 nucleotide base substitutions were found in the coding region of the nmr gene of each of the sister species when compared to the standard N. crassa sequence. However, most of the base changes occurred in third codon positions and were silent. The NMR proteins of N. sitophila and of N. intermedia display only three and four amino acid substitutions, respectively, from the N. crassa protein. Two regions of high variability, which include deletions and insertions of bases, were found in the 5' and 3' noncoding regions of the gene.