Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein.

To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1KO strain.

Mecanismos y regulación de la hidrólisis enzimática de celulosa en hongos filamentosos: casos clásicos y nuevos modelos / Mechanisms and regulation of enzymatic hydrolysis of cellulose in filamentous fungi: classical cases and new models

La celulosa es la fuente de carbono renovable más abundante de la Tierra. Sin embargo, la estructura de este polímero constituye una barrera física y química para acceder al carbono, lo que ha limitado el aprovechamiento del mismo. En la naturaleza, un pequeño porcentaje de microorganismos pueden degradarla a través de la expresión de celulasas. Dentro de estos microorganismos, uno de los grupos más activos y eficientes son los hongos filamentosos. Esta revisión describe las similitudes y diferencias de los mecanismos de acción de las celulasas y los mecanismos de regulación de su expresión para 3 de los modelos de hongos filamentosos celulolíticos más estudiados: Trichoderma reesei, Aspergillus niger y Aspergillus nidulans, y para un modelo recientemente descrito, Neurospora crassa. Se encontró que los mecanismos de acción enzimática son muy similares en todos los modelos estudiados, no así los mecanismos de regulación génica. Entender las particularidades de cada sistema es fundamental en el desarrollo de estrategias para la mejora de la producción de celulasas, ya sea proporcionando el ambiente óptimo (condiciones de fermentación) o aumentando la expresión en estos microorganismos mediante ingeniería genética (AU)

Cellulose is the most abundant renewable carbon source on earth. However, this polymer structure comprises a physical and chemical barrier for carbon access, which has limited its exploitation. In nature, only a few percentage of microorganisms may degrade this polymer by cellulase expression. Filamentous fungi are one of the most active and efficient groups among these microorganisms. This review describes similarities and differences between cellulase activity mechanisms and regulatory mechanisms controlling gene expression for 3 of the most studied cellulolytic filamentous fungi models: Trichoderma reesei, Aspergillus niger and Aspergillus nidulans, and the recently described model Neurospora crassa. Unlike gene expression mechanisms, it was found that enzymatic activity mechanisms are similar for all the studied models. Understanding the distinctive elements of each system is essential for the development of strategies for the improvement of cellulase production, either by providing the optimum environment (fermentation conditions) or increasing gene expression in these microorganisms by genetic engineering (AU)

Isolation, characterization and application of a cellulose-degrading strain Neurospora crassa S1 from oil palm empty fruit bunch.

BACKGROUND: Oil palm empty fruit bunch (EFB) is a lignocellulosic waste produced in palm oil industry. EFB mainly consists of cellulose, hemicellulose (mainly xylan) and lignin and has a great potential to be reused. Converting EFB to fermentable sugars and value-added chemicals is a much better choice than treating EFB as waste. RESULTS: A cellulase-producing strain growing on oil palm empty fruit bunch (EFB) was isolated and identified as Neurospora crassa S1, which is able to produce cellulases using EFB as the sole carbon source. The strain started to secret cellulases into the medium after 24 h of cultivation at 30°C and reached its maximal cellulase activity at 240 h. Mass spectroscopy (MS) analysis showed that more than 50 proteins were secreted into the medium when EFB was used as the sole carbon source. Among them, 7 proteins were identified as putative enzymes associated with cellulose degradation. The whole cell culture of Neurospora crassa S1 was used to hydrolyze acid-treated EFB, giving a total sugar yield of 83.2%, which is comparable with that (82.0%) using a well-known cellulase producer Trichoderma reesei RUT-C30 (ATCC56765). CONCLUSION: Neurospora crassa S1 is a commercially promising native cellulase producer for EFB hydrolysis especially when the sugars obtained are to be fermented to products that require use of non-genetically engineered strains.

Discovering functions of unannotated genes from a transcriptome survey of wild fungal isolates.

Most fungal genomes are poorly annotated, and many fungal traits of industrial and biomedical relevance are not well suited to classical genetic screens. Assigning genes to phenotypes on a genomic scale thus remains an urgent need in the field. We developed an approach to infer gene function from expression profiles of wild fungal isolates, and we applied our strategy to the filamentous fungus Neurospora crassa. Using transcriptome measurements in 70 strains from two well-defined clades of this microbe, we first identified 2,247 cases in which the expression of an unannotated gene rose and fell across N. crassa strains in parallel with the expression of well-characterized genes. We then used image analysis of hyphal morphologies, quantitative growth assays, and expression profiling to test the functions of four genes predicted from our population analyses. The results revealed two factors that influenced regulation of metabolism of nonpreferred carbon and nitrogen sources, a gene that governed hyphal architecture, and a gene that mediated amino acid starvation resistance. These findings validate the power of our population-transcriptomic approach for inference of novel gene function, and we suggest that this strategy will be of broad utility for genome-scale annotation in many fungal systems. IMPORTANCE Some fungal species cause deadly infections in humans or crop plants, and other fungi are workhorses of industrial chemistry, including the production of biofuels. Advances in medical and industrial mycology require an understanding of the genes that control fungal traits. We developed a method to infer functions of uncharacterized genes by observing correlated expression of their mRNAs with those of known genes across wild fungal isolates. We applied this strategy to a filamentous fungus and predicted functions for thousands of unknown genes. In four cases, we experimentally validated the predictions from our method, discovering novel genes involved in the metabolism of nutrient sources relevant for biofuel production, as well as colony morphology and starvation resistance. Our strategy is straightforward, inexpensive, and applicable for predicting gene function in many fungal species.

A factor in a wild isolated Neurospora crassa strain enables a chromosome segment duplication to suppress repeat-induced point mutation.

Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than (approx.) 300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps less than 200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds (approx.) 300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.

Carrefour Mme. Gras: a wild-isolated Neurospora crassa strain that suppresses meiotic silencing by unpaired DNA and uncovers a novel ascospore stability defect.

Ordinarily, RIP-induced erg-3 mutant Neurospora crassa ascospores and their erg(+) siblings do not differ in stability during long-term storage. Consequently, the frequency of RIP-induced erg-3 mutants remains about constant regardless of the time that has elapsed between ascospore harvest and germination. We found, however, that RIP-induced erg-3 mutants were apparently selectively lost with time from among the ascospores stored from a cross with the wild-isolated Carrefour Mme. Gras strain from Haiti. The Haitian strain was also found to exert a dominant suppression of meiotic silencing by unpaired DNA. Similar loss of RIP-induced erg-3 mutant ascospores was seen among the stored ascospores from a subset of crosses heterozygous for the semi-dominant Sad-1 or Sad-2 suppressors of meiotic silencing. Our results suggest that crosses suppressed in meiotic silencing can compromise the stability during storage of ascospores that inherit RIP-induced mutations.

Functions of the small proteins in the TOM complex of Neurospora crasssa.

The TOM (translocase of the outer mitochondrial membrane) complex of the outer mitochondrial membrane is required for the import of proteins into the organelle. The core TOM complex contains five proteins, including three small components Tom7, Tom6, and Tom5. We have created single and double mutants of all combinations of the three small Tom proteins of Neurospora crassa. Analysis of the mutants revealed that Tom6 plays a major role in TOM complex stability, whereas Tom7 has a lesser role. Mutants lacking both Tom6 and Tom7 have an extremely labile TOM complex and are the only class of mutant to exhibit an altered growth phenotype. Although single mutants lacking N. crassa Tom5 have no apparent TOM complex abnormalities, studies of double mutants lacking Tom5 suggest that it also has a minor role in maintaining TOM complex stability. Our inability to isolate triple mutants supports the idea that the three proteins have overlapping functions. Mitochondria lacking either Tom6 or Tom7 are differentially affected in their ability to import different precursor proteins into the organelle, suggesting that they may play roles in the sorting of proteins to different mitochondrial subcompartments. Newly imported Tom40 was readily assembled into the TOM complex in mitochondria lacking any of the small Tom proteins.

A new UV-sensitive mutant that suggests a second excision repair pathway in Neurospora crassa.

In an attempt to understand the relationship between photorepair and dark repair in Neurospora crassa, a new mutant was isolated, which showed defects in both repair processes. The new mutant, mus-38, is moderately sensitive to UV and shows imperfect photoreactivation following UV irradiation. DNA was purified from this mutant and the other UV-sensitive mutants, and analyzed for the removal of cyclobutane pyrimidine dimers (CPDs). UV-specific endonuclease-sensitive sites (ESS) completely disappeared with 1 h of photoreactivation in mus-38 DNA, although the survival recovery with photoreactivation was greatly reduced in this mutant. This suggests that the insufficient survival recovery with photoreactivation in mus-38 does not result from a failure of photo-reversal of CPDs. Removal of ESS during liquid holding (dark repair) was slower in mus-38 compared to wild type. To test the possibility that this mutant was involved in excision repair, the double mutant was made between mus-38 and mus-18, which encodes a UV-damage-specific endonuclease. CPD excision in the mus-18 null mutant was severely affected but not completely inhibited. The double mutant showed a complete loss of the excision activity and was super sensitive to UV. These results indicate that mus-38 participates in an excision pathway that is different from the mus-18 pathway. The mus-38 mutant was sensitive not only to UV but also to some chemical mutagens which make adducts on DNA. Thus, mus-38 is possibly involved in an excision-repair pathway that is related to the Saccharomyces cerevisiae RAD3 pathway.

Isolation and characterization of strains defective in vacuolar ornithine permease in Neurospora crassa.

Vacuolar uptake of ornithine and lysine was characterized in Neurospora crassa using a cupric ion permeabilization system. Michaelis constants were measured as 1.4 mM for lysine and 11.0 mM for ornithine, and maximal velocities were determined. Vacuolar lysine uptake was shown to be inhibited competitively by L-arginine and histidine while ornithine uptake was inhibited by a variety of amino acids. Strains defective in the vacuolar ornithine permease were isolated using a filtration enrichment method. Two isolates--RSC-39 and RSC-63--had a reduced ability to accumulate ornithine. Vacuolar uptake of amino acids was measured using cupric ion-permeabilized mycelia; both strains had reduced ornithine uptake while lysine uptake and arginine uptake were normal. For both isolates, both the Michaelis constant and the maximal velocity for ornithine uptake were reduced compared to those of wild type. These results suggest that both strains are defective in the gene which encodes the vacuolar ornithine permease.

Allelic specificity at the het-c heterokaryon incompatibility locus of Neurospora crassa is determined by a highly variable domain.

In filamentous fungi, the ability to form a productive heterokaryon with a genetically dissimilar individual is controlled by specific loci termed het loci. Only strains homozygous for all het loci can establish a heterokaryon. In Neurospora crassa, 11 loci, including the mating-type locus, regulate the capacity to form heterokaryons. An allele of the het-c locus (het-cOR) of N. crassa has been previously characterized and encodes a nonessential 966 amino acid glycine-rich protein. Herein, we describe the genetic and molecular characterization of two hei-c alleles, het-cPA and het-cOR, that have a different specificity from that of het-cOR, showing that vegetative incompatibility is mediated by multiple alleles at het-c. By constructing chimeric alleles, we show that het-c specificity is determined by a highly variable domain of 34-48 amino acids in length. In this regard, het-c is similar to loci that regulate recognition in other species, such as the (S) self-incompatibility locus in plants, the sexual compatibility locus in basidiomycetes and the major histocompatibility complex (MHC) genes in vertebrates.

A Neurospora crassa mutant altered in the regulation of L-amino acid oxidase.

The isolation and characterization of a Neurospora crassa mutant altered in L-amino oxidase regulation is reported. The previously isolated gln-1bR8 strain, which only synthesizes the glutamine synthetase alpha monomer and lacks the beta monomer, was used as parental strain. A mutant derivative of strain was selected for its ability to grow on minimal medium in the presence of DL-methionine-SR-sulfoximine (MSO), an inhibitor of glutamine synthetase activity. This gln-1bR8;MSOR mutant overcame the inhibitory effect of MSO by increasing the activity of L-amino acid oxidase, an enzyme capable of degrading this compound. In contrast with the wild-type strain, the L-amino acid oxidase of the MSOR mutant was resistant to glutamine repression; in fact, it was induced by this amino acid but repressed by ammonium. This mutant is different from other nitrogen regulatory N. crassa mutants reported and is only altered in the regulation of L-amino acid oxidase. The MSOR mutation is epistatic to nit-2 since the nit2;MSOR double mutant regulated the L-amino acid oxidase in the same way as the MSOR single mutant.

Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.

In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.

Isolation and characterization of a new acetate-requiring mutant strain, ace-9, of Neurospora crassa.

A new acetate-requiring mutant strain of Neurospora crassa, ace-9, has been isolated. The mutant gene was mapped between nuc-2 and arg-12 on the right arm of the second linkage group. The ace-9 mutant strain shows very weak activity of pyruvate dehydrogenase complex (PDHC). Three strains that show no activity of PDHC had already been found, i.e., ace-2, ace-3, and ace-4. Thus the ace-9 is the fourth gene that causes the deficiency in PDHC activity by a mutation. Deficiency of PDHC activity in ace-9 strain seems to be due to defective E1 component, because (1) the activity of E1 component enzyme is very weak in ace-9 mutant strain, and (2) normal PDHC activity was resumed when a preparation of ace-9 was mixed with E1-E2 fraction of wild type or with E1 component of wild type E. coli. Difference in thermostability of both E1 component enzyme and PDHC between ace-9 and the wild type strains supports this conclusion.

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India.

Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5' termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.

Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. Production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. Isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. The lah-1 mutation is mapped between nit-2 and leu-3 on linkage group I, and it behaved as a recessive mutation in a forced heterokaryon. No differences were detected biochemically or immunologically between the laccase protein produced by the lah-1 mutant in the absence of cycloheximide and that induced with cycloheximide in the wild-type strain. This suggests that both laccases (66 kilodaltons) are products of the same structural gene. Relative amounts of laccase in the culture filtrate of the lah-1 mutant were much higher than those induced with cycloheximide in the wild-type strain, demonstrating high efficiency of the lah-1 mutant in production and secretion of laccase. The time course of laccase production by the lah-1 mutant revealed that expression of 66-kilodalton laccase was repressed in conidia and derepressed during vegetative mycelial growth. This suggests that a multiple regulatory mechanism is involved in the production and/or maturation of Neurospora laccase. The lah-1 mutant may be useful for identifying genes that regulate expression of the laccase gene in N. crassa.

Isolation and characterization of cadmium-resistant mutants of Neurospora crassa.

This study identified and characterized four cadmium-resistant mutants of Neurospora crassa. One of these mutants maps to linkage group II and the other three map to linkage group VII, whereas a naturally occurring resistant trait in a strain from Japan resides at a distinct but unmapped locus. Transport of cadmium into Neurospora cells occurs by more than a single uptake system and involves both energy-dependent and -independent components. The resistant mutants transport cadmium in the same manner as does the cadmium-sensitive wild-type strain. Cadmium resistance in these mutants does not appear to result from an increase in cytosolic heat-stable cadmium-binding proteins. Cadmium does not induce the typical heat-shock response in conidia. Under various growth conditions, each of the mutants exhibited morphological alterations, possibly involving the cell wall or plasma membrane.

The isolation and characterization of mutants defective in nitrate assimilation in Neurospora crassa.

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth test that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing cofactor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-y, nit-8 and nit-9), all of which are gentically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar ao the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one lous (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.

[Isolation and certain features of a polyphosphate phosphohydrolase deficient mutant of the fungus Neurospora crassa]. / Vydelenie i nekotorye osobennosti defitsitnogo po polifosfatfosfogidrolaze mutanta griba Neurospora Crassa.

A polyphosphatase deficient mutant of Neurospora crassa has been isolated. The criterion for selecting the mutant was the capacity of the fungus to assimilate polyphosphates as the source of exogenous phosphorus. The mutant like the parent strain ad-6, was an adenine auxotroph but differed from the parent strain by a lower growth rate though, at the stationary stage, its biomass reached the same level as in the strain ad-6. The character of changes in the activity of polyphosphatase in the course of growth was the same in the two cultures, but the activity of the enzyme in the mutant was considerably lower at all the growth stages. The content of polyphosphate fractions with the highest molecular weight increased twofold in the mutant culture. These data suggest that there is a close metabolic and topographic correlation between polyphosphatase and the highest molecular weight fractions of polyphosphates in N. crassa.

Isolation of Neurospora crassa bradytrophs.

A method was developed for the isolation of Neurospora bradytrophs. The bradytrophs (representing lesions in a number of pathways) were resistant to DL-p-fluorophenylalanine when growing in a leaky fashion but were sensitive when grown in the presence of their stimulating supplement.