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Background: Integration of first- and second-generation ethanol processes is one among the alternate approaches that efficiently address the current socio-economic issues of the bioethanol sector. Edible filamentous fungus capable of utilizing pentoses from lignocelluloses and also possessing biomass application as potential animal feed component was used as the fermentation strain for the integration model. This study presents various fermentation aspects of using edible filamentous fungi in the integrated first and second generation ethanol process model., Results: Fermentation of edible strain of N. intermedia on the integrated first and second-generation ethanol substrate (the mixture of dilute acid pretreated and enzymatically hydrolyzed wheat straw and thin stillage from the first-generation ethanol process), showed an ethanol yield maximum of 0.23 ± 0.05 g/g dry substrate. The growth of fungal pellets in presence of fermentation inhibitors (such as acetic acid, HMF and furfural) resulted in about 11 to 45% increase in ethanol production as compared to filamentous forms, at similar growth conditions in the liquid straw hydrolysate. Fungal cultivations in the airlift reactor showed strong correlation with media viscosity, reaching a maximum of 209.8 ± 3.7 cP and resulting in 18.2 ± 1.3 g/L biomass during the growth phase of fungal pellets., Conclusion: N. intermedia fermentation showed high sensitivity to the dilute acid lignocellulose pretreatment process, with improved fermentation performance at milder acidic concentrations. The rheological examinations showed media viscosity to be the most critical factor influencing the oxygen transfer rate during the N. intermedia fermentation process. Mycelial pellet morphology showed better fermentation efficiency and high tolerance towards fermentation inhibitors.

When subjected to rapid drops of temperature (cold shock), Neurospora responds with a temporary shift in its morphology. This report is the first to examine this response genetically. We report here the results of a screen of selected mutants from the Neurospora knockout library for alterations in their morphological response to cold shock. Three groups of knockouts were selected to be subject to this screen: genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes; transcription factors; and genes homologous to E. coli genes known to alter their expression in response to cold shock. A total of 344 knockout strains were subjected to cold shock. Of those, 118 strains were identified with altered responses. We report here the cold shock morphologies and GO categorizations of strains subjected to this screen. Of strains with knockouts in genes associated with hyphal growth or morphology, 33 of 131 tested (25%) showed an altered response to cold shock. Of strains with knockouts in transcription factor genes, 30 of 145 (20%) showed an altered response to cold shock. Of strains with knockouts in genes homologous to E. coli genes which display altered levels of transcription in response to cold shock, a total of 55 of 68 tested (81%) showed an altered cold shock response. This suggests that the response to cold shock in these two organisms is largely shared in common., (Copyright © 2018 Watters et al.)

During ascogenesis in Neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel TNt strains by introgressing four Neurospora crassa insertional translocations (EB4, IBj5, UK14-1, and B362i) into N. tetrasperma. We now show that crosses of all the TNt strains with single-mating-type derivatives of the standard N. tetrasperma pseudohomothallic strain 85 (viz. T Nt a x 85A or T Nt A x 85a) can produce rare eight-spored asci that contain heterokaryotic ascospores, or ascospores with other unexpected genotypes. Our results suggest that these rare asci result from the interposition of additional mitoses between the post-meiotic mitosis and the partitioning of nuclei into ascospores, leading to the formation of supernumerary nuclei that then generate the heterokaryotic ascospores. The rare asci probably represent a background level of ascus dysgenesis wherein the partitioning of ascospores becomes uncoupled from the post-meiotic mitosis. Ordinarily, the severest effect of such dysgenesis, the production of mating-type heterokaryons, would be suppressed by the N. crassa tol (tolerant) gene, thus explaining why such dysgenesis remained undetected thus far.

Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom compost infected by red bread mold, exhibited antagonistic activities against Neurospora sitophila. Antifungal activity against phytopathogenic fungi was also observed. The maximum antifungal activity was reached during the stationary phase. This antifungal activity was stable over a wide pH and temperature range and was not affected by proteases. Assay of antifungal activity in vitro indicated that a purified antifungal substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In addition, treatment with strain B154 in A. bisporus mushroom compost infected with N. sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-associated laser desorption ionization time-of-flight mass spectrometry, and electrospray ionization tandem mass spectrometry analyses revealed a molecular weight consistent with 1498.7633 Da. The antifungal compound might belong to a new type of lipopeptide fengycin.

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC50) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC50. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC50 and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.

Unlabelled: Contamination of food industrial environments and recontamination of finished products by Chrysonilia sitophila and Hyphopichia burtonii have long represented serious problems for the bakery industries. As one of the most common ways to slow down or avoid fungal spoilage on bakery products is the use of ethanol, in the present work the effect of this substance has been assessed on growth of two of the most frequently occurring associated moulds, C. sitophila and H. burtonii, by means of tests on both synthetic media and sliced bread. Test on synthetic media: H. burtonii was less markedly affected in lag-phase duration and radial growth rates by the addition of ethanol to DG18 and the reduction in incubation temperature than C. sitophila that failed to grow at the highest concentrations of ethanol tested (2·0 and 4·0% at 15°C; 4·0% at 25°C). Test on sliced bread: ethanol proved to be effective to prevent spoilage by C. sitophila even at the lowest concentration tested (0·8%, w/w), while higher concentrations (2·0%, w/w) were needed to prevent spoilage by H. burtonii., Significance and Impact of the Study: This study shows that ethanol could represent an effective barrier to prevent spoilage of bakery products by associated moulds such as Chrysonilia sitophila and Hyphopichia burtonii, whose growth on packed and sliced bread was inhibited at very low (0·8%) or medium (2·0%) ethanol concentrations, respectively. The results obtained represent a fundamental point of reference for the bakery industries, as they can apply them in the productive practice to avoid spoilage by C. sitophila and H. burtonii on their products., (© 2013 The Society for Applied Microbiology.)

Neurospora tetrasperma is a pseudohomothallic filamentous ascomycete that has evolved from heterothallic ancestors. Throughout its life cycle, it is predominantly heterokaryotic for mating type, and thereby self-fertile. However, studies of N. tetrasperma have revealed the occasional production of self-sterile asexual and sexual spores of a single-mating type, indicating that it can be functionally heterothallic. Here, we report the extensive sampling and isolation of natural, heterokaryotic, strains of N. tetrasperma from the United Kingdom (UK): 99 strains were collected from Surrey, England, and four from Edinburgh, Scotland. We verified by phylogenetic analyses that these strains belong to N. tetrasperma. We isolated cultures from single germinated asexual spores (conidia) from 17 of these newly sampled UK strains from Surrey, and 16 previously sampled strains of N. tetrasperma from New Zealand (NZ). Our results show that the N. tetrasperma strains from the UK population produced a significantly greater proportion of self-sterile, homokaryotic conidia than the NZ population: the proportion of homokaryotic conidia was 42.6 % (133/312 spores) and 15.3 % (59/386) from the UK and the NZ populations, respectively. Although homokaryons recovered from several strains show a bias for one of the mating types, the total ratio of mat A to mat a mating type in homokaryons (UK: 72/61, NZ 28/31) did not deviate significantly from the expected 1:1 ratio for either of these populations. These results indicate that different populations exhibit differences in their life cycle characteristics, and that a higher degree of outcrossing might be expected from the UK population. This study points to the importance of studying multiple strains and populations when investigating life history traits of an organism with a complex life cycle, as previously undetected differences between populations may be revealed., (Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX(n)HXHX(10)D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF(FWD-1) complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa., Competing Interests: The authors have declared that no competing interests exist.

Meiotic silencing by unpaired DNA is a presumed RNAi-mediated elimination of the transcripts of any gene that is not properly paired with a homolog in meiosis. Eighty wild-isolated strains of Neurospora crassa were classified into three types based on the apparent strength of meiotic silencing of the bml (β-tubulin) and mei-3 genes in crosses with the ::Bml(r) and ::mei-3 tester strains. "OR" and "Sad" type wild-isolates, respectively, did or did not silence both the genes, whereas the "Esm" type (68 strains) silenced bml but not mei-3(+), suggesting an intermediate strength of silencing. Many wild strains, especially of the Esm type, gave fertile crosses with strains bearing the 148 kbp chromosome segment duplication Dp(EB4) but their crosses with the 405 kbp Dp(IBj5) were barren. Larger Dps might include more genes whose sensitivity to silencing is comparable with that of bml, consequently their crosses with the OR and Esm type strains would be about equally barren, whereas small Dps, with presumably few or no bml-like genes suffer little gene silencing in crosses with Esm and Sad strains and thus remain fertile. Using lollipop-shaped asci as indicators of silencing in crosses of the ::act tester with Esm type strains, and white-spored asci in Neurospora tetrasperma self-crosses heterozygous for an ectopic insertion of the asm-1 gene, we found that meiotic silencing was expressed more strongly in the early but not in the late perithecia of these crosses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The Varkud satellite (VS) plasmid, which requires the Varkud (V) plasmid for replication, is found in the mitochondria of several natural isolates of Neurospora. The VS transcript is sufficiently abundant that it might be expected to alter the function of mitochondria; however, previous limited characterization revealed no effect. In this work we have used genetic, biochemical and proteomic approaches to search for effects of the V and VS plasmids. We observed differences in the relative abundance of several mitochondrial proteins between plasmid-containing and plasmid-lacking natural isolates, but subsequently found these not to be due to the plasmids. We constructed a pair of iso-nuclear and iso-mitochondrial strains that differed only by the presence or absence of V and VS, and observed only subtle differences in the abundance of several mitochondrial proteins. We further attempted to detect a cryptic plasmid-related phenotype by growing this pair of strains in the presence of a variety of inhibitors of mitochondrial function or other stress conditions: this also revealed no effect of the plasmids. These observations suggest that, despite the high concentration of VS RNA in the mitochondrion, the V and VS plasmids do not cause substantial changes in the host., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this study, we investigated the genealogies of genes important for sexual identity, i.e. mating-type (mat) and pheromone-receptor (pre) genes, among heterothallic and peudohomothallic taxa of Neurospora. The resulting genealogies were compared with the species phylogeny derived from non-coding sequences. We found conflicting topologies between the reproductive genealogies and the species phylogeny, and these conflicts were supported by both node support analyses and likelihood tests on the relative fit of datasets to alternative phylogenetic hypotheses. We argue that reproductive genes are more permeable to gene flow, i.e. are more often introgressed between species of Neurospora, than other parts of the genome. Certain conflicts between the species phylogeny and both mat genealogies were observed, suggesting that the two mating-type idiomorphs were selectively introgressed into a species from a single ancestral source. Taken together, the results presented here highlight complex evolutionary trajectories of reproductive genes in the fungal kingdom, which may be of importance for reproductive behavior in natural populations., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Dodge's early work (1927-1940) on Neurospora genetics and sexual biology inspired Beadle and Tatum at Stanford to use N.crassa for their landmark discovery that genes specify enzymes. Neurospora has since become a model organism for numerous genetic, cytogenetic, biochemical, molecular and population biology studies. Neurospora is haploid in the vegetative phase with a transient diploid sexual phase. Its meiotic cells (asci) are large, allowing easy examination of dividing nuclei and chromosomes under a light microscope. The haploid meiotic products are themselves the sexual progeny that grow into vegetative cultures, thus avoiding the cumbersome testcrosses and complex dominance -recessive relationships, as in diploid organisms.The Perkins'laboratory at Stanford (1949-2007) played a pivotal role in advancing our knowledge of Neurospora genetics, sexual biology, cytogenetics and population biology. Since 1974, I have taken advantage of various chromosome-staining methods to examine ascus and ascospore development in wild type and in numerous mutant strains. In addition,I have used GFP-tagged genes to visualize the expression or silencing of unpaired genes in a post-transcriptional gene silencing process (meiotic silencing by unpaired DNA) that operates specifically during meiosis. The genome of N. crassa contains over 10 000 protein- coding genes. Gene knockouts or mutations in specific sequences may now be readily correlated with the observed cytological defects in the sexual stage, thus advancing our molecular understanding of complex processes during ascus and ascospore development.

Regulation of Rho GTPase signaling is critical for cell shape determination and polarity. Here, we investigated the role of LRG1, a novel member of the GTPase-activating proteins (GAPs) of Neurospora crassa. LRG1 is essential for apical tip extension and to restrict excessive branch formation in subapical regions of the hypha and is involved in determining the size of the hyphal compartments. LRG1 localizes to hyphal tips and sites of septation via its three LIM domains. The accumulation of LRG1 as an apical cap is dependent on a functional actin cytoskeleton and active growth, and is influenced by the opposing microtubule-dependent motor proteins dynein and kinesin-1. Genetic evidence and in vitro GTPase assays identify LRG1 as a RHO1-specific GAP affecting several output pathways of RHO1, based on hyposensitivity to the glucan inhibitor caspofungin, synthetic lethality with a hyperactive beta1,3-glucan synthase mutant, altered PKC/MAK1 pathway activities, and hypersensitivity to latrunculin A. The morphological defects of lrg-1 are highly reminiscent to the Ndr kinase/RAM pathway mutants cot-1 and pod-6, and genetic evidence suggests that RHO1/LRG1 function in parallel with COT1 in coordinating apical tip growth.

Meiotic silencing by unpaired DNA is a posttranscriptional gene silencing process in Neurospora crassa. Any gene without a homolog in the same chromosomal position during meiotic prophase generates a sequence-specific signal that prevents expression of all copies of that gene, but only during meiosis. Meiotic silencing is epigenetic and involves components of a meiosis-specific RNA silencing machinery. Although N. tetrasperma is closely related to N. crassa, its sexual biology is significantly different. N. tetrasperma was used here to evaluate both the generality of meiotic silencing within the genus and its possible evolutionary significance. A reporter gene for meiotic silencing, a histone H1-GFP fusion construct, was introgressed from N. crassa into various chromosome locations in N. tetrasperma. Whereas we did not observe meiotic silencing in four out of five introgression series, we obtained inconclusive results in the fifth series. Thus, we propose that meiotic silencing in N. tetrasperma is either absent or is substantially reduced when compared to N. crassa, possibly because the sad-1 gene (RNA-directed RNA polymerase, RdRP) is naturally unsynapsed (although "paired") and self-silenced during meiosis by structural differences between N. tetrasperma mating-type chromosomes. In N. crassa, wild-type sad-1 function is essential for meiotic silencing. Many point mutations in or deletion of sad-1 result in self-silencing of RdRP, and consequently suppression of meiotic silencing in heterozygous asci. The apparent absence or reduced meiotic silencing in N. tetrasperma raises the possibility that this form of silencing is not necessarily a major genome defense mechanism or responsible for reproductive isolation among the species of the genus Neurospora.

Background: An open, focal issue in evolutionary biology is how reproductive isolation and speciation are initiated; elucidation of mechanisms with empirical evidence has lagged behind theory. Under ecological speciation, reproductive isolation between populations is predicted to evolve incidentally as a by-product of adaptation to divergent environments. The increased genetic diversity associated with interspecific hybridization has also been theorized to promote the development of reproductive isolation among independent populations. Using the fungal model Neurospora, we founded experimental lineages from both intra- and interspecific crosses, and evolved them in one of two sub-optimal, selective environments. We then measured the influence that initial genetic diversity and the direction of selection (parallel versus divergent) had on the evolution of reproductive isolation., Results: When assayed in the selective environment in which they were evolved, lineages typically had greater asexual fitness than the progenitors and the lineages that were evolved in the alternate, selective environment. Assays for reproductive isolation showed that matings between lineages that were adapted to the same environment had greater sexual reproductive success than matings between lineages that were adapted to different environments. Evidence of this differential reproductive success was observed at two stages of the sexual cycle. For one of the two observed incompatibility phenotypes, results from genetic analyses were consistent with a two-locus, two-allele model with asymmetric (gender-specific), antagonistic epistasis. The effects of divergent adaptation on reproductive isolation were more pronounced for populations with greater initial genetic variation., Conclusion: Divergent selection resulted in divergent adaptation and environmental specialization, consistent with fixation of different alleles in different environments. When brought together by mating, these alleles interacted negatively and had detrimental effects on sexual reproductive success, in agreement with the Dobzhansky-Muller model of genetic incompatibilities. As predicted by ecological speciation, greater reproductive isolation was observed among divergent-adapted lineages than among parallel-adapted lineages. These results support that, given adequate standing genetic variation, divergent adaptation can indirectly cause the evolution of reproductive isolation, and eventually lead to speciation.

In Neurospora, metabolic oscillators coexist with the circadian transcriptional/translational feedback loop governed by the FRQ (Frequency) and WC (White Collar) proteins. One of these, a choline deficiency oscillator (CDO) observed in chol-1 mutants grown under choline starvation, drives an uncompensated long-period developmental cycle ( approximately 60-120 h). To assess possible contributions of this metabolic oscillator to the circadian system, molecular and physiological rhythms were followed in liquid culture under choline starvation, but these only confirmed that an oscillator with a normal circadian period length can run under choline starvation. This finding suggested that long-period developmental cycles elicited by nutritional stress could be masking output from the circadian system, although a caveat was that the CDO sometimes requires several days to become consolidated. To circumvent this and observe both oscillators simultaneously, we used an assay using a codon-optimized luciferase to follow the circadian oscillator. Under conditions where the long-period, uncompensated, CDO-driven developmental rhythm was expressed for weeks in growth tubes, the luciferase rhythm in the same cultures continued in a typical compensated manner with a circadian period length dependent on the allelic state of frq. Periodograms revealed no influence of the CDO on the circadian oscillator. Instead, the CDO appears as a cryptic metabolic oscillator that can, under appropriate conditions, assume control of growth and development, thereby masking output from the circadian system. frq-driven luciferase as a reporter of the circadian oscillator may in this way provide a means for assessing prospective role(s) of metabolic and/or ancillary oscillators within cellular circadian systems.

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.

We analyzed several natural suppressor isolates of the pKalilo-based fungal senescence syndrome of Neurospora intermedia. The pKalilo plasmid did not increase in titer in these isolates. Nor did it show integration "de novo." In at least two of the senescence suppressor isolates, pKalilo had formed stable recombinants with other mitochondrial elements. pKalilo/mtDNA recombination junctions were complete and appeared to have been formed via a nonhomologous recombination mechanism. Further analysis revealed that pKalilo had recombined a novel, 2.6-kb cryptic mitochondrial retroplasmid, similar to the mitochondrial retroplasmid pTHR1 from Trichoderma harzianum and retroplasmids of the "Varkud" homology group. The recombinant molecules consisted of pKalilo, the novel element, and short intervening stretches of mtDNA. The latter stretches clearly corresponded to "in vivo" mitochondrial cDNA, suggesting that the molecules had formed via the action of a template-switching reverse transcriptase. We discuss how different types of mitochondrial plasmids interact and how their detrimental effect on the host may be suppressed.

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

The natural population of Neurospora intermedia from Hawaii is polymorphic for the presence of the linear mitochondrial plasmid pKALILO that is associated with an infectious senescence syndrome. Although inter-specific horizontal transmission is experimentally possible, thus far pKALILO associated senescence has never been found outside N. intermedia in nature. Here, we demonstrate that it is not limited to the natural population of the heterothallic species N. intermedia, but also present in the sympatric population of its close relative, the pseudo-homothallic species Neurospora tetrasperma. We did a comparative analysis of the hallmarks of senescence in both species and show that: (1) Senescence is contagious in both species: the senescent state is efficiently transmitted between vegetatively compatible isolates. (2) All senescent isolates from both species contain the autonomously replicating linear mitochondrial senescence plasmid pKALILO. (3) In both species, senescent cultures contained copies of pKALILO inserted into the mitochondrial genome. Two of these inserts were characterized using semi-random two-step PCR, and were located within the large subunit mitochondrial rRNA gene. (4) However, pKALILO was less frequent in N. tetrasperma than in N. intermedia. (5) Also, the onset of senescence was significantly delayed in N. tetrasperma, compared to that in N. intermedia. We hypothesize how these differences in frequency and effect of pKALILO are connected to the respective life histories of their hosts.

A recessive ascospore mutant of Neurospora tetrasperma, named bud, was isolated from a wild-collected heterokaryotic strain with four different nuclear components. bud segregates as a single mendelian gene. When bud is homozygous, meiosis is apparently normal but postmeiotic events are not. Abnormal orientation of spindles at the postmeiotic mitosis often results in failed pair-wise association of nuclei and their irregular distribution along the length of the ascus prior to spore delimitation. Consequently, many asci cut out more than four ascospores; some contain no nuclei while others contain more than two. The most dramatic effect of bud is on ascospore delimitation itself. Many ascospores are irregularly shaped and are often interconnected, because of incomplete spore delimitation. Ascospores also show one or two lobes or bud-like extensions of varying sizes. Over 75% of ascospores from bud x bud remain white or tan and are inviable. The interaction of bud with a dominant Eight-spore mutant (E) was examined in both heterozygous and homozygous crosses. When both bud and E are heterozygous, bud has no effect on ascospore delimitation or on the phenotype of E because bud is recessive; many asci produce 5-8 ascospores just as in E x E(+). And when bud is homozygous and E is heterozygous, ascospore delimitation is less affected than when E is absent. Moreover, when both bud and E are homozygous, the effect on ascospore development is less extreme than when E is homozygous singly.

Neurospora mitochondrial plasmids are ubiquitous in natural populations, yet many of them are lethal to their host strains or seem to impose a molecular genetic load. Five pairs of strains of Neurospora tetrasperma and N. crassa with and without kalilo-like plasmids were tested under a variety of situations. The purpose was to find possible beneficial effects of plasmids that might offset their disadvantages. We found that, in all cases tested, plasmids conferred an advantage to growth at temperatures close to the top of the range for this fungus. Also, the plasmids improved fertility, as measured by perithecial production. Negative results were obtained for heavy metal resistance and ascospore germination. The results generate the hypothesis that plasmids may have adaptive significance to their hosts., (Copyright 2000 Academic Press.)

The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52-208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.

Whereas the gene stability related considerations are important in bioprocessing with recombinant cultures, bioreactor design and scale-up require attention to the often reduced shear tolerance of the genetically altered biocatalysts relative to the corresponding wild strains. In addition, the peculiarities of expression of the rDNA product impact the downstream recovery methods. As a consequence, a bioprocessing scheme and the process machinery designed for a naturally occurring organism may need significant modifications for use with a genetically modified variety of the same organism. The case studies described highlight some of the processing constraints and consideration of general relevance.

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species of Gelasinospora. The plasmid genome is 8231 bp long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of the Neurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.

This review followed from experiments suggesting that some fungi do not require calcium. It was found that many studies of a calcium requirement in microorganisms had assumed specificity for chelation agents such as EGTA and A23187, which the reagents did not possess. Early studies still cited today often preceded the recognition that microorganisms required manganese and zinc. As a result of both of these misunderstandings, there was rarely any attempt to replace calcium by other important trace elements. In some studies that seem to have been overlooked, the apparent requirement for calcium depended on the growth conditions used. Escherichia coli, Neurospora crassa, and Saccharomyces cerevisiae were then selected for detailed consideration and it is concluded that further experiments are needed before the involvement of calcium is proved.

A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un-1, un-3, and un-17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35 degrees C and comparatively inferior growth at 15-30 degrees C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or O2 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression.

The activated ascospores of Neurospora tetrasperma were inactive in protein synthesis and did not accumulate transcripts for a constitutive protein until after 90 min of incubation. These spores were blocked even longer in the expression of a gene encoding a heat shock protein, hsp30, which could not be induced until after 300 min of spore germination. Early in germination the ascospores were highly susceptible to damage from moderately high temperatures. At the same time that spores became capable of expressing the hsp30 gene, there was a loss of cytosine methylation from the gene.

Heat-pasteurized cassava root, cowpeas, partially defatted peanuts, rice and soybeans were evaluated for their suitability to support growth and sporulation of the tempeh mold, Rhizopus microsporus var. oligosporus, and the oncom mold, Neurospora intermedia, at 25, 30 and 37 degrees C. The molds grew best and sporulated most luxuriantly on cassava and rice incubated at 37 degrees C. Viability of molds remained high for up to 30 weeks when dried (a(w) 0.48), powdered substrates on which the molds had been cultured, were stored at 5, 25 and 37 degrees C. Survival was best when powders were stored at 5 degrees C, although storage at 25 degrees C did not cause a marked decline in CFU/g. R. microsporus var. oligosporus starter cultures produced on rice and then stored for 20 weeks at these temperatures compared most favorably with commercial starter cultures for preparing high-quality soybean tempeh. The simple technology required to prepare these starter cultures enhances the potential for their application in developing countries where the introduction of nontraditional fermented legume foods to low-protein diets could help to ameliorate malnutrition.

Hyperthermia, CdCl2, sodium arsenite, and H2O2 led to the rapid appearance of high levels of peroxidase in Neurospora crassa cultures and induced tolerance toward normally lethal temperatures in 60-h-old colonies. Intracellular superoxide dismutase levels did not correlate with the development of thermotolerance.

The natural death (nd) mutant of the fungus Neurospora crassa, unlike the wild-type, undergoes an aging process, which leads to the cessation of growth. It is shown here that the ATP/ADP ratio of the mutant declines with age to about 3:1 whereas other strains of Neurospora in the same growth medium maintain ratios of about 8 to 9:1. The decline in ATP/ADP ratio is not caused by the cessation of growth of the mutant. The results suggest, rather, that the cessation of growth may be caused, in part or in whole, by the defect in energy metabolism that produces the low ATP/ADP ratio. They support the hypothesis that defects in mitochondrial energy metabolism may be an important contributing factor to the aging process.

Three characteristics of the biochemical genetics of antioxidant enzyme regulation in Neurospora and enteric bacteria are analogous. This paper reports two additional analogies:responsiveness to change in respiratory rate or thermal stress. A negative regulatory Neurospora mutant is defective in those responses. Although several differences are noted, the common denominator of the two organisms is probably an oxy-regulon, a global unit of genetic function. The degree of homology of bacterial and fungal antioxidant enzyme regulatory mechanisms at the molecular-genetic and signal transduction levels of organization remains to be examined. The hypothesis that the genetic control of antioxidant enzymes is a prerequisite for cellular differentiation of Neurospora is discussed.

Cycloheximide (CHX), an inhibitor of cytosolic (80S) protein synthesis in eucaryotes, causes phase shifts of the circadian clock of Neurospora crassa when administered as 4-h pulses to cultures in liquid medium. Differential effects of the pulses at different phases of the circadian cycle were observed and plotted as a phase-response curve (PRC). Nearly all phase shifts observed were phase advances, with maximum sensitivity in the middle of the subjective day. Inhibition of protein synthesis by CHX was the same at both phases of the cycle. The PRC was the same at 20 and 25 degrees C. Dose-response curves for the effects of CHX on phase shifting and inhibition of protein synthesis were determined and showed a striking parallel in the responses of these two phenomena to CHX. These results support the view that synthesis of one or more proteins at specific phases of the circadian cycle is necessary for the normal operation of the circadian clock of Neurospora.

Colonial growth of Neurospora sitophila phenotypically induced by ramihyphin A is accompanied by marked changes in the contents of DNA, RNA and proteins in the mycelium, and in the relative proportion of hexoses in cell wall hydrolysates. The glucosamine/glucose ratio is also characteristic for colonial growth. X-ray analysis of cell walls showed that ramilhyphin A suppresses the crystalline arrangement of chitin in cell walls. A combination of microbiological, biochemical and physico-chemical methods yielded a general picture of the changes accompanying the colonial growth of Neurospora sitophila.

The treatment of undifferentiated mycelia with heavy suspensions of their own conidia triggers protoperithecial development. This effect was also observed with white collar (wc) mutants and suggests that the wc genes are not structural genes necessary for morphogenesis of protoperithecia but that they are probably involved in regulation.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.

Neurospora crassa strain cel, which is deficient in fatty acid synthesis, was grown with phytanic acid supplementation. The temperature dependence of membrane potential is increased by growth on phytanic acid. A temperature change of 40 degrees C produces a change of 184 mV in phytanic acid-grown cells as compared to a 50 mV change for cel grown on palmitic acid or wild-type. Membrane resistance (measured as DC input resistance) of phytanic acid-grown cells did not differ from cel grown on palmitic acid or wild-type. Lipid analysis of cel grown on phytanic acid revealed approximately 7 mole percent phytanic acid incorporation into phospholipids, no change in phospholipid base composition, a reduction of ergosterol content from 80 to 30 percent, and the induction of beta sitosterol, a sterol not usually present in Neurospora. beta sitosterol accounted for approximately 60 percent of the sterol present. Incorporation of 7 mole percent phytanic acid into phospholipids lowers the phase transition temperature by approximately 5 degrees C, and decreases the heat content of the phase transition (delta H) slightly. Results are discussed in relation to Refsum's disease, a human neurological disorder associated with high plasma levels of phytanic acid. It is proposed that high intracellular phytanic acid concentration induces novel sterol synthesis and that the incorporation of the novel sterol into the membrane is responsible for the increased temperature sensitivity of membrane potential. The excitable membrane deficits observed in patients with Refsum's disease may be explained by such a mechanism.

The kinetic parameters of [8-(14)C]adenosine transport by a general nucleoside uptake system were studied in germinated conidia of the ad 8 strain of Neurospora crassa. The apparent K(m) for adenosine uptake by this system was found to be 6.2 muM. The apparent K(i) values for other nucleosides competing with adenosine for uptake were measured by using Dixon plots. Nucleosides which were efficient competitive inhibitors of adenosine transport were found to inhibit severely the rate of growth of strain ad-8 on adenosine-supplemented medium. Xanthosine and thymidine did not inhibit [8-(14)C]adenosine uptake as severely as other nucleosides, nor did they cause significant inhibition of ad-8 growth rate on adenosine.

Studies using the morphological mutant "amycelial"--a conditional, aconidial strain of N. crassa--have revealed certain consistent functional and biochemical differences from wild type strains showing normal morphology and conidia formation. Thus it was found that the mutant strain showed an abnormal utilization of protein reserves at a time when there is extensive degradation during conidiogenesis in wild type. In addition, the mutant exhibited abnormally low oxygen consumption and free amino acid pools depressed nearly 3/4 with respect to wild type. All of these findings are consistent with earlier work linking conidiogenesis to aerobic oxidative processes and suggest that catabolic reactions particularly regarding protein breakdown may be important to conidiogenesis from the standpoint of the synthesis of new conidial proteins and for the supply of endogenous energy reserves during the process. The aconidial character of "amycelial" could be partially alleviated on solid acetate medium supplemented with amino acids, especially tryptophan or histidine. Conidiation was also induced by cyclic-AMP, even on sucrose medium. A polymer strongly antigenic in rabbits was found to be released into growth medium by the mutant but not by wild type cultures, providing a possible clue to the immediate cause of the abnormal morphology seen in "amycelial". This substance appears to be a glycopeptide containing a few hexoses and amino acids associated with polyphosphate.

Six nuclear suppressors of the (poky) cytoplasmic mutant (sup-1, sup-3, sup-4, sup-5, sup-10, sup-14) have been obtained in Neurospora crassa. The sup genes suppress the slow growth phenotype of (poky), and alleviate, at least partially, the deficiency of cyanide sensitive respiratory activity in the mycelium of this cytoplasmic mutant. The six suppressors are nonallelic, suppress the phenotypic effects of (stp-Bl) in addition to (poky), but have no effect on the phenotype expression of the (mi-3) cytoplasmic mutant. On the basis of experimentally established molecular defects in (poky) and on the basis of hypothetical consideration, it is proposed that the sup mutations affect the structure and properties of mitochondrial ribosomal proteins.

The extensional growth rate of wild-type 74A8 N. crassa in the presence of various concentrations of 19 amino acid analogs was measured. Seven analogs were not inhibitory at concentrations in the range of one to 10mM. Of the remaining 12 analogs, nine inhibited growth in a novel way. The kinetics of growth in the presence of these analogs at 30 degrees were characterized by seven sequential phases: (1)lag; (2) acceleration of growth rate; (3) steady-state growth rate; (4) exponential rate of decline of growth rate; (5) no growth or growth rate less than or equal to 0.1 mm h-1; (6) accleration of growth rate; and (7) steady state. At 33 degrees, phases 6 and 7 did not occur and irreparable death of the clones occurred. The mechanism by which the clones acquired resistance at 30 degrees appeared to involve a combination of physiological adaptation and cellular selection. Dietary application of either free radical scavengers or surface-active membrane 'stabilizers' alleviated or prevented the inhibition and deterioration of growth rate which occurred in the presence of the nine amino acid analogs. Culture with either 4-fluorophenylalanine or ethionine led to an increase of the activities of antioxygenic enzymes glutathione peroxidase, glutathione reductase and superoxide dismutase. The amino acid analogs that cause senescence and death of growing cells are known to be incorporated into proteins and such proteins are generally abnormal. Because a substantial fraction of cellular protein occurs in membranes and the proteins synthesized by mitochondria are exclusively intrinsic membrane proteins, we suggest that a primary consequence of errors in protein synthesis is the production of faulty membranes. The deterioration of such membranes with associated lipid autoxidation and free radical production proceeding as a chain reaction at an exponential rate may in itself contribute to the exponential rate of cellular deterioration which is characteristic of the ageing process. According to this hypothesis, dietary membrane stabilizers, free radical scavengers and antioxygenic enzymes protect cells from error catastrophy arising from the chain of events leading from membrane deterioration.

We report initial attempts to define the role of Ca2+ in the polarized extension of Neurospora crassa. Growth of the organism was diminished in media containing less than 1 mM-Ca2+; extension was more severely impaired than biomass synthesis, resulting in the formation of stubby, bulbous hyphae, even of spherical cells. Reduced extension and abnormal morphology were correlated with the loss of surface-bound Ca2+, probably associated with the cell wall. Intracellular Ca2+ may be represented by material that fluoresces brightly in the presence of chlortetracycline. Punctate fluorescent bodies and diffuse fluorescence were both arrayed in a longitudinal gradient, maximum apically. Addition of the calcium ionophore A23187 induced dissipation of the fluorescence; concurrently, the hyphae lost as much as one half of their Ca2+ content. Extension continued almost unabated, but multiple branches quickly emerged from the apex. The observations suggest that a cytoplasmic Ca2+ gradient is not required for polarized extension, but may play a role in ensuring the dominance of the apex.

A temperature-sensitive mutant of Neurospora crassa was found to undergo rapid death on minimal medium at 35 degrees C. The loss of viability in this mutant was prevented by various factors which retard growth, including deprivation of carbon sources or interruption of protein synthesis. Synthesis of nucleic acids and protein in this mutant was normal at the early stages of germination and then depressed at 35 degrees C. The active transport of glucose and the respiration rate in this mutant were depressed at 35 degrees C. Phopholipid synthesis was significantly repressed at 35 degrees C. The possible significance of the characteristics of this mutant is discussed in terms of membrane biosynthesis.