Complementation among cytoplasmic mutants of Neurospora crassa

Summary Heteroplasmons with normal growth rates are formed when the slow-growing, female fertile, group I or II extranuclear mutants of Neurospora crassa are combined by forced heterokaryosis with the female sterile, stopper mutants of group III. Different mutants from the same growth and fertility group do not complement each other, and the poky-like strains of group I do not interact synergistically with [mi-3], the only known group II mutant. The mitochondrial cytochrome system of the complementing heteroplasmons are as abnormal as the cytochrome complements of the component extranuclear mutants, indicating that defects in the electron transport system represented by those mutants are related inconsequentially to growth. The observed functional complementation indicates the expression of the mitochondrial genome is not restricted to the specific organelle of which it is a part.Contribution No. 1255 Department of Agronomy; Contribution No. 1148, Division of Biology, Kansas Agriculture Experiment Station, Manhattan, Kansas.     

Cyanide-Resistant Respiration of a Neurospora crassa Membrane Mutant

Choline starvation of the Neurospora crassa chol-1 mutant leads to a decrease in respiration through the cytochrome chain and a concomitant induction of the alternate oxidase.     

Acetate-nonutilizing Mutants of Neurospora crassa I. Mutant Isolation, Complementation Studies, and Linkage Relationships

Sixty mutants of Neurospora crassa unable to grow on acetate as sole source of carbon, but able to utilize sucrose, were isolated. On the basis of complementation tests, they were divided into seven groups, each group representing a different gene. Six of the genes have been mapped; no two are closely linked. These loci have been designated acu-1 to acu-7. Mutations at four of these loci result in poor germination of ascospores.     

Altered Mitochondrial Respiration in a Chromosomal Mutant of Neurospora crassa

A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.     

Multiple Alterations in Metabolite Uptake in a Mutant of Neurospora crassa

A temperature-conditional lethal mutant of Neurospora crassa, un-t (55701), was resistant to neutral amino acid analogues by virtue of a decreased ability to transport these analogues and their natural congeners across the cell membrane. The uptake of acidic, but not basic, amino acids was also impaired, as was the uptake of potassium ions. After preincubation above the tolerated temperature, the ability to take up a still wider variety of metabolites was greatly reduced. Protoplasts of the mutant were more osmotically fragile than those of wild type. The possibility that the mutant has a generalized membrane defect is discussed.     

Disruption of an Amino Acid Transport Mutant of Neurospora crassa by KCl

A double amino acid transport-deficient mutant (Pm (-)NB) of Neurospora crassa is shown to be altered in the molecular structure of its cell wall or membrane. This alteration was revealed by a high degree of cellular disruption and cell-cell interaction following extraction by a high molar concentration of KCl.     

Circadian Nature of a Rhythm Expressed by an Invertaseless Strain of Neurospora crassa

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25 degrees ); B) the period is relatively temperature-independent (Q(10) is between 0.95 and 1.21 for temperature range of 18 to 35 degrees ); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.     

Uptake of Nitrite by Neurospora crassa

Like the nitrate transport system, the nitrite uptake system in Neurospora crassa is induced by either nitrate or nitrite. This induction is prevented by cycloheximide, puromycin, or 6-methyl purine. The K(m) for nitrite of the induced nitrite uptake system is 86 muM, and the V(max) is 100 mumol of nitrite per g (wet weight) per h. Nitrite uptake is inhibited by metabolic poisons such as arsenate, dinitrophenol, cyanide, and antimycin A. No repression or inhibition of the nitrite transport system by ammonia, nitrate, or Casamino Acids was observed.     

Metabolism of ricinoleate by Neurospora crassa

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and of 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate of 14 M–3 mM [1-¹⁴C]ricinoleate. The fate of the [¹⁴C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.     

Expression of a plant protein by Neurospora crassa.

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.     

Peptide utilization by nitrogen-starved Neurospora crassa

Peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. Under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. Growth of a leu-2 mutant of Neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a leu-2; gltR mutant on these same peptides resulted in induction of peptidohydrolytic activity. The induced extracellular proteolytic activity was shown to be analogous to that inducible by growth on proteins, e.g., bovine serum albumin.     

Histidine Uptake in Strains of Neurospora crassa with Normal and Mutant Transport Systems

Kinetic parameters for three systems of active histidine uptake by germinated conidia of Neurospora crassa have been measured. Each system appears to follow typical Michaelis-Menten kinetics when studied separately from the other systems. Under the conditions studied, the general amino acid transport system was found to account for the major portion of histidine uptake from low concentrations. Three types of transport mutants with altered growth inhibition patterns were selected in a histidine auxotroph. Growth of one mutant, type bas(a), could be inhibited by the addition of methionine to a histidine-supplemented medium, and another type, neu(a), could be inhibited by the addition of arginine. These mutants were shown to be lacking active histidine uptake by the basic amino acid and neutral amino acid transport systems, respectively. Another type of double mutant (his-3, neu(r)) could be inhibited only by the addition of very high concentrations of methionine in the presence of arginine and histidine, and the mutation appeared to have altered the specificity of the neutral amino acid permease.     

Neurospora crassa Cytoplasmic Ribosomes: Isolation and Characterization of a Cold-Sensitive Mutant Defective in Ribosome Biosynthesis

Twenty-seven cold-sensitive mutants of Neurospora crassa were isolated by mutagenesis of wild-type conidia followed by filtration enrichment in complete medium at the nonpermissive temperature (10 C). Zone sedimentation analyses of cytoplasmic ribosomes isolated from the wild-type strain and from 14 of the mutant strains grown at 10 C indicate that one cold-sensitive mutant is defective in ribosome biosynthesis at that temperature: instead of the 2.3:1 mass ratio of 60S:37S ribosomal subunits characteristic of wild type, the mutant strain PJ30201 (called crib-1 for cytoplasmic ribosome biosynthesis) exhibits a mass ratio of approximately 7.2:1. Ribosomal subunits synthesized by strain PJ30201 at 25 C are present in wild-type proportions. The cold-sensitive and ribosomal phenotypes segregate together in tetrads isolated from crosses between strain PJ30201 and the wild type indicating that a single nuclear gene mutation is probably responsible for both mutant phenotypes. The crib-1 locus lies near the centromere in linkage group IV.     

A Folylpolyglutamate Synthetase Lesion in the met(S2706) Mutant of Neurospora crassa

The met(S2706) mutant (FGSC 4248) of Neurospora crassa was culturedin Vogel's minimal medium with and without L-methionine supplementation.Methionine Stimulated growth but significant mycelial productionalso occurred in minimal medium. The mutant had ability to generatefree and protein methionine from [³⁵S]-cysteine in vivo butthe rate of this synthesis was below that shown by a wild typestrain (Lindegren A, FGSC 853). Cytosolic and mitochondrialfractions of met(S2706) also generated methionine from [3-¹⁴C]-serine.Following dialysis, this reaction was stimulated by additionsof tetrahydrofolate and its diglutamate derivative. Folate analysesshowed that the mutant had a folylpolyglutamate concentrationthat was only 10% of that detected in the wild type. Despitethis, mitochondria of met(S2706) contained folates that werelargely polyglutamates and the total mitochondrial folate concentration(ng/mg protein) was comparable to that of the wild type. Assays of folylpolyglutamate synthetase showed that met(S2706)had a lesion affecting a cytosolic, tetrahydrofolate diglutamate-formingactivity. Cytosolic protein had ability to catalyze a diglutamate hexaglutamate reaction. Mitochondria of the mutant catalyzedthe formation of methylenetetrahydrofolate triglutamate fromthe corresponding methylene monoglutamate. It is suggested thatthe limited folylpolyglutamate synthesis of met(S2706) may involvecytosolic and mitochondrial folylpolyglutamate synthetase activities. (Received September 10, 1984; Accepted December 17, 1984)     

Microconidia of Neurospora crassa

Neurospora crassa produces two types of vegetative spores-relatively small numbers of uninucleate microconidia and very large numbers of multinucleate macroconidia (blastoconidia and arthroconidia). The microconidia can function either as spermatia (male gametes) or as asexual reproductive structures or both. In nature they probably function exclusively in fertilization of protoperithecia. The environmental conditions favoring their formation and the pattern of their development are quite distinct from those of macroconidia. Mutants of N. crassa have been isolated in which macroconidiation is selectively blocked without affecting microconidiation, showing that these two types of conidial differentiation involve distinct developmental pathways. Unlike microconidia of some related ascomycetes, those of Neurospora are capable of germination, providing viable uninucleate haploid cells which are desired in several types of investigations. A technique of selectively removing macroconidia from culture initiated on cellophane overlying agar medium allows pure microconidia to be obtained even from the wild-type strains of Neurospora. The conditional microcyclic strain, mcm, allows either macroconidia or microconidia to be obtained at will, depending on the conditions of culture. The new methods of obtaining pure microconidia from normal laboratory strains will make it quick and easy to purify heterokaryotic transformants following introduction of DNA into multinucleate protoplasts. Moreover, these methods allow the detection of genetic variability that remains hidden within an individual fungus and the estimation of the frequency of nuclear types in laboratory-constructed heterokaryons. The discovery, function, and development of microconidia are described and their research applications are discussed in this review.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.