A temperature-sensitive mutant of Neurospora crassa deficient in cytochrome b.

Summary A nuclear gene mutant of Neurospora crassa designated cyb-3 is deficient in cytochrome b and coenzyme QH₂-cytochrome c reductase. Nearly normal when grown at 25°C, the strain expresses a mutant phenotype at 38°C. Mitochondria from cybr-3 mycelium, which has undergone 3–4 mass doublings at the elevated temperature, possess 3-fold less cytochrome b, 2-fold more cytochrome, c, 5-fold less coenzyme QH₂-cytochrome c reductase activity, and require 3-fold less antimycin A per milligram of protein to inhibit NADH oxidation than do wild type mitochondria. The activity of coenzyme QH₂-cytochrome c reductase declines rather slowly in cultures of cyb-3 transferred to 38° C, and the in vitro thermostability of the enzyme is very similar in wild type and mutant mitochondria. Therefore, the mutation may decrease synthesis or impair integration into the membrane of cytochrome b and perhaps other proteins of the enzyme complex.Contribution No. 1294-j, Division of Biology, Agricultural Experiment Station, Kansas State University, Manhattan, Kansas.     

An acetate-sensitive mutant of Neurospora crassa deficient in acetyl-CoA hydrolase.

The predicted amino acid sequence of the product of the acetate-inducible acu-8 gene of Neurospora crassa, previously of unknown function, has close homology to the recently published sequence of Saccharomyces cerevisiae acetyl-CoA hydrolase. An acu-8 mutant strain, previously characterized as acetate non-utilizing, shows strong growth-inhibition by acetate, but will use it as carbon source at low concentrations. The mutant was shown to be deficient in acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with acetate. As in Saccharomyces, the Neurospora enzyme is acetate-inducible.     

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity.

A mutant of Neurospora crassa (pm-nbg27) was isolated on the basis of its resistance of p-fluoro-phenylalanine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport activity of all amino acids. Genetic analysis of pm-nbg27 by crossing it to wild-type (74A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types. The above observations confirm the postulated general amino acid permease system as well as a single genetic locus control of that activity.     

Genetic and enzymatic analysis of a glycerol kinase deficient mutant in Neurospora crassa.

Summary Genetic analysis showed that the glycerol non-utilizing isolate gly-u(234) of Neurospora crassa is derived by mutation in a nuclear gene situated in the right arm of linkage group I, about 2.2 crossover units distal to ad-9 and 11 units proximal to nit-1.Enzymatic testings using a radiochemical method indicate that the mutant is deficient for the enzyme glycerol kinase. The radiochemical testings further indicate that the mutation has inactivated an inducible glycerol kinase, while a low residual activity may be due to a second, basal and non-inducible glycerol kinase, in accordance with a proposal by North (1973, 1974) that Neurospora has two glycerol kinases with these properties.     

Isolation and certain features of a polyphosphate phosphohydrolase deficient mutant of the fungus 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.     

Neurospora crassa mutants deficient in asparagine synthetase

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.     

A mutant of Neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria

The nuclear cyt-2-1 mutant of Neurospora crassa is characterized by a gross deficiency of cytochrome c (Bertrand, H., and Collins, R. A. (1978) Mol. Gen. Genet. 166, 1-13). The mutant produces mRNA that can be translated into apocytochrome c in vitro. Apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. Mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mRNA and convert it to holocytochrome c. This conversion depends on the addition of heme by cytochrome c heme lyase and is coupled to translocation of cytochrome c into the intermembrane space. Mitochondria from the cyt-2-1 strain are deficient in the ability to bind apocytochrome c. They are also completely devoid of cytochrome c heme lyase activity. These defects explain the inability of the cyt-2-1 mutant to convert apocytochrome c to the holo form and to import it into mitochondria.     

Characteristics of a glycerol utilization mutant of Neurospora crassa.

A mutant of Neurospora crassa able to grow on liquid minimal glycerol medium without evidence of conidiation and with high cell yields has been isolated and shown to be allelic to ff-1. The glycerol-specific induction of glycerokinase and glycerol-3-phosphate dehydrogenase was similar in both wild-type and mutant cells, although higher specific activities as well as higher glycerokinase cross-reacting material levels were found in fully induced mutant cells. After growth in minimal glycerol medium there is a significant reduction in wild-type cells of the activities of both pyruvate dehydrogenase and dihydrolipoyl transacetylase. This evidence indicates a relationship between the conditional acetate requirement by wild-type cells grown on glycerol medium and the levels of the pyruvate dehydrogenase complex.     

Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase.

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.     

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.     

A perithecial color mutant of Neurospora crassa

Summary A mutation, per-1, was found in N. crassa which caused orange instead of black perithecia when the protoperithecial parent contained the per-1 allele. No effect of per-1 was found on vegetative morphology or nutritional requirements. The per-1 locus was mapped proximal to iv (?6201) in the right arm of linkage group V.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Electron transport in the cni-1 mutant of Neurospora crassa.

1. Mitochondria from the nuclear mutant cni-1 have no optically detectable cytochrome aa3 in early log phase growth. These mitochondria have a high level of respiration that is not inhibited by cyanide but is inhibited by salicylhydroxamic acid. They also show a substantial amount of cyanide-sensitive respiration. 2. As cultures of mutant cni-1 age, flux through the hydroxamate-sensitive pathway decreases markedly while flux through the cytochrome chain remains constant. 3. Growth studies with mutant cni-1 indicate that the cytochrome chain in this mutant is more important in supporting growth than the hydroxamate-sensitive pathway. 4. Measurements of the steady-state level of reduction of cytochrome c in mutant cni-1 indicate that the rate-limiting step in the cytochrome chain is at the position occupied by cytochrome oxidase. 5. Electron spin resonance studies with cni-1 mitochondria show normal cytochrome oxidase signals in the g approximately 6 region although there is little or no optically detectable cytochrome aa3.     

Germination-defective mutant of Neurospora crassa that responds to siderophores.

A conditionally germination-defective mutant of Neurospora crassa has been found to be partially curable by ferricrocin and other siderophores. The mutant conidia rapidly lose their membrane-bound siderophores when suspended in buffer or growth media. Germination is consequently delayed unless large numbers of conidia are present (positive population effect). This indicates that the mutant has a membrane defect involving the siderophore attachment site.     

Glycine synthesis in a Neurospora mutant deficient in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), commonly implicatedin the glycine synthesis of eucaryotes, was examined in Neurosporacrassa, wild type (FGSC 853) and a formate-requiring mutant(FGSC 9). The mutant was SHMT-deficient, containing only 15%of the total activity found in the wild type. Differential anddensity gradient centrifugations showed the mutant to be deficientin soluble SHMT activity. Both strains contained particulateSHMT which sedimented with mitochondrial marker enzymes. The origins of glycine were examined by a combination of enzyme,growth and ¹⁴C feeding experiments. Growth of the mutant wasstrongly inhibited by the isocitrate lyase-directed inhibitoritaconate. This inhibition was reduced when exogenous glycinewas supplied. Itaconate (up to 30 mM) did not inhibit growthof the wild type but in both strains isocitrate lyase activitieswere reduced. The mutant contained more lyase and glyoxylateaminotransferase than the wild type. In feeding experiments,[2-¹⁴C]acetate and [l-¹⁴C]glyoxylate were more readily incorporatedinto glycine in the mutant than the wild type. Itaconate (30mM) reduced the flow of acetate carbon into glycine by up to70% in the mutant. It is concluded that deficiency in solubleSHMT necessitates glycine synthesis via an isocitrateglyoxylateglycinesequence. (Received December 24, 1979; )