Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

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.     

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.     

Ascus development in two temperature-sensitive four-spore mutants of Neurospora crassa

Two nonallelic Four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. Expression depends on temperature. The Four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees C) for Fsp-1 or to low temperatures (15-20 degrees C) for Fsp-2. Heterozygous Fsp-1 X Fsp-1+ crosses make eight-spored asci at 15-20 degrees C but produce many four-spored asci at 25 degrees C and mostly four-spored asci at 30 degrees C. Homozygous Fsp-1 X Fsp-1 crosses respond similarly to increasing temperature but make 40-50% four-spored asci even at 20 degrees C. Heterozygous Fsp-2 X Fsp-2+ crosses produce almost exclusively four-spored asci at 15 degrees C but a mixture of four- and eight-spored asci at 20, 25, and 30 degrees C. Homozygous Fsp-2 X Fsp-2 crosses make all four-spored asci at 15 and 20 degrees C and a mixture of four- and eight-spored asci at 25 and 30 degrees C. When both Fsp-1 and Fsp-2 are present in a cross, either homozygous or heterozygous, no asci contain more than four ascospores at any temperature. Limited temperature-shift experiments with Fsp-1 and Fsp-2 show that the sensitive period for Four-spore expression is sometime after meiotic prophase, possibly at interphase II.     

Unsaturated fatty acid mutants of Neurospora crassa.

Unsaturated fatty acid (ufa) auxotrophs of Neurospora crassa were obtained by treatment of conidia with N-methyl-N'-nitro-N-nitrosoguanidine followed by isolation on media containing polyunsaturated fatty acids suspended in Tergitol NP-40. The 24 mutants for which reisolates were obtained from crosses with wild type were assigned to two complementation classes, ufa-1 and ufa-2, located on linkage group V. Unsaturated fatty acids with varying degrees of unsaturation, chain length, and double-bond position as well as different steric configurations were tested for growth requirements.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.     

Deoxyglucose-resistant mutants of Neurospora crassa: isolation, mapping, and biochemical characterization.

Neurospora crassa mutants resistant to 2-deoxyglucose have been isolated, and their mutations have been mapped to four genetic loci. The mutants have the following characteristics: (i) they are resistant to sorbose as well as to 2-deoxyglucose; (ii) they are partially or completely constitutive for glucose transport system II, glucamylase, and invertase, which are usually repressed during growth on glucose; and (iii) they synthesize an invertase with abnormal thermostability and immunological properties, suggesting altered posttranslational modification. All of these characteristics could arise from defects in the regulation of carbon metabolism. In addition, mutants with mutations at three of the loci lack glucose transport system I, which is normally synthesized constitutively by wild-type N. crassa. Although the basis for this change is not yet clear, the mutants provide a way of studying the high-affinity system II uncomplicated by the presence of the low-affinity system I.     

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.     

Isolation and characterization of new fluoroacetate resistant/acetate non-utilizing mutants of Neurospora crassa.

Sixty-two mutants of the filamentous fungus Neurospora crassa were isolated on the basis of resistance to the antimetabolite fluoroacetate. Of these, 14 were unable to use acetate as sole carbon source (acetate non-utilizers, acu) and were the subject of further genetic and biochemical analysis. These mutants fell into four complementation groups, three of which did not complement any known acu mutants. Mutants of complementation group 3 failed to complement acu-8, demonstrated similar phenotypic properties and proved to be closely linked (less than 2% recombination) but not allelic. Representatives of groups 2 and 4 were mapped to independent loci; the single representative of group 1 could not be mapped. The four complementation groups were therefore designated as genes acu-10 to acu-13 respectively. All the mutants demonstrated normal acetate-induced expression of acetyl-CoA synthetase and the unique enzymes of the glyoxylate cycle and gluconeogenesis. The nature of these mutations is therefore quite different to those reported for other fungal species. Mutant acu-11 was unable to fix labelled acetate, indicating the loss of an initial transport function; partial enzyme lesions were observed for acu-12 (acetyl-CoA hydrolase) and acu-13 (acetate-inducible NAD(+)-specific malate dehydrogenase).     

Urease defective mutants in Neurospora crassa

Summary A method for isolating urease mutants was developed. It is based on the use of microconidial strains with small and compact colonies. Mutants are detected by their inability to change the color of a pH indicator when they are brought in contact with a solution of urea. The assay is performed in the absence of growth conditions so that the colonies remain separate. Two isolated urease mutants are unable to grow on urea as the sole source of nitrogen, but grow as well as the wild type on other sources of nitrogen. The same two mutants give rise to different acidities in liquid growth medium. The two mutants are also genetically different (K?lmark, 1969 b). The finding that two genetically and physiologically distinct loci participate in the control of one enzyme is discussed.     

Selection of succinic dehydrogenase mutants of Neurospora crassa.

A method is described which permits the selection of mutants of Neurospora crassa that are deficient in succinic dehydrogenase activity. The method relies on the observation that succinic dehydrogenase-deficient strains fail to reduce the dye nitrotetrazolium blue when overlaid with the dye in the presence of succinate and phenazine methosulfate. Wild-type colonies reduced the dye and turned blue, whereas mutant colonies remained colorless. In this communication we present studies of a mutant, SDH-1, isolated by this method. The mutant had 18% of the succinic dehydrogenase activity of the parent strain used in the mutation experiments as determined from the ratio of Vmax activities obtained from Lineweaver-Burk plots. The SDH-1 mutant segregated in a Mendelian manner when back-crossed to its parent strain. Succinate oxidase activity in SDH-1 was low and was markedly inhibited by adenosine 5'-diphosphate. The succinate oxidase activity of the parent strain was high and was not affected by the presence of adenosine 5'-diphosphate.     

Isolation and characterization of Neurospora crassa mutants impaired in feedback control of ornithine synthesis.

Thirty-two independent mutants were isolated which overcame the proline requirement of pro-3 mutations in Neurospora crassa. The mutations were not revertants, appeared to be allelic, were closely linked or allelic to arg-6, and in strains unable to degrade ornithine no longer suppressed the proline requirement. The suppressor mutations did not alter the levels of biosynthetic or catabolic enzymes, yet allowed accumulation of ornithine. Suppressed strains unable to degrade arginine still produced ornithine (as detected by growth) in arginine-supplemented medium. The results suggest that the suppressor mutants were impaired in the feedback inhibition of ornithine synthesis by arginine. The activity of the appropriate biosynthetic enzyme was less sensitive to inhibition by arginine. The potential usefulness of such mutations is discussed.     

Genetic and kinetic analysis of Neurospora crassa mtr mutants.

Allelic complementation occurs at the mtr (methyltryptophan resistance) locus. Kinetic properties of neutral amino acid transport are altered for mtr mutants.     

Nuclear cytochrome-deficient mutants of Neurospora crassa: isolation, characterization, and genetic mapping.

Summary We have isolated twenty-six nuclear, singlegene cytochrome-deficient mutants of Neurospora crassa as an initial step toward the study of the structural components and regulatory mechanisms involved in the biogenesis of the mitochondrial cytochrome system. These mutants, together with two previously described mutants, cyt-1 and cyt-2, have been classified into six distinct groups on the basis of cytochrome phenotype: a) cytochrome aa ₃ deficiency (due to mutations affecting loci designated cya); b) cytochrome b deficiency (cyb-1 locus); c) cytochrome b deficiency with a partial deficiency of cytochrome aa ₃ (cyb-2 locus); d) deficiency of both cytochromes aa ₃ and b (cyt loci); e) deficiency of both cytochromes aa ₃ and c (cyt-2 locus); and f) partial deficiency of cytochromes aa ₃ and c (cyt-12 locus).Four of seven mutations affecting cya loci have been mapped and are located on linkage groups I, II, V, and VI. It is not yet known whether these genes code for structural components of cytochrome oxidase or have a regulatory function that affects synthesis or assembly of the enzyme. The cyb-1 and cyb-2 genes are located on linkage groups V and VI, respectively, and appear to code for regulatory elements that control the biogenesis of cytochromes b and aa ₃ . The positions of the cyt mutations that cause a simultaneous deficiency of cytochromes aa ₃ and b are dispersed throughout the genome, except for two gene clusters on the left arm of linkage group I. Some of these mutants may be deficient in mitochondrial protein synthesis. Two mutations, cyt-2 and cyt-12, are located on linkage groups VI and II, respectively, and appear to affect genes that code for components of a regulatory system that controls the biogenesis of cytochromes aa ₃ and c.     

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.