Uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of Neurospora crassa.

Seven mutant strains defective for utilization of glycerol, glyceraldehyde or dihydroxyacetone were isolated. One strain was deficient for NAD-linked glycerol-3-phosphate dehydrogenase, two for glycerol kinase, and four had no detected enzymatic deficiency, although one of the latter strains was deficient in glycerol uptake. Glycerol uptake was increased by incubation in glycerol, glycerol-3-phosphate, erythritol, and propanediol, and was protein-mediated below 0.14 mM glycerol, but at higher concentrations free diffusion predominated. Glycerol uptake was decreased by cycloheximide and was more sensitive to sodium azide than to iodoacetate.

Extraction and quantification of solutes in solidified agar culture media.

A method is described for determining the concentration of certain solutes in solidified culture media. The method is based upon the finding that under specified conditions the concentration of solute in an agar gel (C(g)) is related to the concentration of solute in a centrifugally extracted gel supernatant (C(s)) by the ratio, C(g)/C(s), which is characteristic for each solute. The method avoids direct assays of the gels and instead involves assaying the supernatants from inoculated and uninoculated (control) gels with conventional liquid assay techniques and then calculating solute concentrations in the inoculated gels by use of the C(g)/C(s) ratios determined from the controls. Uninoculated agar gels containing known concentrations of various solutes and similar gels inoculated with Neurospora crassa or Escherichia coli were centrifuged at various times, and the supernatants were assayed for solute concentrations. The solute concentrations in the supernatants from the inoculated gels multiplied by the C(g)/C(s) ratios for those solutes determined at the same times for the uninoculated controls gave calculated solute concentrations in the inoculated gels. The differences between these calculated solute concentrations and those initially present in the inoculated gels indicated the amounts of solutes utilized from the gels by the microorganisms at various incubation times.

Reversion and interallelic complementation at four urease loci in Neurospora crassa.

Analysis of heat stability of urease in extracts of 24 revertants, six for each of four ure loci, revealed that at least one revertant for each locus had a heat stability about one-third that of wild type. Similar results were obtained with urease formed by interallelic complementation at the ure-2 and ure-4 loci, but interallelic complementation at the ure-1 and ure-3 loci produced insufficient urease activity for analysis. The data are interpreted to suggest, as a tentative model, a structural function for each of the four ure loci.

Effect of carbon source on enzymes involved in glycerol metabolism in Neurospora crassa.

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains of Neurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater efficiency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.

Characterization of glycerol nonutilizing and protoperithecial mutants of Neurospora.

Mutants defective in polyol metabolism and/or in protoperithecial development were selected in Neurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed into N. crassa to facilitate genetic analysis. One, glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, between ad-1 and rib-1; the other, glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal to ad-9. Another mutant, gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal to ad-9.

Phenotypic diversity among alleles at the per-1 locus of Neurospora crassa.

Comparison of 11 perithecial color mutants suggested that all were alleles at the per-1 locus but nonetheless separable into two groups because of phenotypic differences. Three of the mutant strains produced orange perithecia and black ascospores, and eight produced paler, yellow perithecia and white ascospores. Perithecial phenotype was dependent upon the genotype of the protoperithecial parent; ascospore phenotype, upon the genotype of the individual ascospore. No evidence was found that the white ascospores were due to chromosomal rearrangements. No separation of the perithecial and ascospore phenotypes by recombination was observed in a cross between one of the mutants and a per-1+ strain. However, apparent low levels of recombination in crosses between some of the mutants indicated possible genetic complexity at the per-1 locus. The phase specificity of the per-1 mutations and the possible nature and mode of expression of the orange and yellow perithecial pigments are discussed.

Scanning electron microscopy of surface and internal features of developing perithecia of Neurospora crassa.

Stages in the development of perithecia of Neurospora crassa, designated by the time elapsed after crossing, were investigated with the scanning electron microscope, from protoperithecia through perithecia. The usual examination of external features of whole specimens with this instrument was augmented by a freeze-fracture technique which allowed the viewing of development internally as well. Rapid increases in perithecial size soon after crossing were followed by the appearance, in section, of a centrum, at first undifferentiated but subsequently developing ascogenous hyphae. The perithecial beak appeared as a compact mass easily distinguishable in whole specimens from the surrounding hyphae by means of texture as well as shape. Two ascospores were photographed during emergence from an ostiole, but ostioles were found more frequently closed than open.

Nutrient-dependent inhibition of perithecial development due to sequential crosses on the same mycelium of Neurospora tetrasperma.

An explanation of perithecial inhibition in the second of two sequential crosses at different locations on the same mycelium of Neurospora tetrasperma was sought by (1) assaying media that had supported inhibited and uninhibited portions of the mycelium which contained no developing perithecia, (2) determining the effect of these media on perithecial development, (3) adding nutrients to inhibited portions of the mycelium, and (4) assaying carbon sources in media that had supported portions of the mycelium which contained developing perithecia, and portions, both inhibited and uninhibited, which contained no developing perithecia. Different kinds and volumes of media and various intervals of time between sequential crosses were used to aid in determining limits of perithecial inhibition. Perithecial inhibition was observed to be independent of volatile metabolites and pH, independent of non-volatile metabolites, reversible by addition of nutrients, dependent upon nutrient volume, and correlated with the concentration of the carbon source in the medium. It is proposed that second crosses are inhibited because of a previous lowering of the concentration of nutrients in the medium in second-cross locations, owing to prior demand upon those nutrients by the developing perithecia in first-cross locations. The possibility of an activation signal between first- and second-cross locations is discussed. No inhibitory substance in inhibited locations was detected.