Extranuclear mutant ofSchizophyllum commune

A mutant of the hymenomyceteSchizophyllum commune was isolated which, owing to an extranuclear mutation, did not utilize acetate as the sole carbon source for growth. The growth of the mutant on glucose minimal medium was completely inhibited by sodium azide but was resistant to the effect of 2,4-dinitrophenol or oligomycin. Its endogenous respiration was cyanide-sensitive and was stimulated by 2,4-dinitrophenol to a considerably smaller degree than that of the wild-type strain. The experimental results obtained with this mutant suggest a defect in aerobic phosphorylation.     

Respiration of a puff morphological mutant of Schizophyllum commune

Summary The growth kinetics of wild-type mycelium and a puff morphological mutant of Schizophyllum commune revealed greater acid production and slower growth by this mutant. The compact mycelium growth habit of puff in defined liquid medium facilitated manometric studies of cellular respiration during culture aging. Basal oxygen consumption was highest in young, 2-day cultures as was exogenous glucose stimulation while both responses declined rapidly as the mycelial pellets aged. Respiratory stimulation by certain l-amino acids including histidine, arginine and serine was only demonstrated in aged cultures of puff mycelium. A qualitative shift in terminal respiration was considered unlikely because the metabolic poison sodium azide was a potent inhibitor of mycelial oxygen consumption regardless of either the culture age or the respective exogenous substrates employed.     

Morphogenesis of Schizophyllum commune III.Activity of inhibitors of energy metabolism

The effect of azide, 2,4-dinitrophenol, malonate and 2-deoxyglucoseon the physiology and morphology of normal and mutant strainsof Schizophyllum commune was studied. Normal, thin and puffstrains showed a decrease in dry weight and an increase in glucoseutilization in response to azide, dinitrophenol and 2-deoxyglucose.Streak was less sensitive to azide and dinitrophenol and almostcompletely resistant to 2-deoxyglucose. Numerous morphogeneticeffects were caused by the inhibitors. These included the inductionof certain aspects of the morphology of the common-A sexualreaction in normal and thin homokaryons by 2-deoxyglucose, areversion to normal morphology by thin strains in response toazide and dinitrophenol, and a morphogenetic insensitivity tothe inhibitors by the streak mutants. A discussion of the relationshipof these effects to the control of morphology in S. communeis given. ¹This investigation was supported in part by Research GrantAI-06570 from the National Institute of Allergy and InfectiousDiseases of the U. S. Public Health Service and by Grant No.GB-3613 from the National Science Foundation (U.S.A.). ²Present Address: Department of Biology, State College at Bridgewater,Bridgewater, Massachusetts 02324, U. S. A.     

Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.     

Hyphal ultrastructure in fruit-body primordia of the basidiomycetesSchizophyllum commune andCoprinus cinereus

Summary Light and electron microscopic investigations of fruit-body primordia of the basidiomycetesSchizophyllum commune andCoprinus cinereus showed that hyphal fusions are common in specific areas of the fruit-body primordia of both species. In these areas conspicuous amounts of a mucilaginous substance occur between the closely packed hyphae. A possible function of this mucilage is to aid the adhesion of hyphae prior to fusion. In the fruit-body primordium ofCoprinus cinereus the dolipore/parenthesome septum is surrounded by an additional hemispherical membranous cap (outer cap). Such a cap was never found inSchizophyllum commune fruit-body primordia.     

A potent specific inhibitor of 6-phosphogluconate dehydrogenase ofCryptococcus neoformans and of certain other fungal enzymes

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases fromCryptococcus neoformans andSchizophyllum commune and glucose-6-phosphate dehydrogenase fromSchizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than theCryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.     

Septal involvement in nuclear migration inSchizophyllum commune

Summary A system, utilizing thedwarf andpuff morphological mutants ofSchizophyllum commune, was devised to select specific hyphal segments that were in different stages of septal dissolution and nuclear migration. These stages were observed with the electron microscope. Direct evidence of the dissolution of complex septal during nuclear migration was obtained. Initially there was a disruption of the septal swelling, followed by erosion of the remaining cross wall. Complex septa were therepy converted to simple septa through which nuclei migrated. These septa were covered with secondary cell wall material. Following nuclear migration only vacuolate hyphae with remnant membrane structures remained. Occasionally, intact hyphae were observed within these vacuolate hyphae.     

Isotopic studies of carbohydrate metabolism during basidiospore germination inSchizophyllum commune

Summary Uptake and respiration of radioactive glucose, mannitol and arabitol were studied during basidiospore germination of the woodrotting mushroomSchizophyllum commune. Glucose uptake was rapid and immediate, depressed at 4° C and unaffected by the protein synthesis antagonist cycloheximide. In contrast, uptake of either mannitol or arabitol exhibited a lag phase while the induction of this process was sensitive to cycloheximide. Prior incubation of basidiospores in unlabelled mannitol induced uptake processes for either labelled mannitol or arabitol. Respiratory rates for either arabitol or mannitol increased markedly upon germination in glucose-asparagine minimal culture medium.This research was supported by Public Health Service Grant AI-04603-09 to Donald J. Niederpruem.     

Mitosis in hyphae ofSchizophyllum commune Fr.

Summary In hyphae of the basidiomyceteSchizophyllum commune Fr. mitosis occurs through a longitudinal division of a strand of chromatin, which is followed by a parallel separation of the daughter strands.     

Enzymic dissolution of hyphal septa in a Basidiomycete

Treatment of hyphal-wall preparations ofSchizophyllum commune with chitinase and R-glucanase results in the dissolution of septa but the walls retain their structural integrity.     

Cyanide-insensitive respiration of Candida lipolytica

Whole cells of the yeast Candida lipolytica exhibited a high, cyanide-sensitive endogenous respiration which became completely cyanide-insensitive under certain physiological circumstances namely (1) in the stationary phase of growth and (2) upon aeration in the resting state. This cannot be due to a change in permeability of the cell wall as the respiration of protoplasts showed the same (in)sensitivity to cyanide as the cells from which they were obtained.The cyanide-insensitive respiration of C. lipolytica was located in the mitochondria and coexisted with the normal respiratory chain, as the mitochondria isolated from cyanide-insensitive cells exhibited at the same time a cyanidesensitive respiration of ascorbate and N,N,N,N-tetramethyl-p-phenylenediamine and a cyanide-insensitive respiration of succinate.The alternate respiratory pathway was sensitive to benzyl- and salicylhydroxamic acids. In this respect it resembles the alternate mitochondrial pathway described in the literature for various plants.The cyanide-insensitive respiration did not appear in the resting state when the cells were aerated in the presence of cycloheximide nor at 0 C instead of at room temperature. These facts suggest some form of induction involving new protein synthesis. The induction process depends on the presence of molecular oxygen as the cyanide-insensitive endogenous respiration did not appear during agitation of yeast cells in the resting state if the gaseous atmosphere lacked oxygen.     

Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune

Isolation and identification of substances having an activity to stimulate the fruiting body formation of Schizophyllum commune were attempted. The active principles in its mycelia were divided into four fractions by sequential purification with silica gel column and reverse-phase HPLC column chromatography. By infrared spectra and thin-layer chromatography, the active substances in these four fractions were revealed as cerebrosides. About 0.1 μg of the cerebroside fractions showed a discriminative stimulating activity on S. commune when tested by the method these authors adopted. The active substance in the fraction II was N-2′-hydroxypalmitoyl-1-O-glucosyl-nonadecasphingadienine. The cerebrosides from pea seeds and Fusicoccum amygdali showed the similar activity on S. commune, but some commercial synthetic cerebrosides and cerebrosides from bovine and porcine brains exhibited no stimulating activity. Only definite cerebrosides with special structures seem to be able to induce the fruiting of S. commune.     

m-Chlorobenzhydroxamic Acid–an Inhibitor of Cyanide-Insensitive Respiration in Trypanosoma brucei*

Cyanide-insensitive respiration of bloodstream and culture forms of T. brucei was inhibited by m-chlorobenz-hydroxamic acid (m-CLAM). The cyanide-sensitive respiration of culture forms of this organism was not affected by m-CLAM. This compound is the 1st really effective inhibitor to be described that acts specifically on the cyanide-insensitive respiration of the T. brucei group of trypanosomes; as such it may be of some importance as a trypanocidal drug. Evidence is adduced which suggests that a branched electron transport chain is present in culture forms of T. brucei and that the cyanide-insensitive terminal oxidase found in these stages is the same as that found in bloodstream forms.     

The respiratory system of the aquatic phycomyceteAllomyces macrogynus wild-type and sexual mutants

A series of predominantly male mutants induced with ethidium bromide were isolated fromAllomyces macrogynus (Emerson) Emerson and Wilson strain Burma 3–35 (35°C) and crossed to the wild-type female. Gametangia and sporangia from ethidium bromide-induced mutants, predominantly male and female Mendelian mutants, and double mutants constructed from these Mendelian sexual mutants, were analyzed for their response to cyanide, propyl gallate, and CCCP. Reduced minus oxidized room temperature cytochrome difference spectra were made on homogenized mycelium from wild-type and sexual mutants. The ethidium bromide-induced male mutants showed a non-Mendelian segregation pattern when crossed to wild type. Mendelian and non-Mendelian mutants have lower respiratory rates than the wild type. Neither the mutants nor the wild type were sensitive to propyl gallate. Mutants and wild type have the same response to CCCP. The concentrations of the cytochromes from the mutants were generally the same as in the wild type, but the ratio ofa-type cytochromes toc- andb-type cytochromes was lowered in many mutants. Both higher and lower relative amounts of cyanide insensitive respiration (compared to total respiration) were observed among the sexual mutants when the sexual mutants were compared to wild type. The relative amount of cyanide-insensitive respiration of the double mutants can be related to the relative amount of cyanide-insensitive respiration of the single mutants from which the double mutants were constructed. It is concluded that the respiratory deficiency of the sexual mutants disturbs the respiratory system in a complex manner.     

Meiosis in Schizophyllum commune: the effect of hydroxyurea on basidiospore sporulation, germination, and nuclear number

Summary In a search for an experimental procedure to synchronize meiosis in fruit bodies of Schizophyllum commune, the effect of hydroxyurea on sporulation was tested. Results indicate that hydroxyurea has an immediate and reversible effect on basidiospore sporulation, germination, and nuclear number. A tentative time schedule for meiosis is presented.     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.     

Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus: Inhibition by Cyanide or Antimycin and Salicylhydroxamic Acid or Propyl Gallate

Gametangia of the aquatic phycomycete Allomyces macrogynus have a cyanide- and antimycin A-insensitive respiration, which is sensitive to salicylhydroxamic acid (alternative respiration). Propyl gallate is also an inhibitor of this alternative pathway, and propyl gallate is more efficient than hydroxamic acid. Gametangial respiration is insensitive to propyl gallate, but propyl gallate sensitivity is gradually established when the gametangia are titrated with cyanide. Carbonyl cyanide m-chlorophenyl hydrazone stimulates the cyanide-sensitive respiration and engages the alternative sensitive respiration. Sodium azide inhibits both the alternative and the cyanide-sensitive respiration, but the cyanide-sensitive respiration is inhibited 10 times more efficiently than the alternative respiration. Rotenone inhibits the total respiration and the propyl gallate-insensitive respiration by 33% and the cyanide-insensitive respiration by 43%.

The kinetic results reported here are discussed with respect to the models of de Troostembergh and Nyns (1977 Arch Int Physiol Biochem 85:404-406; 1978 Eur J Biochem 53:423-432) and of Bahr and Bonner (1973 J Biol Chem 248:3446-3450) for the partitioning of electrons between cyanide-insensitive and propyl gallate-insensitive respiration. The results reported here do not agree with the model of de Troostembergh and Nyns.

     

Chlamydospores of Schizophyllum commune

Summary The detection of chlamydospores of Schizophyllum commune in liquid medium is described. The short thick walled cells are formed by intercalary septation which leads also to modification of the septal complex. The chemical composition of the cell walls of chlamydospores is similar to the composition of the vegetative mycelium.     

Selectively recombining B incompatibility factors of Schizophyllum commune

Summary Analysis of genetic crosses among strains of Schizophyllum commune carrying recombining B factors has revealed that not all heteroallelic pairs of B factors are able to recombine with each other. This suppression of recombination is highly specific and appears to be determined by the B factors themselves.     

Selection of biochemical mutants of Aspergillus niger with enhanced extracellular ribonuclease production

Combined with u.v. irradiation and the nitrosoguanidine method, selection of biochemical mutants resistant to metabolic inhibitors (2-deoxy-D-glucose, antimycin A, sodium orthovanadate and sodium azide) was a very efficient method for improvement of ribonuclease production by Aspergillus niger. Resistance to sodium azide produced highest RNase production, greatest frequency of positive mutation and shortest sporulation time. The most active strain, Aspergillus niger SA-13-20 resistant to sodium azide, was obtained, which had a 433% increase in RNase production in comparison with the parent strain and had good subculturing stability. Its dynamic characters were similar to those of the parent strain.