Glutamine metabolism during aerial mycelium growth of Neurospora crassa

During vegetative growth, glutamine is accumulated in the mycelium of Neurospora crassa. This high pool of glutamine seems to be required for aerial mycelium growth. Enzymes responsible for the synthesis and catabolism of glutamine were measured before and during the partial transformation of a mycelial mat into aerial mycelium. In the transforming mycelial mat,considerable activities of the biosynthetic NADP-glutamate dehydrogenase and glutamine synthetase (predominantly β polypeptide) and also some activity of glutamate synthase were observed. In the aerial mycelium, glutamine synthetase (predominantly β polypeptide) was detected, but very low activities of NADP-glutamate dehydrogenase and glutamate mycelium could derive from glutamine. No glutaminase activity could be detected. It is suggested that glutamate is formed through the activities of the glutamine transaminase-ω -amidase pathway and another transaminase. High activities of glutamine and alanine transaminases were observed in the aerial mycelium. These results are discussed in terms of the possible role of glutamine as a nitrogen carrier from the mycelium to the growing aerial hyphae.     

Adenine uptake in Neurospora crassa mycelium]

The uptake of 8-C14-adenine in N. crassa strain Lindegren (+) was studied. The ability of N. crassa cells to uptake adenine from the medium reaches maximum at the very beginning of the logarithmic stage of growth. Adenine enters the mycelium against the concentration gradient. The uptake of adenine is maximal at 25-30 degrees C, pH 4,6-4,8, and adenine concentration in the medium about 2-15X10(-6) M. The entry of adenine into the cells follows normal Michaelis-Menten kinetics, the apparent Km=0.83+/-0.02 micron. The uptake is inhibited at higher concentrations (10(-3)-10(-4) M) of adenine. 2,6-Diaminopurine, hypoxanthine, guanine, 8-azaadenine and 8-azaguanine inhibit the transport of adenine into the cell. Xanthine and cytosine do not affect the uptake of adenine. Adenine taken up into the cell is rapidly metabolized to AMP, ADP and ATP.     

Glutamine Metabolism and Cycling in Neurospora crassa

[This corrects the article on p. toc in vol. 54.].     

Glutamine metabolism and cycling in Neurospora crassa.

Evidence for the existence of a glutamine cycle in Neurospora crassa is reviewed. Through this cycle glutamine is converted into glutamate by glutamate synthase and catabolized by the glutamine transaminase-omega-amidase pathway, the products of which (2-oxoglutarate and ammonium) are the substrates for glutamate dehydrogenase-NADPH, which synthesizes glutamate. In the final step ammonium is assimilated into glutamine by the action of a glutamine synthetase (GS), which is formed by two distinct polypeptides, one catalytically very active (GS beta), and the other (GS alpha) less active but endowed with the capacity to modulate the activity of GS alpha. Glutamate synthase uses the amide nitrogen of glutamine to synthesize glutamate; glutamate dehydrogenase uses ammonium, and both are required to maintain the level of glutamate. The energy expended in the synthesis of glutamine drives the cycle. The glutamine cycle is not futile, because it is necessary to drive an effective carbon flow to support growth; in addition, it facilitates the allocation of nitrogen or carbon according to cellular demands. The glutamine cycle which dissipates energy links catabolism and anabolism and, in doing so, buffers variations in the nutrient supply and drives energy generation and carbon flow for optimal cell function.     

Glutamine metabolism in nitrogen-starved conidia of Neurospora crassa.

During nitrogen deprivation, de novo synthesis of glutamine synthetase was induced in non-growing conidia of Neurospora crassa. When ammonia or glutamine was added to conidia which had been deprived of nitrogen, glutamine and arginine accumulated at a higher rate than in condia not deprived of nitrogen. The degradation of exogenous glutamine to glutamate is apparently a necessary step in the accumulation of glutamine and arginine within the conidia. In non-growing conidia, a cycle probably operates in which glutamine is degraded and resynthesized. The advantages of such a cycle would be that the carbon and nitrogen could be used to synthesize amino acids in general, as well as for the synthesis and accumulation of arginine and/or glutamine in particular.     

Glutamine Metabolism in Neurospora crassa under Conditions of Copper Toxicity

Rao, S. and Venkateswerlu, G. 1986. Glutamine metabolism inNeurospora crassa under conditions of copper toxicity.—J.exp. Bot. 37: 947–955. The enzyme, glutamine synthetase, of Neurospora crassa was inhibitedby copper in a non-competitive manner. Nitrate reductase activityincreased with an increase in copper concentration in the culturemedium probably as a consequence of decreased glutamine synthetaseactivity. The hexosamine content was low, whereas DNA and RNAcontents were high in cultures of N. crassa inhibited by copper.A slight accumulation of arginine and 20% less arginase activitywere observed in such cultures. Iron counteracted the toxicityof copper. Key words: Glutamine metabolism, copper toxicity, Neurospora crassa, glutamine synthetase     

Presence of an actin-like protein in mycelium of Neurospora crassa.

Addition of ATP, CaCl2, and KCl to supernatants prepared from mycelia of Snowflake (strain 507), a morphological mutant of Neurospora crassa, results in the formation of filaments 70 nm in diameter. The "decorated" appearance of these filaments after incubation with heavy meromyosin from rabbits suggests they are actin-like.     

Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.     

Dual roles for calcium ions in apical growth of Neurospora crassa

We report initial attempts to define the role of Ca2+ in the polarized extension of Neurospora crassa. Growth of the organism was diminished in media containing less than 1 mM-Ca2+; extension was more severely impaired than biomass synthesis, resulting in the formation of stubby, bulbous hyphae, even of spherical cells. Reduced extension and abnormal morphology were correlated with the loss of surface-bound Ca2+, probably associated with the cell wall. Intracellular Ca2+ may be represented by material that fluoresces brightly in the presence of chlortetracycline. Punctate fluorescent bodies and diffuse fluorescence were both arrayed in a longitudinal gradient, maximum apically. Addition of the calcium ionophore A23187 induced dissipation of the fluorescence; concurrently, the hyphae lost as much as one half of their Ca2+ content. Extension continued almost unabated, but multiple branches quickly emerged from the apex. The observations suggest that a cytoplasmic Ca2+ gradient is not required for polarized extension, but may play a role in ensuring the dominance of the apex.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.     

Hyphal wall growth in Neurospora crassa and Geotrichum candidum.

Growth of the walls of hyphae of Neurospora crassa and Geotrichum candidum was studied using longitudinal and serial transverse sectioning methods. Rigidification of the hyphal wall below the extension zone did not appear to involve the gross formation of a secondary wall since the transition from extensible to non-extensible wall was not associated with an increase in thickness. However, behind the extension zone the walls leading hyphae of N. crassa increased in thickness until eventually they attained a thickness which was up to five times that of the tip wall. A hypothesis of hyphal wall growth is proposed.     

Protease secretion in Neurospora crassa

Secreted and constitutive intracellular proteases of $\text{Neurospora crassa}$ differ with regard to inhibitor sensitivity, substrate specificity, isoelectric points and other properties. Upon the induction of protease secretion the enzymes released from the mycelium are formed $\text{de novo}$ as demonstrated by density labelling with D₂O. Vesicles which contain the constitutive intracellular proteases are, therefore, not involved in the secretion of proteases.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.