In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins. However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression. Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain. Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa. Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

https://mmbr.asm.org/content/mmbr/61/1/17.full.pdf

Genetic regulation of nitrogen metabolism in the fungi.

SUMMARY
Measurements of the chitin content of the rooting horizons of a typical mor-humus heathland soil, indicate that chitin can contain in excess of 20% of the total nitrogen in the litter (L) horizon and 30% in the fermentation (F) horizon. Much of this chitin-nitrogen is thought to be contained in the mycelial walls of soil fungi.

Experiments were therefore designed to test the hypothesis that such sources of N could be rendered accessible to the ericaceous plants by their fungal endophytes. Mycelium of the ericoid endophyte Hymenoscyphus ericae (Read) Korf & Kernan and of the ectomycorrhizal fungus Suillus bovinus (Fr.) O. Krantze were grown in liquid culture before being killed and added either in the intact condition, or after fractionation, as sole sources of N to sterile media upon which were grown H. ericae in pure culture, or mycorrhizal and non-mycorrhizal plants of Vaccinium macrocarpon Ait. and Calluna vulgaris L. The abilities of the test organisms to utilize the nitrogen contained in the intact mycelial necromass, or in its fractions, were assessed by determining their yields and nitrogen concentrations of their tissues.

It was revealed that H. ericae was able to produce significantly higher yield when grown on intact fungal necromass than when provided with equivalent concentrations of N in the form of ammonium. Its yields on mycelial fractions were lower, but still significantly greater than those obtained in the controls lacking N. Significantly greater yields and N contents were also found in the ericaceous plants grown with these nitrogenous substrates in the mycorrhizal condition. Without H. ericae they had no access to the substrates. The possible ecological implications of these results are discussed.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.1973.tb02110.x

The biology of mycorrhiza in the Ericaceae

Comparative biochemistry of the proteinases of eucaryotic microorganisms.

Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency.

Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions, is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers. The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa molecular mass range.

Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation, production and characterization

Neurospora crassa strain 74A grown on Vogel's medium containing bovine serum albumin (BSA) as principal carbon source secretes proteolytic enzymes which appear in the culture filtrate. Low concentrations of sucrose (0.1%) are necessary for growth from conidia, as conidia will not germinate on BSA alone. Once growth is initiated, however, protease production begins and at 5 to 6 hr growth and enzyme production are parallel. Higher concentrations of sucrose (0.5-2%) repress protease synthesis. Other metabolizable materials (sugars, amino acids, peptide mixtures) also repress protease synthesis. Some sugars will not sustain growth but allow germination and full induction of protease in the presence of protein. A material found in culture fluids of cells during induction of protease synthesis when added to repressed cultures causes a five-fold increase in the amount of protease production, although this is still approximately half that of normally induced cells. This material appears to be produced by induced cells in as little as 2 hr of culture, which is before detectable levels of protease can be found. It is heat-stable, of low molecular weight, and is not a simple product of protein digestion by the N. crassa proteases.

Regulation of Exocellular Proteases in Neurospora crassa: Induction and Repression of Enzyme Synthesis

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.

H. Drucker

Papers

Regulation of Exocellular Proteases in Neurospora crassa: Induction and Repression of Enzyme Synthesis

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction