Nitrogen Limited Growth of Wood-decaying fungi

Schizophyllum commune as a model system. Schizophyllum commune is a widely distributed wood-decaying basidiomycete. Over 50 years of cytological, physiological, biochemical, and genetic studies provide a strong basis for research using this organism. It has proven to be a tractable model system to understand mechanisms of mating, fruit body formation, signal transduction , biodegradation, and nutrient recycling and translocation. In the past decade, the organism has also been demonstrated to be an opportunistic human pathogen of some importance.

Basidiomycete fungi that inhabit wood produce fruit-body structures in which meiotic cell divisions produce haploid spores that are dispersed into the environment. Each haploid spore germinates and produces a haploid mycelium in which all of the nuclei are identical. The mating of haploid strains is dependent upon mating type loci. In S. commune these loci are known as MATA and MATB. A compatible mating occurs between homokaryons with different MATA and MATB alleles. The roles of the mating type loci have been substantially elucidated. Briefly, the MATA locus encodes two genes, Y and Z. In a compatible mating, the product of the Y gene of one nucleus interacts with the product of the Z gene of the other nucleus. This interaction (the products have homeodomains) results in the A-on phenotype necessary for growth of the dikaryon. The MATB locus encodes pheromones and a pheromone receptor that can interact in a heterospecific manner with those encoded by an alternative MATB allele . The products of the MATB locus can complement mutations in the pheromone and receptor genes in Saccharomyces cerevisiae. Two sub-loci exist for both MATA and MATB. A mating can occur between two homokaryons that differ in one sub-locus of each mating type locus. Given the large number of alleles for the MATA and MATB loci, literally thousands of mating types can exist in nature. This suggests that in nature a homokaryon would not grow very large before encountering a compatible mate. Following mating, the nuclei from each homokaryon undergo numerous divisions and invade the other homokaryon via a process involving septal dissolution and nuclear migration . The haploid nuclei stay independent, but closely associated in each cell, establishing a dikaryotic mycelium. Production of new cells in this mycelium results from apical cell divisions in which the nuclei divide more or less simultaneously, and are sorted between the new apical and sub-apical cells using structures called clamp connections. Clamp connections, then, are diagnostic of a growing dikaryotic mycelium. The vegetative dikaryotic mycelium can persist indefinitely in culture. Appropriate environmental conditions, including light, can lead to the production of fruit-body primordia. During the fruiting process, changes in several gene activities occur, the best studied of which are the hydrophobin genes. A fertile layer of cells, the hymenium, is produced in fruit-bodies, and in specific cells of the hymenial layer known as basidia, karyogamy occurs producing a diploid nucleus. Meiosis occurs in the basidia and this ultimately leads to formation of haploid spores.

The work on understanding the role of mating type genes and the genes that are controlled by them, including hydrophobins, has been done in homogeneous nitrogen-rich medium. The genetic/biochemical mechanisms described above undoubtedly will be the same regardless of environment. However, they do not shed light on the post-mating nutritional physiology of the organism in the natural environment. Indeed, S. commune dikaryons will not produce fruiting bodies on homogeneous medium containing nitrogen concentrations typical of wood.

Nitrogen nutrition in wood-inhabiting fungi. The heterotrophic nature of fungal growth requires the uptake of nutrients from the environment. In the case of wood-decayers such as S. commune, the bulk of nutrients present in the growth substrate are sequestered in macromolecules consisting primarily of celluloses, hemicelluloses and lignin. The remaining molecules are principally hydrophobics and a very small proportion of small soluble molecules and proteins. Effective exploitation of this nutrient base requires the secretion of a diverse complement of hydrolytic enzymes. The generally accepted model for nitrogen assimilation from wood begins with secretion of a set of highly active endopeptidases of general substrate specificity. A permease system transports amino acids into the mycelium upon their hydrolytic release, where they may be used directly for building proteins or metabolized as a nitrogen source for other biosynthetic processes. Wood does not provide an amenable substratum for testing much of this model. Therefore, most work has been done in pure culture systems, and components of the model have been tested indirectly. Several proteolytic activities can be found in the growth medium supporting actively growing fungi. These activities are generally serine or metalloproteases. The nitrogen status of the nutrient base affects the amounts (and perhaps type) of protease activity in the growth medium.

A central characteristic of fungal inhabitation of wood is that wood is generally a very poor source of nutrients other than carbon. Nitrogen constitutes only 0.01 to 0.3% of the dry weight of wood, with a concentration estimated at 0.0075 M. This does not take into account that a portion of the nitrogen is sequestered in lignin and other aromatics and is not available to fungi lacking the ability to metabolize these molecules. Furthermore, few wood-decayers can utilize oxidized forms of nitrogen such as nitrate (n.b. S. commune does not utilize nitrate). Indeed, most data suggest that nitrogen is the limiting nutrient in fungal growth in wood. The inability of homokaryons of some wood-decaying fungi such as S. commune to produce asexual spores places a demand on endogenous sources to sustain mycelial growth in the absence of high levels of exogenous substrate. Thus, sustained apical growth results from the recycling of nutrients from established parts of the mycelium.

An often-overlooked aspect of fungal growth in wood substrates is that the nitrogen in wood is not uniformly distributed. Greater concentrations of nitrogen are found in parenchyma rays, pockets of other invading organisms, and cambial regions. Exploitation of these regions can lead to localized increases in growth of the mycelium with translocation of nitrogen to hyphal apices. In addition, the formation of dikaryotic hyphae from compatible homokaryons using disparate nitrogen pools may result in redistribution of nitrogen between the mating mycelia. With the exception of the important experiments of Boddy and Watkinson on nutrient exploitation and translocation in strand-forming fungi, nearly all physiological studies of fungal nitrogen metabolism have been done in homogeneous media. As a result virtually nothing is known about the biochemistry of fungal growth on heterogeneous sources.

S. commune proteases. For several years we have worked to characterize the cellular proteolytic system of S. commune, making it the best characterized such system among the basidiomycetes. Several aminopeptidases and carboxypeptidases, as well as at least 10 gelatinase activities, are produced by S. commune mycelia. Both long-term and short-term nitrogen starvation of S. commune colonies result in an increase in proteolytic activity against non-native substrates. Furthermore, an increase in the number of proteases detectable by electrophoresis in gelatin-containing gels occurs during nitrogen starvation. Further study revealed that major proteolytic activities in mycelial extracts were serine proteases and metalloproteases, and that much of this activity appeared to result from the two gelatinases ScPrA (serine) and ScPrB (metallo). While the increase in activity seen in these in vitro assays suggests that in vivo proteolysis is occurring in response to nitrogen starvation, this has not been measured directly.

Physiological and biochemical data suggest that ScPrB is a principal enzyme of autolysis. In 24 hours at room temperature, ScPrB (in the presence of PMSF to block ScPrA activity) can digest ca. 90 % of the total protein in a S. commune crude extract. The enzyme is a 58 kDa Zn²⁺-dependent metalloprotease. Using quantitative gelatin-containing SDS-PAGE, we demonstrated the role of ScPrB in nitrogen-limitation induced autolysis and localized the ScPrB-mediated autolytic process to the region of mycelia directly behind the expanding margin. The activity of a serine protease, ScPrA, is also greatly increased during nitrogen deprivation. It has proven especially difficult to study, appearing to be much more labile than ScPrB. We also have purified and characterized a small serine protease, ScPrI, which also shows increased activity during nitrogen deprivation. Another protease with increased activity in S. commune under nitrogen deprivation is APF, a novel, Zn²⁺ -dependent, phenylalanine-specific aminopeptidase. It is unclear whether these proteases are transcriptionally regulated; however, we have yet to isolate a protease gene transcript from cDNA from nitrogen-limited colonies. Furthermore, there are no other reports in the basidiomycete literature of proteases that are transcriptionally up-regulated in response to low nitrogen.

Amino acid recycling during nitrogen-limited growth. When exponentially-growing S. commune colonies are transferred to media low in nitrogen, radial expansion continues at nearly the same rate as in colonies transferred to high nitrogen media. We have shown that at least two sources of recyclable nitrogen exist in this organism. One of these is the pool of free amino acids stored in older portions of the colony. This pool is mobilized and transferred by an intramycelial transport mechanism to colony margins. While translocation of the free amino acid pool may provide short-term support for continued mycelial expansion, the size of the pool is limited. The other source is protein-bound amino acids present in older portions of the colony. Increased proteolytic activity, brought on by nitrogen deprivation, results in increased levels of free amino acids in colonies and the subsequent transport of these amino acids to colony margins. Autolysis and recycling of mycelial components have the potential to support expansion for considerably longer.

Reassimilation of translocated nitrogen. We have demonstrated that amino acids released by proteolysis and translocated to the mycelial margin are incorporated into newly synthesized proteins in apical cells. In addition, we have recently found that free ammonium concentration increases in mycelia that are nitrogen starved. If ammonium is translocated to the apices, then one would expect nitrogen assimilatory enzymes such as glutamine synthetase (GS) and glutamate synthase (GOGAT) to be involved. It has been demonstrated that ammonium assimilation in the closely related basidiomycete Agaricus bisporus occurs almost exclusively via the GS/GOGAT pathway. GS has been shown to be transcriptionally regulated in response to nitrogen source in A. bisporus, though it is also regulated post-transcriptionally.