Nucleases in the autolysis of filamentous fungi

RNase and DNase activities were studied in seven fungi of the subdivisions Ascomycotina, Zygomycotina and Basidiomycotina during their autolysis, and extracellular and intracellular RNase and DNase were found. RNase specific activity reached higher levels than DNase specific activity in the culture liquid and mycelial extract, except in Aspergillus nidulans. Generally maximal RNase specific activities were observed at the onset of autolysis in the culture liquid. In the mycelial extract an increase in this activity with the incubation time was observed, except in A. nidulans and Coriolus versicolor. The highest values of DNase specific activities were found at the third day of autolysis in A. nidulans culture liquid and at the thirtieth day of autolysis in Schizophyllum commune mycelial extract. A possible relationship between the culture liquid pH during the autolysis of the studied fungi and the levels of DNase specific activity was observed.     

The autolysis of industrial filamentous fungi

Fungal autolysis is the natural process of self-digestion of aged hyphal cultures, occurring as a result of hydrolase activity, causing vacuolation and disruption of organelle and cell wall structure. Previously, authors have considered individual aspects of fungal lysis, in terms of either an enzyme, a process or an organism. This review considers both the physiology and morphology of fungal autolysis, with an emphasis on correlations between enzymological profiles and the morphological changes occurring during culture degeneration. The involvement of the main groups of autolytic hydrolases is examined (i.e., proteases, glucanases, and chitinases), in addition to the effects of autolysis on the morphology and products of industrial bioprocesses. We call for a concerted approach to the study of autolysis, as this will be fundamental for research to progress in this field. Increased understanding will allow for greater control of the prevention, or induction of fungal autolysis. Such advances will be applicable in the development of antifungal medicines and enable increased productivity and yields in industrial bioprocesses. Using paradigms in existing model systems, including mammalian cell death and aging in yeast, areas for future study are suggested in order to advance the study of fungal cell death.     

Lytic enzymes in the autolysis of filamentous fungi

The degrees of autolysis attained by five different genera of filamentous fungi during an incubation period of 60 days, under the same culture conditions were: 87.3% for Penicillium oxalicum; 65.9% for Neurospora crassa; 62.7% for Polystictus versicolor; 51.7% for Aspergillus niger and 23.5% for Nectria galligena. N. crassa, A. niger and P. versicolor reached the end of the autolysis during this incubation period (60 days), whereas P. oxalicum and N. galligena did not.The excretion of the lytic enzymes -N-acetyl-glucosaminidase, -1–3 glucanase, chitinase, invertase and acid phosphatase into the culture medium during growth and autolysis was investigated. The excretion of these enzymes was consistent with the degree of autolysis reached, the maximum excretion belonging to P. oxalicum and the minimum to N. galligena. The N. crassa invertase was excreted into the culture liquid at levels very much higher than the other enzymes studied, and at levels very much higher than the invertases excreted by the other fungi.     

Comparative study of acid and alkaline phosphatase during the autolysis of filamentous fungi

Acid and alkaline phosphatase activities in culture liquid and mycelial extract during autolysis were studied in seven fungi of the general Ascomycotina, Basidiomycotina and Zygomycotina. High activities of extracellular and mycelial extract acid phosphatase and lower activities of alkaline phosphatase were found in Ascomycotina, and acid phosphatase was present in Basidiomycotina. In Zygomycotina only mycelial extract alkaline phosphatase activity was detected. A correlation between degree of autolysis, pH and acid phosphatase activity was demonstrated.     

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.     

Proteomics of filamentous fungi

Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.     

Nuclear movement in filamentous fungi

One of the most striking features of eukaryotic cells is the organization of specific functions into organelles such as nuclei, mitochondria, chloroplasts, the endoplasmic reticulum, vacuoles, peroxisomes or the Golgi apparatus. These membrane-surrounded compartments are not synthesized de novo but are bequeathed to daughter cells during cell division. The successful transmittance of organelles to daughter cells requires the growth, division and separation of these compartments and involves a complex machinery consisting of cytoskeletal components, mechanochemical motor proteins and regulatory factors. Organelles such as nuclei, which are present in most cells in a single copy, must be precisely positioned prior to cytokinesis. In many eukaryotic cells the cleavage plane for cell division is defined by the location of the nucleus prior to mitosis. Nuclear positioning is thus absolutely crucial in the unequal cell divisions that occur during development and embryogenesis. Yeast and filamentous fungi are excellent organisms for the molecular analysis of nuclear migration because of their amenability to a broad variety of powerful analytical methods unavailable in higher eukaryotes. Filamentous fungi are especially attractive models because the longitudinally elongated cells grow by apical tip extension and the organelles are often required to migrate long distances. This review describes nuclear migration in filamentous fungi, the approaches used for and the results of its molecular analysis and the projection of the results to other organisms.     

Meiotic recombination in filamentous fungi

The exchange of genes by crossing over and by gene conversion is a basic process in eukaryotes. Fungi have played a special role in the study of this process because they permit tetrad analysis, which provides complete information on the distribution of genes and chromosomes in meiosis. Recombination is detected by new combinations of genetic markers. The first observation gave only the simple picture of a crossover provided by two segregating loci far apart on the chromosome. Later the discovery of recombination between sites within a gene led to a revolution in our knowledge of this process. Today we carry the resolution a step further with RFLP markers, which can detect the details of recombination down to nucleotide distances. I review here observations on filamentous fungi, which have contributed to this pursuit at each stage of the emerging synthesis.     

Mitochondrial dynamics in filamentous fungi

Mitochondria are essential organelles of eukaryotic cells. They grow continuously throughout the cell cycle and are inherited by daughter cells upon cell division. Inheritance of mitochondria and maintenance of mitochondrial distribution and morphology require active transport of the organelles along the cytoskeleton and depend on membrane fission and fusion events. Many of the molecular components and cellular mechanisms mediating these complex processes have been conserved during evolution across the borders of the fungal and animal kingdoms. During the past few decades, several constituents of the cellular machinery mediating mitochondrial behavior have been identified and functionally characterized. Here, we review the contributions of fungi, with special emphasis on the filamentous fungus Neurospora crassa, to our current understanding of mitochondrial morphogenesis and inheritance.     

Biodiversity amongst filamentous fungi

Diversities in fungi are manifold. Fungi themselves are heterogeneous and constitute at least three unrelated major taxa. Structural diversity reflects, in most cases, adaptive and functional strategies. Diversity in nucleic acids and chemical compounds is very high in several fungal taxa. Fungi play an essential role in the function of ecosystems. The diversity of plant parasites is extremely high and species-dependent associations exist. Saprobic fungi are most important in wood and litter decay and diverse taxa comprise the main decomposers in specific successional niches. Two dominating symbiotic systems have evolved convergently in various fungal groups, notably lichens and mycorrhizas, both remarkably diverse in their heterotrophic partners.     

Decoding the mannitol enigma in filamentous fungi

Mannitol is a 6-carbon polyol that is among the most abundant biochemical compounds in the biosphere. Mannitol has been ascribed a multitude of roles in filamentous fungi including carbohydrate storage, reservoir of reducing power, stress tolerance and spore dislodgement and/or dispersal. The advancement of genetic manipulation techniques in filamentous fungi has rapidly accelerated our understanding of the roles and metabolism of mannitol. The targeted deletion of genes encoding proteins of mannitol metabolism in several fungi, including phytopathogens, has proven that the metabolism of mannitol does not exist as a cycle and that many of the postulated roles are unsupported. These recent studies have provided a much needed focus on this mysterious metabolite and make this a fitting time to review the roles and metabolism of mannitol in filamentous fungi.     

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.