Abundant Small Protein ICARUS Inside the Cell Wall of Stress-Resistant Ascospores of Talaromyces macrosporus Suggests a Novel Mechanism of Constitutive Dormancy.

Ascospores of Talaromyces.macrosporus belong to the most stress resistant eukaryotic cells and show a constitutive dormancy, i.e., no germination occurs in the presence of rich growth medium. Only an extreme trigger as very high temperature or pressure is able to evoke synchronized germination. In this study, several changes within the thick cell wall of these cells are observed after a heat treatment: (i.) a change in its structure as shown with EPR and X-ray diffraction; (ii.) a release of an abundant protein into the supernatant, which is proportional to the extent of heat activation; (iii.) a change in the permeability of the cell wall as judged by fluorescence studies in which staining of the interior of the cell wall correlates with germination of individual ascospores. The gene encoding the protein, dubbed ICARUS, was studied in detail and was expressed under growth conditions that showed intense ascomata (fruit body) and ascospore formation. It encodes a small 7-14 kD protein. Blast search exhibits that different Talaromyces species show a similar sequence, indicating that the protein also occurs in other species of the genus. Deletion strains show delayed ascomata formation, release of pigments into the growth medium, higher permeability of the cell wall and a markedly shorter heat activation needed for activation. Further, wild type ascospores are more heat-resistant. All these observations suggest that the protein plays a role in dormancy and is related to the structure and permeability of the ascospore cell wall. However, more research on this topic is needed to study constitutive dormancy in other fungal species that form stress-resistant ascospores.

Structural analysis of novel trehalose-based oligosaccharides from extremely stress-tolerant ascospores of Neosartorya fischeri (Aspergillus fischeri).

Different fungi, including the genera Neosartorya, Byssochlamys and Talaromyces, produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. Here, the NMR analysis of a series of trehalose-based oligosaccharides, being compatible solutes that are accumulated to high levels in ascospores of the fungus Neosartorya fischeri, is presented. These oligosaccharides consist of an α,α-trehalose backbone, extended with one [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp; isobemisiose], two [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp] or three [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp] glucose units. The tetra- and pentasaccharide, dubbed neosartose and fischerose, respectively, have not been reported before to occur in nature.

A decrease in bulk water and mannitol and accumulation of trehalose and trehalose-based oligosaccharides define a two-stage maturation process towards extreme stress resistance in ascospores of Neosartorya fischeri (Aspergillus fischeri).

Fungal propagules survive stresses better than vegetative cells. Neosartorya fischeri, an Aspergillus teleomorph, forms ascospores that survive high temperatures or drying followed by heat. Not much is known about maturation and development of extreme stress resistance in fungal cells. This study provides a novel two-step model for the acquisition of extreme stress resistance and entry into dormancy. Ascospores of 11- and 15-day-old cultures exhibited heat resistance, physiological activity, accumulation of compatible solutes and a steep increase in cytoplasmic viscosity. Electron spin resonance spectroscopy indicated that this stage is associated with the removal of bulk water and an increase of chemical stability. Older ascospores from 15- to 50-day-old cultures showed no changes in compatible solute content and cytoplasmic viscosity, but did exhibit a further increase of heat resistance and redox stability with age. This stage was also characterized by changes in the composition of the mixture of compatible solutes. Mannitol levels decreased and the relative quantities of trehalose and trehalose-based oligosaccharides increased. Dormant ascospores of N. fischeri survive in low-water habitats. After activation of the germination process, the stress resistance decreases, compatible solutes are degraded and the cellular viscosity drops. After 5 h, the hydrated cells enter the vegetative stage and redox stability has decreased notably.

Functionality and prevalence of trehalose-based oligosaccharides as novel compatible solutes in ascospores of Neosartorya fischeri (Aspergillus fischeri) and other fungi.

Ascospores of Neosartorya, Byssochlamys and Talaromyces can be regarded as the most stress-resistant eukaryotic cells. They can survive exposure at temperatures as high as 85°C for 100 min or more. Neosartorya fischeri ascospores are more viscous and more resistant to the combined stress of heat and desiccation than the ascospores of Talaromyces macrosporus which contain predominantly trehalose. These ascospores contain trehalose-based oligosaccharides (TOS) that are novel compatible solutes, which are accumulated to high levels. These compounds are also found in other members of the genus Neosartorya and in some other genera within the order Eurotiales that also include Byssochlamys and Talaromyces. The presence of oligosaccharides was observed in species that had a relatively high growth temperature. TOS glasses have a higher glass transition temperature (Tg ) than trehalose, and they form a stable glass with crystallizing molecules, such as mannitol. Our data indicate that TOS are important for prolonged stabilization of cells against stress. The possible unique role of these solutes in protection against dry heat conditions is discussed.

VeA of Aspergillus niger increases spore dispersing capacity by impacting conidiophore architecture.

Aspergillus species are highly abundant fungi worldwide. Their conidia are among the most dominant fungal spores in the air. Conidia are formed in chains on the vesicle of the asexual reproductive structure called the conidiophore. Here, it is shown that the velvet protein VeA of Aspergillus niger maximizes the diameter of the vesicle and the spore chain length. The length and width of the conidiophore stalk and vesicle were reduced nearly twofold in a ΔveA strain. The latter implies a fourfold reduced surface area to develop chains of spores. Over and above this, the conidial chain length was approximately fivefold reduced. The calculated 20-fold reduction in formation of conidia by ΔveA fits the 8- to 17-fold decrease in counted spore numbers. Notably, morphology of the ΔveA conidiophores of A. niger was very similar to that of wild-type Aspergillus sydowii. This suggests that VeA is key in conidiophore architecture diversity in the fungal kingdom. The finding that biomass formation of the A. niger ΔveA strain was reduced twofold shows that VeA not only impacts dispersion capacity but also colonization capacity of A. niger.

Fungal spores for dispersion in space and time.

Spores are an integral part of the life cycle of the gross majority of fungi. Their morphology and the mode of formation are both highly variable among the fungi, as is their resistance to stressors. The main aim for spores is to be dispersed, both in space, by various mechanisms or in time, by an extended period of dormancy. Some fungal ascospores belong to the most stress-resistant eukaryotic cells described to date. Stabilization is a process in which biomolecules and complexes thereof are protected by different types of molecules against heat, drought, or other molecules. This review discusses the most important compounds that are known to protect fungal spores and also addresses the biophysics of cell protection. It further covers the phenomena of dormancy, breaking of dormancy, and early germination. Germination is the transition from a dormant cell toward a vegetative cell and includes a number of specific changes. Finally, the applied aspects of spore biology are discussed.

Microcolonial fungi on rocks: a life in constant drought?

Black microcolonial fungi (MCF) and black yeasts are among the most stress-resistant eukaryotic organisms known on Earth. They mainly inhabit bare rock surfaces in hot and cold deserts of all regions of the Earth, but some of them have a close phylogenetic relation to human pathogenic black fungi which makes them important model organisms also with respect to clinical mycology. The environment of those fungi is especially characterized by extreme changes from humidity to long periods of desiccation and extreme temperature differences. A key to the understanding of MCF ecology is the question about metabolic activity versus dormancy in the natural environments. In this study, the time lag from the desiccated state to rehydration and full metabolic activity and growth was measured and defined in accordance with simulated environmental conditions. The ability to survive after desiccation and the speed of rehydration as well as changes of the whole cell protein pattern are demonstrated. Whereas both mesophilic strains--Exophiala jeanselmei and Knufia perforans (=Coniosporium perforans)--show a clear reaction toward desiccation by production of small proteins, Cryomyces antarcticus--the extremotolerant MCF--does not show any response to desiccation but seems just to down-regulate its metabolism. Data on intracellular sugar suggest that both trehalose and mannitol might play a cell protective role in those fungi.