Rapid detection of lytic antimicrobial activity against yeast and filamentous fungi.

A rapid method for assessing the lytic activity of antimicrobial agents against yeast and fungi has been developed. The assay is based on the release of the intracellular enzyme, maltase (alpha-glucosidase). The released maltase activity was measured colorimetrically by the production of p-nitrophenol from p-nitrophenyl-alpha-D-glucopyranoside (PNPG). The lytic activity of different antimicrobial compounds was measured against yeast cells or germinating spores of filamentous fungi. Lytic anti-yeast activity could be detected within 20 min incubation at 30 degrees C against Saccharomyces cerevisiae, Candida albicans, and Cryptococcus neoformans. Lytic anti-fungal activity appeared after 2 h of incubation at 30 degrees C against germinating spores of Aspergillus niger and Botrytis cinerea. Whole cells of either yeast or fungi did not hydrolyze sufficient PNPG within 3 h at 30 degrees C to yield a detectable color change. Lytic activity of enzymes (e.g., Lyticase), antibiotics (e.g., Amphotericin B), and an antibiotic-producing strain of bacteria were detected using the assay. The anti-yeast assay has been adapted to a 96-well microtiter format. Both assays provided a rapid, sensitive, and reproducible detection of lytic anti-yeast and anti-fungal activity.     

Polarity in filamentous fungi: moving beyond the yeast paradigm

Filamentous fungi grow by the polar extension of hyphae. This polar growth requires the specification of sites of germ tube or branch emergence, followed by the recruitment of the morphogenetic machinery to those sites for localized cell wall deposition. Researchers attempting to understand hyphal morphogenesis have relied upon the powerful paradigm of bud emergence in the yeast Saccharomyces cerevisiae. The yeast paradigm has provided a useful framework, however several features of hyphal morphogenesis, such as the ability to maintain multiple axes of polarity and an extremely rapid extension rate, cannot be explained by simple extrapolation from yeast models. We discuss recent polarity research from filamentous fungi focusing on the position of germ tube emergence, the relaying of positional information via RhoGTPase modules, and the recruitment of morphogenetic machinery components including cytoskeleton, polarisome and ARP2/3 complexes, and the vesicle trafficking system.     

Survival strategies of yeast and filamentous fungi against the antifungal protein AFP

The activities of signaling pathways are critical for fungi to survive antifungal attack and to maintain cell integrity. However, little is known about how fungi respond to antifungals, particularly if these interact with multiple cellular targets. The antifungal protein AFP is a very potent inhibitor of chitin synthesis and membrane integrity in filamentous fungi and has so far not been reported to interfere with the viability of yeast strains. With the hypothesis that the susceptibility of fungi toward AFP is not merely dependent on the presence of an AFP-specific target at the cell surface but relies also on the cell's capacity to counteract AFP, we used a genetic approach to decipher defense strategies of the naturally AFP-resistant strain Saccharomyces cerevisiae. The screening of selected strains from the yeast genomic deletion collection for AFP-sensitive phenotypes revealed that a concerted action of calcium signaling, TOR signaling, cAMP-protein kinase A signaling, and cell wall integrity signaling is likely to safeguard S. cerevisiae against AFP. Our studies uncovered that the yeast cell wall gets fortified with chitin to defend against AFP and that this response is largely dependent on calcium/Crz1p signaling. Most importantly, we observed that stimulation of chitin synthesis is characteristic for AFP-resistant fungi but not for AFP-sensitive fungi, suggesting that this response is a successful strategy to protect against AFP. We finally propose the adoption of the damage-response framework of microbial pathogenesis for the interactions of antimicrobial proteins and microorganisms in order to comprehensively understand the outcome of an antifungal attack.     

A current view on long noncoding RNAs in yeast and filamentous fungi

Applied Microbiology and Biotechnology - Long noncoding RNAs (lncRNAs) are crucial players in epigenetic regulation. They were initially discovered in human, yet they emerged as common factors...     

Absence of spermine in filamentous fungi.

Polyamines were examined in several yeasts and filamentous fungi. Whereas putrescine, spermidine, and spermine were present in the yeasts, spermine was not detected in any of the filamentous fungi.     

Characterization of yeast and filamentous fungi isolated from cryoconite holes of Svalbard,Arctic

Cryoconite holes have biogeochemical, ecological and biotechnological importance. This communication presents results on culturable psychrophilic yeast and filamentous fungi from cryoconite holes at Midre Lovénbreen glacier. The identification of these microbes was achieved through conventional and DNA sequencing techniques. Effect of temperature, salt and media on growth of the cultures was studied. Measurements on the bioavailability of nutrients and trace metals were made through different methods including ICPMS (Inductively Coupled Plasma Mass Spectrometry). Colony forming unit (CFU) per gram of sediment sample was calculated to be about 7 × 10³–1.4 × 10⁴ and 4 × 10³–1.2 × 10⁴ of yeast and filamentous fungi, respectively. Based on morphology and sequence data, these were identified as Cryptococcus gilvescens, Mrakia sp., Rhodotorula sp., Phialophora alba and Articulospora tetracladia. Amongst these, Phialophora alba, Cryptococcus gilvescens and Mrakia sp. zhenx-1 are reported for the first time from Svalbard Arctic, while Rhodotorula sp. (95% gene similarity) is a new species, yet to be described. Rhodotorula sp. expressed high amylase, while Cryptococcus gilvescens showed high lipase activity. Mrakia sp. showed phosphate solubilization between 4 and 15°C, which is a first record. Chemical analysis revealed the presence of organic carbon, nitrogen and phosphorus in substantial amounts in the sediments. Filamentous fungi and yeast in the cryoconite holes drive the process of organic macromolecule degradation through cold-adapted enzyme secretion, thereby assisting in nutrient cycling in these subglacial environments. Further, these cold-adapted enzymes may provide an opportunity for the prospect of biotechnology in Arctic. This is the first report on mycological investigation into cryoconite holes from Midre Lovénbreen glacier.     

Possible involvement of pleiomorphic vacuolar networks in nutrient recycling in filamentous fungi

Morphological analyses of vacuoles in filamentous fungi in the past decade have led to the remarkable finding that they are highly pleiomorphic organelles. Among them, tubular vacuoles have been implicated in nutrient transport between hyphal tips and the host plant surface in mycorrhizal fungi. However, a series of works suggested the presence of tubular vacuoles in other fungi that are not mycorrhizal, including Aspergillus oryzae, hinting at more general roles of the tubular vacuoles. Recently, we made two key observations by using the fusion protein of enhanced green fluorescent protein (EGFP) with a putative vacuolar t-SNARE in A. oryzae; tubular vacuoles formed more extensively in hyphae that were not in contact with nutrients, and vacuoles that were interconnected by tubules in the mature mycelial region displayed traces of microautophagy-mediated degradation of cytoplasm. The aim of this addendum is to discuss the possible involvement of vacuoles in degrading, transporting, and recycling nutrients from the mature mycelial region to hyphal tips, to support the continuous tip growth.     

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.     

Gene expression systems for filamentous fungi.

The extraordinary capacity of filamentous fungi to produce large quantities of extracellular protein, together with the advent of DNA-mediated fungal transformation, has resulted in rapid advances in the development of gene expression systems for filamentous fungi. This review focuses on recent developments in the expression of both fungal and non-fungal genes and improvements to the host.     

Heterologous expression of genes in filamentous fungi.

Isolation of some biologically important proteins from natural sources was found to be too expensive or scarcely possible (human proteins). The problem could be solved by expression of heterologous genes. Many biologically active proteins have been successfully expressed in filamentous fungi, some of them, however, at a low level. Thus, improvement of this technique appears to be a very important task. The process comprises several steps. Some of them, such as efficient transformation, vector construction, processing of signal sequences, post-translational modifications and secretion of the expressed proteins, have been intensively investigated. This review presents obstacles and problems encountered in expression of heterologous genes and discusses strategies of development in this area.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

丝状真菌的细胞凋亡

凋亡是一种程序性细胞死亡类型,为多细胞生物发育和维持生命所必需的,也普遍存在于细菌等原核生物和酵母、丝状真菌等真核生物中。丝状真菌既具有酵母和哺乳动物共有的凋亡同源蛋白,也具有酵母所不具备的哺乳动物凋亡同源蛋白,所以其凋亡机制较酵母更为复杂,而又较哺乳动物简单。凋亡在丝状真菌的发育、繁殖、衰老等过程中具有重要的作用。近年,丝状真菌作为新的凋亡研究的模式生物被广泛研究,而且进展迅速。综述丝状真菌的凋亡现象和检测方法,丝状真菌中凋亡的生物学功能,丝状真菌凋亡的诱导条件,以及丝状真菌凋亡相关基因的功能研究进展。     

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.     

Genomics of the filamentous fungi - moving from the shadow of the bakers yeast

Fungi have now well and truly entered the genomic age. We currently know the complete DNA sequence for 18 fungal species and many more fungal genome sequencing projects are in progress. Whilst yeasts dominated the early genomic years, recently there has been a dramatic increase in filamentous fungal genome projects. The implications of this wealth of genetic information for mycologists worldwide is immense. In this review we summarise the background to fungal genome projects with an emphasis on the filamentous fungi. We discuss efforts to determine gene function and to compare genomes from different species. Since this is such a fast-moving field, useful web sites are listed that will enable the reader to keep up to date with developments.     

Morphology and productivity of filamentous fungi

Cultivation processes involving filamentous fungi have been optimised for decades to obtain high product yields. Several bulk chemicals like citric acid and penicillin are produced this way. A simple adaptation of cultivation parameters for new production processes is not possible though. Models explaining the correlation between process-dependent growth behaviour and productivity are therefore necessary to prevent long-lasting empiric test series. Yet, filamentous growth consists of a complex microscopic differentiation process from conidia to hyphae resulting in various macroscopically visible appearances. Early approaches to model this morphologic development are recapitulated in this review to explain current trends in this area of research. Tailoring morphology by adjusting process parameters is one side of the coin, but an ideal morphology has not even been found. This article reviews several reasons for this fact starting with nutrient supply in a fungal culture and presents recent advances in the investigation of fungal metabolism. It illustrates the challenge to unfold the relationship between morphology and productivity.