Quantifying metabolic activity of filamentous fungi using a colorimetric XTT assay

The filamentous nature and robust cell walls of many fungi render traditional measurements of active biomass (e.g., turbidity, dry cell weight) ineffective for most fungal bioprocesses. To overcome this challenge, an assay for quantification of overall metabolic activity is developed using 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), which in the presence of active mitochondria is converted to a water-soluble formazan derivative that absorbs light in the visible spectrum (430-490 nm). Tests on the model fungus Aspergillus nidulans show that in actively growing cultures XTT absorbance is linearly related to dry cell weight below 0.2 g/kg broth. Validation through growth rate testing shows the developed XTT assay is able to accurately quantify reductions in culture metabolism during damaging physical treatment (heat, high shear, microwaving). Experiments in batch culture demonstrate that the developed XTT assay is capable of reporting on metabolic activity where dry cell weight is not. The developed assay is inexpensive, relatively rapid, and easy to conduct, making it ideally suited for assessment of fungal processes in the biotechnology industry.     

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.     

Chitinolytic activity of filamentous fungi

The chitinolytic activity of nine species of filamentous fungi, classified with seven genera (specifically, Aspergillus, Penicillium, Trichoderma, Paecilomyces, Sporotrichum, Beaueria, and Mucor), was studied. When cultured in liquid medium containing 1% crystalline chitin, all fungi produced extracellular chitosans with activity varying from 0.2 U/mg protein (Sporotrichum olivaceum, Mucor sp., etc.) to 4.0-4.2 U/mg protein (Trichoderma lignorum, Aspergillus niger).     

A new continuous biofilm bioreactor for immobilized oil-degrading filamentous fungi

A new type of horizontal biofilm bioreactor for continuous bioconversion of emulsified oily substrate by immobilized growing biofilm of filamentous fungi was designed, constructed, and feasibility tested. The new reactor design provides "self"-immobilization of homogenized mycelium leading to even biofilm development. This was accomplished by using stainless steel screens of optimal mesh, mounted in parallel and stretching outward from a main rotating axis of a biological rotating contractor. Each screen was equipped with a pair of stainless steel blades mounted on supports allowing for continuous biofilm "shaving" beyond a predetermined thickness, thus retaining freshly growing active biofilm surface. The feasibility of the new bioreactor was demonstrated by decalactone production from emulsified castor oil by immobilized filamentous fungi (Tyromyces sambuceus). The combination of oriented metal screens and moving blades was found to be highly effective for a model system in maintaining stable substrate emulsion in the reactor in either batchwise or continuous processing, as well as maintaining biofilm thickness with continuous removal of excess growing hyphae. (c) 1996 John Wiley & Sons, Inc.     

A new assay to measure RNA N-glycosidase activity

We have developed a convenient procedure to measure the activity of Ricin A-chain and other enzymes with RNA N-glycosidase activity. The method is based on the use of a tritiated oligoribonucleotide as a substrate. The enzymatic activity is directly determined by measuring the release of adenine from the substrate. This method should prove useful in the study of the molecular mechanism of action of Ricin A and other RNA N-glycosidases.     

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.     

Approaches to functional genomics in filamentous fungi

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.     

A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi

Background  

Gene loss, inversions, translocations, and other chromosomal rearrangements vary among species, resulting in different rates of structural genome evolution. Major chromosomal rearrangements are rare in most eukaryotes, giving large regions with the same genes in the same order and orientation across species. These regions of macrosynteny have been very useful for locating homologous genes in different species and to guide the assembly of genome sequences. Previous analyses in the fungi have indicated that macrosynteny is rare; instead, comparisons across species show no synteny or only microsyntenic regions encompassing usually five or fewer genes. To test the hypothesis that chromosomal evolution is different in the fungi compared to other eukaryotes, synteny was compared between species of the major fungal taxa.     

Morphogenic effects of ramihyphin A in filamentous fungi

Ramihyphin A at subfungistatio concentrations stimulates ramification of hyphae of filamentous fungi. Stimulation of terminal ramification of hyphae that can be observed particularly in phytopathogenic fungi is most frequent. Hyphae ofMicrosporon canis, Trichophyton mentagrophytes, Blastomyces dermatitidis, Coccidioides immitis andHistoplasma capsulatum ramify intensively laterally. Stimulation of the lateral ramification was observed inMonilia fructigena, Penicillium marneffei andPenicillium chrysogenum. The antibiotic induces also formation of vesicular structures in phytopathogens. Due to the substantial ramification of hyphae, both terminal and lateral, the growth of colonies is interrupted. The addition of the antibiotic to a growing colony ofBotrytis cinerea induces dichotomic ramification of terminal hyphae after 3 h of growth. Lateral hyphae begin to grow later and further ramify dichotomically. Dense bundles of ramified hyphae are formed after 24 h due to the unbalanced ramification and the colony no longer increases its size.     

A simple assay with dyed substrates to quantify cellulase and hemicellulase activity of fungi

Summary A simple assay is described for the quantitative measurement of cellulase and hemicellulase activities. It is based on the enzymatic release of stained cleavage products originating from dyed substrates such as different celluloses, mannan and xylan, diffusing from an upper into a lower agar layer. The amount of cleavage products is assessed by direct absorbance measurement in the agar tubes employing an ordinary filter photometer. The method proved to be applicable to various species of basidiomycete fungi.     

A simple histochemical assay to detect cancer cells

Oxygen insensitivity of cancer cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity enables detection of cancer cells in unfixed cell smears or cryostat sections of biopsies. The assay is based on reduction of the tetrazolium salt neotetrazolium. It is a cheap assay that is easy to perform. It takes only 30 min at the most. The test discriminates between adenomas and carcinomas of colon and rectum with a certainty higher than 80% and is the best prognosticator of survival of colorectal cancer patients. Pancreatic cancer can be discriminated from pancreatitis with 100% certainty. Therefore, the assay is an excellent tool for the pathologist to provide additional information in difficult cases of diagnosis of cancer and for prognosis.     

Genome mining for the discovery of new nitrilases in filamentous fungi

Purpose of work  

Our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles.     

A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts

To determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. This assay enables us to discriminate intact non-fluorescent and depolarized fluorescent bacteria after exposure to antimicrobial peptides by measurement at the direct target, the cytoplasmic membrane and the membrane potential. The feasibility of the flow cytometric assay was evaluated with recombinant human beta-defensin 3 (HBD-3) against 25 bacterial strains representing 12 species. HBD-3 showed a broad-spectrum dose dependent activity and the minimal dose to cause depolarization ranged from 1.25 to >15 microg/ml HBD-3, depending on the species tested. The antibacterial effect was diminished with sodium chloride or dithiothreitol and could be abrogated with a HBD-3 antibody. Additionally, isolated cationic extracts from human intestinal biopsies showed a strong bactericidal effect against Escherichia coli K12, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923, which was diminished towards E. coli at 150 mM NaCl, whereas the activity towards S. aureus ATCC 25923 remained unaffected at physiological salt concentrations. DTT blocked the bactericidal effect of biopsy extracts completely.     

Critical view on the monochlorodimedone assay utilized to detect haloperoxidase activity

The current study aimed to identify the halogenating enzymes involved in the biosynthesis of the ambigols A, B, C and tjipanazole D, isolated from the cyanobacterium Fischerella ambigua. Haloperoxidase (HPO) activity within F. ambigua was therefore assayed spectrophotometrically by using monochlorodimedone (MCD) during protein purification. This strategy revealed the isolation of a protein positive in the MCD-assay, but an involvement in halogenating processes could not be verified. N-terminal sequencing rather demonstrated homology to cytochrome c(6) from other cyanobacteria and green algae. From our findings it thus has to be concluded that the spectrophotometrical MCD-assay routinely used to detect HPO activity may yield false positive results, mainly since the assay focuses on the decline of the educt and not on the formation of the product. Our data indicate that the reaction of MCD with proteins of the cytochrome c- family leads to unspecific products.     

Population genetics of filamentous fungi

Population genetics aims to understand causes and consequences of the genetic structure of pupulations, i.e. distributions of genetic variants in space and time. Among the most important determinants of the genetic population structure is the genetic system itself, which is the collection of processes and mechanisms responsible for the transmission of genetic information.Filamentous fungi offer excellent opportunities for studying the effects of the genetic system on genetic population structure. Apart from their advantage as laboratory organisms, they exhibit a wide variety of genetic systems. In particular, their inherent capacity for anastomosis provides unique possibilities for investigating rates and consequences of horizontal gene transfer. Furthermore, the temporary confinement of the products of meiosis in a common structure (the ascus) enables the study of competitive and antagonistic interactions between the meiotic products. An intriguing example of the latter is the phenomenon of spore killing, resulting in distorted meiotic segregation.This paper concentrates on population level research of the occurrence of vegatative incompatibility inAspergillus andNeurospora species and to what extent this will inhibit horizontal transmission of genetic information, and on spore killing inPodospora anserina.