Filamentous Fungal Cell Research Articles

Effect of Dissolved Oxygen Changes on Activated Sludge Fungal Bulking during Lab-Scale Treatment of Acidic Industrial Wastewater

The cloning and sequencing of fungal 18S rRNA genes followed by the identification of filamentous fungal species by fluorescent in situ hybridization (FISH) and the enumeration of filamentous fungal cells by flow cytometry-FISH (FC-FISH) were used to investigate the effect of dissolved oxygen (DO) changes on activated sludge (AS) fungal bulking during a lab-scale treatment of acidic industrial wastewater. By increasing DO levels from < .5 to > 2 mg L⁻¹, bulking started to occur due to the outbreak of fungal filaments, whereas the chemical oxygen demand (COD) removals sharply increased from < 40 to > 70%. Clone library analyses revealed that all clonal fungal sequences were of yeast origin, and that only one and four yeast species were individually detected in AS at two DO levels. Subsequent FISH identification of filamentous yeast species within bulking sludge using self-designed oligonucleotide probes suggested that all probe-reactive cells of Trichosporon asahii had a filamentous morphology and were the dominating filamentous microorganism in the AS. The FC-FISH analyses of bacteria and two main yeast species showed that the DO shift resulted in a sharp increase of T. asahii, by a factor of 48-60, which caused filamentous yeast bulking. Subsequently, the restoration of DO levels to <0.5 mg L⁻¹ effectively restored the sludge settlement and yeast community, as well as unacceptable COD removals.

Effect of Rapamycin on Filamentous Fungal Cell Walls

Filamentous fungi are widely used in the bioprocess industry to produce a variety of products resulting in billion dollar returns. Often times in industrial bioprocesses, fungi experience nutrient limitation, and we hypothesize that autophagy, a nutrient starvation response, is induced. We further hypothesize that autophagy leads to changes in the mechanical properties of filamentous fungal cell walls, resulting in thinner, weaker and stiffer walls. We test this hypothesis here, using rapamycin (an immunosuppressant drug) to gratuitously induce autophagy in the model fungi Aspergillus nidulans. Atomic force microscopy (AFM) is used to assess the mechanical properties of the cell wall, electron microscopy is used to assess wall thickness and a novel fragmentation assay is used to determine relative tensile strength of the culture. We will report on these studies and how they support our hypotheses.

Erratum: Control of filamentous fungal cell shape by septins and formins

Nature Reviews Microbiology 4, 223–229 (2006); doi: 10.1038/nrmicro1345 In the above article, the legend to Figure 1 did not match what was shown in the figure. The correct legend is shown below. We wish to apologize to the author, and to readers, for any confusion caused.

Control of filamentous fungal cell shape by septins and formins

Studies in various model systems have identified two protein families that are crucial for shaping cell morphology: the septins and the formins. Both families are conserved in most eukaryotes, but the functions and regulation of individual homologues can vary depending on their precise cellular context. The rich array of cell geometries found in different filamentous fungal species provides a powerful experimental canvas for studying the evolution and regulation of septins and formins. Here, I assimilate what is known about the function of these protein families in filamentous fungi and propose that further studies in these organisms could answer some open mechanistic questions that pertain in general to eukaryotic cells.

Enhanced separation of filamentous fungi by ultrasonic field: possible usage in repeated batch processes

Usage of ultrasonic field-based filters in retention of filamentous fungal cells was assessed using Rhizopus arrhizus NRRL 1526 as a model organism. Effects of operating conditions, such as power input, harvest pump flow rate, run time and stop time, on the system's separation efficiency (SE) were evaluated by modulating the variables according to a Central Composite Design (CCD). The standard pump with which the ultrasonic filter was equipped was shown to be unsuitable and was, therefore, substituted for with a prime rate reverse pump that made possible separation and recycle of the mycelial biomass. The operating conditions were optimised (run time, 300 s; stop time, 3 s; power input, 6 W; harvest pump flow rate, 4 l per day) and a repeated batch process (three batches for a total of 192 h) was performed during which the SE was maintained always higher than 88%.

Proteins associated with the cell envelope of Trichoderma reesei: a proteomic approach.

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.

Proteins associated with the cell envelope of Trichoderma reesei: A proteomic approach

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.

Proteins associated with the cell envelope ofTrichoderma reesei: A proteomic approach

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism

The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.