Synthesis of cell wall glucan, chitin, and protein by regenerating protoplasts and mycelia of Trichoderma reesei

1990 ◽  
Vol 36 (3) ◽  
pp. 211-217 ◽  
Author(s):  
Robert Messner ◽  
Christian P. Kubicek
Keyword(s):  
Cell Wall ◽  
Trichoderma Reesei ◽  
Cell Walls ◽  
Cell Wall Protein ◽  
Protoplast Regeneration ◽  
Secretory Proteins ◽  
Soluble Cell ◽  
Cell Wall Biogenesis ◽  
The Individual ◽  
Specific Labelling

The synthesis of constituent polymers of the cell wall by either growing mycelia or regenerating protoplasts of Trichoderma reesei QM 9414 was investigated by following the incorporation of radioactive precursors of the individual polymers (i.e., N[14C] acetyl-glucosamine, [3H] and [14C]glucose, [14C]mannose, and [35S]methionine) into individual fractions. N-Acetyl-glucosamine and glucose were found to become specifically incorporated into cell wall chitin and glucan by both mycelia and regenerating protoplasts, indicating the activity of chitin and glucan synthases in both systems. Cell wall glucan from regenerating protoplasts, however, consisted only of α-glucan and specifically lacked β-glucan which is found in mycelial cell walls. Mannose became metabolized to glucose before its label appeared in the cell wall, and was thus unsuitable for specific labelling. [35S]Methionine was found in a small (< 21 kDa) polypeptide from the first alkali-soluble cell wall fraction, but also in cell wall bound secretory proteins, i. e., β-glucosidase. These results indicate that cell wall biogenesis in T. reesei, in contrast to a report by other authors, is similar to that of other filamentous fungi. Key words: Trichoderma reesei, protoplast regeneration, cell wall polymer, β-glucan, cell wall protein.

scholarly journals Identification of Cell Wall-Associated Proteins from Phytophthora ramorum

2006 ◽  
Vol 19 (12) ◽  
pp. 1348-1358 ◽  
Author(s):  
Harold J. G. Meijer ◽  
Peter J. I. van de Vondervoort ◽  
Qing Yuan Yin ◽  
Chris G. de Koster ◽  
Frans M. Klis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Plant Pathogens ◽  
Sodium Dodecyl ◽  
Phytophthora Ramorum ◽  
Extracellular Proteins ◽  
Glycoside Hydrolases ◽  
Secretory Proteins ◽  
Plant Infection ◽  
Associated Proteins

The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble secreted proteins or proteins associated with the cell wall, play important roles in the interaction with host plants. Cell walls of P. ramorum contain 1 to 1.5% proteins, the remainder almost exclusively being accounted for by glucan polymers. Here, we present an inventory of cell-wall-associated proteins based on mass spectrometric sequence analysis of tryptic peptides obtained by proteolytic digestion of sodium dodecyl sulfate-treated mycelial cell walls. In total, 17 proteins were identified, all of which are authentic secretory proteins. Functional classification based on homology searches revealed six putative mucins or mucin-like proteins, five putative glycoside hydrolases, two transglutaminases, one annexin-like protein, the elicitin protein RAM5, one protein of unknown function, and one Kazal-type protease inhibitor. We propose that the cell wall proteins thus identified are important for pathogenicity.

scholarly journals A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

2004 ◽  
Vol 3 (5) ◽  
pp. 1297-1306 ◽  
Author(s):  
Hiroshi Kitagaki ◽  
Kiyoshi Ito ◽  
Hitoshi Shimoi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Cell Walls ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Homologous Proteins ◽  
Aberrant Cell ◽  
Cell Wall Biogenesis

ABSTRACT Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Δdcw1 Δdfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Δdfg5). When DC61 cells were incubated at 37°C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37°C. At 37°C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37°C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37°C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Δdcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

Composition and structure of tuber cell walls affect in vitro digestibility of potato (Solanum tuberosum L.)

2016 ◽  
Vol 7 (10) ◽  
pp. 4202-4212 ◽  
Author(s):  
Jovyn K. T. Frost ◽  
Bernadine M. Flanagan ◽  
David A. Brummell ◽  
Erin M. O'Donoghue ◽  
Suman Mishra ◽  
...  
Keyword(s):  
Cell Wall ◽  
Solanum Tuberosum ◽  
New Zealand ◽  
Cell Walls ◽  
Solanum Tuberosum L ◽  
In Vitro Digestibility ◽  
Soluble Cell ◽  
Composition And Structure

Higher amount of 4 M KOH-soluble cell wall pectic galactan is associated with lowerin vitrodigestibility in three New Zealand potato lines.

Repression of N-glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Aspergillus fumigatus

Microbiology ◽  
2011 ◽  
Vol 157 (7) ◽  
pp. 1968-1979 ◽  
Author(s):  
Kai Li ◽  
Haomiao Ouyang ◽  
Yang Lü ◽  
Jingnan Liang ◽  
Iain B. H. Wilson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Unfolded Protein Response ◽  
Cell Wall Protein ◽  
Signalling Pathway ◽  
Cell Wall Biosynthesis ◽  
Unfolded Protein ◽  
Cell Wall Biogenesis ◽  
The Unfolded Protein Response ◽  
Protein Response

Aspergillus fumigatus is the most common airborne fungal pathogen, causing fatal invasive aspergillosis in immunocompromised patients. The crude mortality is 60–90 % and remains around 29–42 % even with treatment. The main reason for patient death is the low efficiency of the drug therapies. As protein N-glycosylation is involved in cell wall biogenesis in A. fumigatus, a deeper understanding of its role in cell wall biogenesis will help to develop new drug targets. The Afstt3 gene encodes the essential catalytic subunit of oligosaccharyltransferase, an enzyme complex responsible for the transfer of the N-glycan to nascent polypeptides. To evaluate the role of N-glycosylation in cell wall biosynthesis, we constructed the conditional mutant strain CPR-stt3 by replacing the endogenous promoter of Afstt3 with the nitrogen-dependent niiA promoter. Repression of the Afstt3 gene in the CPR-stt3 strain led to a severe retardation of growth and a slight defect in cell wall integrity (CWI). One of the most interesting findings was that upregulation of the cell wall-related genes was not accompanied by an activation of the MpkA kinase, which has been shown to be a central element in the CWI signalling pathway in both Saccharomyces cerevisiae and A. fumigatus. Considering that the unfolded protein response (UPR) was found to be activated, which might upregulate the expression of cell wall protein and chitin, our data suggest that the UPR, instead of the MpkA-dependent CWI signalling pathway, is the major compensatory mechanism induced by repression but not abolition of N-glycosylation in A. fumigatus. Our finding is a key to understanding the complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development.

The effect of growth environment on the chloroform–methanol and alkali-extractable cell wall and cytoplasm lipid levels of Mucor rouxii

1975 ◽  
Vol 21 (1) ◽  
pp. 79-84 ◽  
Author(s):  
S. Safe ◽  
J. Caldwell
Keyword(s):  
Fatty Acids ◽  
Cell Wall ◽  
Cell Walls ◽  
Lipid Levels ◽  
Mucor Rouxii ◽  
Growth Environment ◽  
Lipid Fractions ◽  
Filamentous Cell ◽  
The Individual ◽  
Chloroform Methanol

The distribution of chloroform–methanol and alkali-extractable lipids in the cell walls of aerobically grown filamentous cells from Mucor rouxii has been determined. The results have been compared with the corresponding lipid composition of yeast-like cells from M. rouxii, which can be produced in two ways: by growth under anaerobic conditions and by aerobic growth in the presence of 0.22% phenethyl alcohol (PEA). It was observed that in most cases the crude cytoplasmic fraction contained higher levels of several lipids (i.e., squalene, sterols, triterpenes, and fatty acids) than did the corresponding cell walls. The cell walls did, however, contain both "free" (chloroform–methanol extractable) and "bound" (alkali extractable) lipids although the relative amounts were markedly dependent on the cell growth environment. The aerobically grown filamentous cell walls contained higher levels of squalene, sterols, triterpenes, and fatty acids than did aerobically grown yeast-like PEA-induced cell walls and there was also considerable variation in the "free''/"bound" ratios of the various lipid components. The lipid levels in both the cell walls and cytoplasm of the anaerobically grown cells were considerably lower than those of the cells grown under aerobic conditions. In addition, the differences in the growth environment were also reflected in the compositions of the individual lipid fractions from both the cell wall and the cytoplasm fraction.

Transglutaminase-like activity participates in cell wall biogenesis in Saccharomyces cerevisiae

Biologia ◽  
2007 ◽  
Vol 62 (2) ◽  
Author(s):  
Marián Mazáň ◽  
Vladimír Farkaš
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Walls ◽  
Insoluble Fraction ◽  
Amide Bond ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Wall Biogenesis ◽  
Cross Links ◽  
Wall Construction ◽  
Isolated Cell

AbstractTransglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [3H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.

scholarly journals Sed1p Is a Major Cell Wall Protein ofSaccharomyces cerevisiae in the Stationary Phase and Is Involved in Lytic Enzyme Resistance

1998 ◽  
Vol 180 (13) ◽  
pp. 3381-3387 ◽  
Author(s):  
Hitoshi Shimoi ◽  
Hiroshi Kitagaki ◽  
Hisanobu Ohmori ◽  
Yuzuru Iimura ◽  
Kiyoshi Ito
Keyword(s):  
Cell Wall ◽  
Stationary Phase ◽  
Cell Walls ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Sodium Dodecyl ◽  
Cell Wall Protein ◽  
Lytic Enzyme ◽  
Cell Wall Proteins ◽  
Structural Cell

ABSTRACT A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidusprotease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the SED1 gene.SED1 was formerly identified as a multicopy suppressor oferd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a β-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNA increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of SED1 had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.

scholarly journals Multicomponent carbohydrase system from Trichoderma reesei: A toolbox to address complexity of cell walls of plant substrates in animal feed

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0251556
Author(s):  
Ninfa Rangel Pedersen ◽  
Morten Tovborg ◽  
Abdoreza Soleimani Farjam ◽  
Eduardo Antonio Della Pia
Keyword(s):  
Cell Wall ◽  
Confocal Microscopy ◽  
Trichoderma Reesei ◽  
Cell Walls ◽  
Animal Feed ◽  
Raw Material ◽  
Microscopy Imaging ◽  
Diverse Range ◽  
Cell Wall Components ◽  
Wall Components

A diverse range of monocot and dicot grains and their by-products are commonly used in the animal feed industry. They all come with complex and variable cell wall structures which in turn contribute significant fiber to the complete feed. The cell wall is a highly interconnected matrix of various polysaccharides, proteins and lignin and, as such, requires a collaborative effort of different enzymes for its degradation. In this regard, we investigated the potential of a commercial multicomponent carbohydrase product from a wild type fermentation of Trichoderma reesei (T. reesei) (RONOZYME® MultiGrain) in degrading cell wall components of wheat, barley, rye, de-oiled rice bran, sunflower, rapeseed and cassava. A total of thirty-one different enzyme proteins were identified in the T. Reesei carbohydrase product using liquid chromatography with tandem mass spectrometry LC-MS/MS including glycosyl hydrolases and carbohydrate esterases. As measured by in vitro incubations and non-starch polysaccharide component analysis, and visualization by immunocytochemistry and confocal microscopy imaging of immuno-labeled samples with confocal microscopy, the carbohydrase product effectively solubilized cellulolytic and hemicellulolytic polysaccharides present in the cell walls of all the feed ingredients evaluated. The T. reesei fermentation also decreased viscosity of arabinoxylan, xyloglucan, galactomannan and β-glucan substrates. Combination of several debranching enzymes including arabinofuranosidase, xylosidase, α-galactosidase, acetyl xylan esterase, and 4-O-methyl-glucuronoyl methylesterase with both GH10 and GH11 xylanases in the carbohydrase product resulted in effective hydrolyzation of heavily branched glucuronoarabinoxylans. The different β-glucanases (both endo-β-1,3(4)-glucanase and endo-β-1,3-glucanase), cellulases and a β-glucosidase in the T. reesei fermentation effectively reduced polymerization of both β-glucans and cellulose polysaccharides of viscous cereals grains (wheat, barley, rye and oat). Interestingly, the secretome of T. reesei contained significant amounts of an exceptional direct chain-cutting enzyme from the GH74 family (Cel74A, xyloglucan-specific β-1,4-endoglucanase), that strictly cleaves the xyloglucan backbone at the substituted regions. Here, we demonstrated that the balance of enzymes present in the T. reesei secretome is capable of degrading various cell wall components in both monocot and dicot plant raw material used as animal feed.

scholarly journals THE CHEMICAL COMPOSITION OF THE CELL WALL OF CHLAMYDOMONAS GYMNOGAMA AND THE CONCEPT OF A PLANT CELL WALL PROTEIN

1974 ◽  
Vol 63 (2) ◽  
pp. 420-429 ◽  
Author(s):  
David H. Miller ◽  
Ira Seth Mellman ◽  
Derek T. A. Lamport ◽  
Maureen Miller
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Chemical Composition ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Cell Wall Protein ◽  
Ultrastructural Examination ◽  
Widespread Occurrence ◽  
Glycoprotein Complex ◽  
Sexual Mating

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.

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