RESUMO
Trichoderma is a well-known soil-borne fungus, highly efficient producer of extracellular enzymes including chitinases. The aim of this study was to recover a chitinase from fermentation waste after harvesting Trichoderma koningiopsis Th003 conidia and assess its potential as an enhancer of Beauveria bassiana insecticidal activity against Diatraea saccharalis. T. koningiopsis was produced by solid fermentation, conidia were harvested, and a crude extract (CE) was recovered by washing the residual substrate (rice:wheat bran). The partially purified chitinase (PPC) (75 kDa product) with N-acetyl-ß-glucosaminidase activity was obtained by chromatography to 29.3-fold with optimal activity at pH 5 and 55°C. Both the CE and the PPC were mixed with B. bassiana Bv062 conidia and assessed in a bioassay against D. saccharalis larvae. The CE and PPC from T. koningiopsis Th003 did not affect the germination or viability of B. bassiana conidia and enhanced its insecticidal activity when used at 0.06 U/ml enzymatic activity with a 24.5% reduction in B. bassiana lethal time (LT90 ). This study demonstrated the potential of chitinases produced by T. koningiopsis in solid fermentation to be recovered from the waste substrate and used as an additive to enhance B. bassiana, adding value to the main waste from the Trichoderma biopesticide/biofertilizer industries.
Assuntos
Beauveria/fisiologia , Quitinases/farmacologia , Hypocreales/enzimologia , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Animais , Agentes de Controle Biológico , Fermentação , Controle Biológico de Vetores/métodos , Esporos Fúngicos/enzimologiaRESUMO
Conidia from Metarhizium spp. are used for integrated pest control; however, environmental factors diminish the effectivity of these programs. Several approaches tried to improve conidia resistance to overcome this limitation, although little is known about the mechanisms involved in this effect. Here we measured the activity of antioxidant enzymes and conidia virulence, comparing the proteomic profiles of Metarhiziumlepidiotae CP-OAX conidia produced under normal (21% O2) and high oxygen atmospheres (pulses with 30% O2). We detected a higher virulence against Tenebrio molitor larvae, in addition to an increase in ultraviolet light tolerance in conidia produced under 30% O2, which correlates with increased glutathione reductase activity. Two-dimensional gel electrophoresis (2D SDS-PAGE) of proteins extracted in conidia harvested from both experimental conditions revealed a group of proteins that was observed only in conidia from oxidant atmospheres. Some of those proteins were directly involved in oxidative stress responses, whereas others were involved in conidial virulence, thermo-tolerance, and the central metabolism. Thus, a high atmospheric oxygen concentration (30%) activates antioxidant defence and general stress response mechanisms involved in conidia resistance to adverse environmental factors, which can ultimately translate into higher effectivity for the use of entomopathogenic fungi conidia in pest control.
Assuntos
Metarhizium/patogenicidade , Estresse Oxidativo , Oxigênio/metabolismo , Tenebrio/microbiologia , Animais , Glutationa Redutase/metabolismo , Larva/microbiologia , Metarhizium/enzimologia , Oxigênio/análise , Controle Biológico de Vetores , Esporos Fúngicos/enzimologia , Esporos Fúngicos/patogenicidade , VirulênciaRESUMO
Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis.
Assuntos
Antifúngicos/farmacologia , Proteínas Fúngicas/metabolismo , Metaloproteases/metabolismo , Fenantrolinas/farmacologia , Phialophora/efeitos dos fármacos , Phialophora/enzimologia , Esporos Fúngicos/crescimento & desenvolvimento , Humanos , Micoses/microbiologia , Phialophora/crescimento & desenvolvimento , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/enzimologiaRESUMO
Metarhizium anisopliae is an entomopathogenic fungus with the ability to infect a broad range of arthropods, and have evolved distinct strategies for their attachment to hosts. Here, we describe the characterisation of ecto-phosphatase activity on the conidia surface of M. anisopliae and its relevance in the host interaction process. Ecto-phosphatase activity was linear for 60 min and during this time, was linear with the increase of cell density. The optimum pH was in the acidic range and some divalent metals, such as Cu(2+), Cd(2+) and Zn(2+), inhibited ecto-phosphatase activity. The activity was also reduced by phosphatase inhibitors. Importantly, the inhibition of phosphatase activity in conidia reduced the adhesion to Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) integument and, consequently and indirectly, M. anisopliae infection. The results herein presented show, for the first time, the importance of ecto-phosphatase activity in M. anisopliae conidia and provide the first evidence of its direct involvement in adhesion and host infection.
Assuntos
Heterópteros/microbiologia , Metarhizium/metabolismo , Metarhizium/patogenicidade , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Esporos Fúngicos/enzimologia , Animais , Adesão Celular , Ativação Enzimática , Interações Hospedeiro-Patógeno , Concentração de Íons de Hidrogênio , Metarhizium/crescimento & desenvolvimento , VirulênciaRESUMO
Lipases are widely used in the industry for different purposes. Although these enzymes are mainly produced by submerged fermentation, lipase production by solid-state fermentation (SSF) has been gaining interest due to the advantages of this type of culture. Major advantages are higher production titers and productivity, less catabolite repression, and use of the dried fermented material as biocatalyst. This chapter describes a traditional methodology to produce fungal (Rhizopus homothallicus) lipases by SSF using perlite as inert support. The use of different devices (glass columns or Erlenmeyer flasks) and type of inoculum (spores or growing mycelium) is considered so that lipase production by SSF could be easily performed in any laboratory.
Assuntos
Lipase/biossíntese , Micélio/enzimologia , Rhizopus/enzimologia , Esporos Fúngicos/enzimologia , Óxido de Alumínio/química , Reatores Biológicos , Biotecnologia , Contagem de Colônia Microbiana , Fermentação , Vidro/química , Concentração de Íons de Hidrogênio , Dióxido de Silício/químicaRESUMO
Lipases secreted by Metarhizium anisopliae, an important biological control agent, could potentially be involved in the host infection process. Here, we present the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity and prevented the infection of the Rhipicephalus (Boophilus) microplus host. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process. The filamentous fungus Metarhizium anisopliae is one of the most important and studied biological agents for the control of several arthropod pests, including the cattle tick Rhipicephalus (Boophilus) microplus. Lipases secreted by M. anisopliae could potentially be involved in the host infection process. This work presents the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. During the course of tick exposure to spores (6-120 h) lipase activity increased from 0.03 ± 0.00 U to 0.312 ± 0.068 U using rho NP palmitate as substrate. In zymograms, bands of lipase activity were detected in ticks treated with spores without inhibitor. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity, at all times specified, and prevented the infection of the R. microplus host. Spores treated with the inhibitor did not germinate on the tick, although this effect was not observed in the culture medium. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process.
Assuntos
Interações Hospedeiro-Patógeno , Lipase/metabolismo , Metarhizium/enzimologia , Metarhizium/fisiologia , Rhipicephalus/microbiologia , Animais , Lipase/antagonistas & inibidores , Microscopia Eletrônica de Varredura , Controle Biológico de Vetores , Rhipicephalus/ultraestrutura , Esporos Fúngicos/enzimologia , Esporos Fúngicos/fisiologiaRESUMO
Catalases and peroxidases are the most important enzymes that degrade hydrogen peroxide into water and oxygen. These enzymes and superoxide dismutase are the first lines of cell defense against reactive oxygen species. Metarhizium anisopliae displays an increase in catalase-peroxidase activity during germination and growth. To determine the importance of catalase during the invasion process of M. anisopliae, we isolated the cat1 gene. cat1 cDNA expression in Escherichia coli and the subsequent purification of the protein confirmed that the cat1 gene codes for a monofunctional catalase. Expression analysis of this gene by RT-PCR from RNA isolated from fungus grown in liquid cultures showed a decrease in the expression level of the cat1 gene during germination and an increase during mycelium growth. The expression of this gene in the fungus during the infection process of the larvae of Plutella xylostella also showed a significant increase during invasive growth. Transgenic strains overexpressing the cat1 gene had twice the catalase activity of the wild-type strain. This increase in catalase activity was accompanied by a higher level of resistance to exogenous hydrogen peroxide and a reduction in the germination time. This improvement was also observed during the infection of P. xylostella larvae. M. anisopliae transgenic strains overexpressing the cat1 gene grew and spread faster in the soft tissue of the insect, reducing the time to death of the insect by 25% and the dose required to kill 50% of the population 14-fold.
Assuntos
Catalase/genética , Proteínas Fúngicas/genética , Expressão Gênica , Metarhizium/enzimologia , Metarhizium/patogenicidade , Esporos Fúngicos/crescimento & desenvolvimento , Animais , Catalase/metabolismo , Proteínas Fúngicas/metabolismo , Metarhizium/genética , Metarhizium/crescimento & desenvolvimento , Mariposas/microbiologia , Micélio/enzimologia , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/patogenicidade , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética , Esporos Fúngicos/patogenicidadeRESUMO
The conidia-mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62-48 and 22-18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis.
Assuntos
Regulação Fúngica da Expressão Gênica , Micélio , Scedosporium , Esporos Fúngicos , Albuminas/metabolismo , Biomarcadores , Fibronectinas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hemoglobinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Laminina/metabolismo , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Mucinas/metabolismo , Micélio/enzimologia , Micélio/genética , Fenantrolinas/farmacologia , Inibidores de Proteases/farmacologia , Scedosporium/enzimologia , Scedosporium/genética , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genéticaRESUMO
The osmotically-sensitive os-1 mutant of Neurospora crassa overproduced conidial alkaline phosphatase. The enzyme was purified by Phenyl-Sepharose CL-4B chromatography and Sephadex G-200 gel filtration. PAGE analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. The enzyme is a glycoprotein containing 16% saccharide, with apparent molar mass of 137 kDa. Two protein bands (36 and 62 kDa) were observed in SDS-PAGE, suggesting that the native enzyme was a trimer. The pI was estimated to be 2.7, and optima of pH and temperature were 9.5 and 65 degrees C, respectively. The enzyme showed broad substrate specificity, hydrolyzing preferentially 4-nitrophenyl phosphate, O-phosphoamino-acids and 2-phosphoglycerate. The hydrolysis of 4-nitrophenyl phosphate was stimulated by Co(II) (26%), Ni(II) (23%) and Mg(II) ions (80%). The enzyme was stable for up to 6 months at 4 degrees C in 5 mmol/L Tris-HCl buffer and also upon storage at 25 degrees C for 10 d. The kinetic and structural properties of the conidial enzyme purified from the os-1 mutant were quite different from those of the wild type strain. The enzyme overproduction observed in the mutant may be related to cell wall alterations that affect the process of enzyme secretion.
Assuntos
Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Neurospora crassa/enzimologia , Esporos Fúngicos/enzimologia , Fosfatase Alcalina/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Neurospora crassa/genética , Pressão Osmótica , Especificidade por Substrato , TemperaturaRESUMO
In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 84.6 +/- 11.3 nmol Pi h(-1) mg(-1) mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 +/- 27.6 nmol Pi h(-1) mg(-1)) in the presence of 5 mM MgCl2, with values of Vmax and apparent Km for Mg-ATP(2-) corresponding to 541.9 +/- 48.6 nmol Pi h(-1) mg(-1) cellular dry weight and 1.9 +/- 0.2 mM, respectively. The Mg2+-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1(V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg2+ -stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P2 purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Ascomicetos/enzimologia , Ascomicetos/citologia , Ascomicetos/fisiologia , Coenzimas/farmacologia , Ácido Edético/farmacologia , Inibidores Enzimáticos/farmacologia , Fluoretos/farmacologia , Levamisol/farmacologia , Macrolídeos/farmacologia , Cloreto de Magnésio/farmacologia , Oligomicinas/farmacologia , Esporos Fúngicos/enzimologia , Vanadatos/farmacologiaRESUMO
Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H(2)O(2) concentration range (0.01-3.0 M), non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to high temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is structurally different from the non-oxidized one, although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase could have been selected through evolution to assure an active enzyme under oxidative stress conditions.
Assuntos
Catalase/química , Neurospora crassa/enzimologia , Oxigênio Singlete/química , Esporos Fúngicos/enzimologia , Heme/química , OxirreduçãoRESUMO
The influence of the spore concentration in the inoculum preparation on microorganism morphology and glucoamylase synthesis by submerged cultures of Aspergillus awamori was investigated. A series of fermenter runs were perfomed, using an initial total reducing sugar concentration of 40 g/L. The inocula were prepared in a rotatory shaker, at 35§C 200 rev/min for 24 hours, varying spore concentration in the flasks from 9.5 x 10(3) to 1.8 x 10 (7) spores/mL. The inocula prepared with a spore concentration between 9.5 x 10(3) spores/mL and 5.0 x 10(6) spores/mL were composed by a cell suspension mainly in the pellet form, which led to a filamentous growth in the fermenter. Glucoamylase production was not affected by this range of spore concentration, reaching values between 2,100 U/L and 2,300 U/L. However, a higher spore concentration in the inoculum preparation (1.8 x 10(7) spores/mL) led to a inoculum formed by a cell suspension in the filamentous form containing many spore agglomerates (non-germinated spores). The kind of inoculum led to a growth predominantly in the pellet form in fermenter, which reduced the glucoamylase production in 30 (per cent).
Assuntos
Aspergillus , Ensaios Enzimáticos Clínicos , Enzimas , Técnicas In Vitro , Esporos Fúngicos/enzimologiaRESUMO
A glyceraldehyde-3-phosphate dehydrogenase (gpd) cDNA was isolated from the filamentous fungus Trichoderma harzianum in the course of a search for light-regulated genes in this organism. There is apparently only one copy of gpd in the T. harzianum genome, and its sequence is most similar to that of other filamentous ascomycetes. Trichoderma grows in the soil as a saprophyte or mycoparasite. A brief pulse of blue light, or nutrient depletion, induces sporulation, which is accompanied by altered patterns of abundance of specific polypeptides. Mycoparasitic development is also accompanied by changes in gene expression. The abundance of gpd mRNA decreased strongly during sporulation, and was lowest in samples consisting o mature conidiophores and conidia. When T. harzianum was grown in the presence of cell walls of the phytopathogen Rhizoctonia solani, the gpd mRNA level was much lower than in similar cultures grown on glucose. The repression of gpd, which is usually considered a constitutively expressed gene, may be part of the switch to sporulation or to the simulated mycoparasitic state. The implications of these findings for the use of gpd promoters to confer high constitutive expression are discussed.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Trichoderma/enzimologia , Trichoderma/genética , Sequência de Aminoácidos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Regulação para Baixo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Luz , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética , Esporos Fúngicos/efeitos da radiação , Trichoderma/fisiologiaRESUMO
Levels of protein kinase A (PKA) subunits and of cAMP have been measured during aerobic germination of the sporangiospores of the dimorphic fungus Mucor rouxii; further, the holoenzyme and its catalytic (C) and regulatory (R) subunits have been visualized through sucrose gradient centrifugation. Sporangiospores contain around 0.06 microM of a dimeric holoenzyme species of 5.5 S and a sixfold excess of a free R subunit of 2.7 S. Both these species are proposed to be derived by proteolysis from the native forms. Enzymic activity at this stage is highly inhibited, as demonstrated with permeabilized cells. Immediately upon germination, and after a transient increase in cAMP concentration from 10 microM to 90 microM, C-subunit levels fall to 30%. After the onset of germination, the specific activity and concentration of both the 5.5 S holoenzyme species and the 2.7 S species of free R subunit decrease in parallel to the increase in total protein and volume. Net synthesis of C and R subunits to form a native holoenzyme species of 8.8 S is apparent 4 h onwards after germination. A significant increase in cellular concentration is observed at 6 h. At 7 h of growth, when germ-tube emission is complete, the holoenzyme concentration is around 0.23 microM; there is almost no free R subunit and the intracellular concentration of cAMP is around 3 microM. A role for PKA during germination and morphogenesis is discussed.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mucor/enzimologia , Permeabilidade da Membrana Celular , Centrifugação com Gradiente de Concentração , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Cinética , Substâncias Macromoleculares , Mucor/crescimento & desenvolvimento , Mucor/fisiologia , Esporos Fúngicos/enzimologia , Esporos Fúngicos/fisiologiaRESUMO
Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.
Assuntos
Blastocladiella/metabolismo , Regulação Fúngica da Expressão Gênica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , Fosfoproteínas Fosfatases/metabolismo , Blastocladiella/enzimologia , Blastocladiella/crescimento & desenvolvimento , Parede Celular/metabolismo , Quitina/metabolismo , Éteres Cíclicos/farmacologia , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Esporos Fúngicos/enzimologia , Esporos Fúngicos/metabolismoRESUMO
The Phycomyces developmental mutant S356 elaborates spores which show a much poorer viability and a higher affinity for Calcofluor White than the wild-type spores. Protease-activated extracts of the mutant spores showed higher levels of chitin synthetase activity than the parental strain-derived spores. High levels of enzyme activity in the mutant extracts, but not in the corresponding wild-type extracts, could be detected in the absence of an exogenous protease. The high basal active chitin synthetase is not the result of activation by endogeneous proteases during cell breakage since protease inhibitors did not reduce, but rather increased, the activity levels. The analysis of cell wall composition in the mutant spores revealed significant changes in the proportion of uronic acids and protein but not in chitin. The mutant phenotype is discussed in relation to the developmental stage at which the alterations connected with cell wall metabolism occurred.