How to enter the state of dormancy? A suggestion by Trichoderma atroviride conidia.

It is generally accepted that conidia, propagules of filamentous fungi, exist in the state of dormancy. This state is defined mostly phenomenologically, e.g., by germination requirements. Its molecular characteristics are scarce and are concentrated on the water or osmolyte content, and/or respiration. However, a question of whether conidia are metabolic or ametabolic forms of life cannot be answered on the basis of available experimental data. In other words, are mature conidia open thermodynamic systems as are mycelia, or do they become closed upon the transition to the dormant state? In this article, we present observations which may help to define the transition of freshly formed conidia to the putative dormant forms using measurements of selected enzyme activities, 1H- and 13C-NMR and LC-MS-metabolomes, and 14C-bicarbonate or 45Ca2+ inward transport. We have found that Trichoderma atroviride and Aspergillus niger conidia arrest the 45Ca2+ uptake during the development stopping thereby the cyclic (i.e., bidirectional) Ca2+ flow existing in vegetative mycelia and conidia of T. atroviride across the cytoplasmic membrane. Furthermore, we have found that the activity of α-ketoglutarate dehydrogenase was rendered completely inactive after 3 weeks from the conidia formation unlike of other central carbon metabolism enzymes. This may explain the loss of conidial respiration. Finally, we found that conidia take up the H14CO3- and convert it into few stable compounds within 80 d of maturation, with minor quantitative differences in the extent of this process. The uptake of H13CO3- confirmed these observation and demonstrated the incorporation of H13CO3- even in the absence of exogenous substrates. These results suggest that T. atroviride conidia remain metabolically active during first ten weeks of maturation. Under these circumstances, their metabolism displays features similar to those of chemoautotrophic microorganisms. However, the Ca2+ homeostasis changed from the open to the closed thermodynamic state during the early period of conidial maturation. These results may be helpful for studying the conidial ageing and/or maturation, and for defining the conidial dormant state in biochemical terms.

Functional dissection of gamma-aminobutyric acid metabolism in Neurospora crassa.

The GABA shunt is one of the metabolic pathways that is ubiquitous in prokaryotes and eukaryotes. γ-aminobutyric acid (GABA) in fungi is required in the stress responses, virulence and development. The number of genes encoding glutamate decarboxylase (gad), GABA transaminase (gta) and succinic semialdehyde dehydrogenase (ssadh) varies between fungal species. The genome-wide analysis in Neurospora crassa resulted in the identification of a gta and a ssadh. Disruption of either gta or ssadh decreased respiration rate and biomass accumulation, reduced growth on GABA and beta-alanine. The gta and ssadh mutants exhibited aberrant hyphal morphology and displayed differential transcription of the GABA shunt genes. In the gta mutant, protoperithecia and perithecia formation was almost completely suppressed in the presence of GABA and beta-alanine, indicating GTA requirement for the turnover of these amino acids. The strains displayed differential metabolic dysregulations in response to different nitrogen sources. The phenotypic differences between the gta and ssadh mutants could be contributed to accumulation of intermediates of the GABA shunt and/or GABA shunt-independent functions. Together, our data suggest that the GABA shunt could function as a moderate modulator of multiple biological events, including respiration, energy metabolism, carbon and nitrogen metabolism, growth, as well as sexual development in N. crassa.

The study of intracellular and secreted high-molecular-mass protease(s) of Trichoderma spp., and their responses to conidiation stimuli.

We continued our study of high-molecular-mass proteases (HMMPs) using several strains of the genus Trichoderma, and other filamentous fungi (Botrytis cinerea, Aspergillus niger, Fusarium culmorum, and Penicillium purpurogenum). We found that five Trichoderma strains secreted HMMPs into the media after induction with bovine serum albumin. Botrytis cinerea and F. culmorum secreted proteases in the absence of inducer, while A. niger or P. purpurogenum did not secrete proteolytic activity (PA). The activity of HMMPs secreted by or intracellularly located in Trichoderma spp. represents the predominant part of cellular PA, according to zymogram patterns. This observation allowed the study of HMMPs' physiological role(s) independent from the secretion. In studying conidiation, we found that illumination significantly stimulated PA in Trichoderma strains. In the T. atroviride IMI 206040 strain, we demonstrated that this stimulation is dependent on the BLR1 and BLR2 receptors. No stimulation of PA was observed when mechanical injury was used as an elicitor of conidiation. Compounds used as inhibitors or activators of conidiation exerted no congruent effects on both PA and conidiation. These results do not favour a direct role of HMMPs in conidiation. Probably, HMMP activity may be involved in the process of the activation of metabolism during vegetative growth, differentiation, and aging-related processes.

Light-induced conidiation of Trichoderma spp. strains is accompanied by development-dependent changes in the Ca2+ binding to cell walls.

The effect of light on the binding of Ca2+ to mycelia and to cell walls isolated from aerial mycelia of three strains of Trichoderma spp. was studied. Two independent methods were used to measure the total Ca2+ content in mycelia and the Ca2+ bound to cell walls isolated from aerial mycelia. The results of these methods showed that the light-induced formation and maturation of conidia in Trichoderma spp. is accompanied by increased Ca2+ deposition in mycelia and cell walls. Moreover, the cultivation of Trichoderma atroviride F-534 in the presence of 45Ca2+ under circadian illumination showed that radioactivity was exclusively localized in the light-induced conidial rings of aerial mycelia. The fluorescence microscopy of chlortetracycline-stained mycelia showed that the major fraction of Ca2+ was accumulated in conidia and fructification structures, or some intracellular compartments in T. atroviride F-534 grown under circadian illumination, while only a limited amount of Ca2+ was associated with hyphal surfaces. In addition, the study of 45Ca2+ binding to cell walls revealed that T. atroviride F-534 displays both increased 45Ca2+ binding capacity and elevated affinity to 45Ca2+ binding upon illumination. The results indicate that conidia formation and (or) maturation is associated with changes in Ca2+ homeostasis.

Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains.

Pancreatic lipase inhibitors, such as tetrahydrolipstatin (orlistat), are used in anti-obesity treatments. Orlistat is the only anti-obesity drug approved by the European Medicines Agency (EMA). The drug is synthesized by saturation of lipstatin, a ß-lactone compound, isolated from Streptomyces toxytricini and S. virginiae. To identify producers of novel pancreatic lipase inhibitors or microbial strains with improved lipstatin production and higher chemical purity remains still a priority. In this study, a high-throughput screening method to identify Streptomyces strains producing potent pancreatic lipase inhibitors was established. The assay was optimized and validated using S. toxytricini NRRL 15443 and its mutants. Strains grew in 24-well titer plates. Lipstatin levels were assessed directly in culture medium at the end of cultivation by monitoring lipolytic activity in the presence of a chromogenic substrate, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 6-methylresorufin ester (DGGR). The lipase activity decreased in response to lipstatin production, and this was demonstrated by accumulation of red-purple methylresorufin, a product of DGGR digestion. The sensitivity of the assay was achieved by adding a lipase of high lipolytic activity and sensitivity to lipstatin to the reaction mixture. In the assay, the fungal lipase from Mucor javanicus was used as an alternative to the human pancreatic lipase. Many fungal lipases preserve high lipolytic activity in extreme conditions and are not colipase dependent. The assay proved to be reliable in differentiation of strains with high and low lipstatin productivity.

Properties of anaerobic fungi isolated from several habitats: complexity of phenotypes.

Isolates of anaerobic fungi from rumen, animal faeces and compost displayed morphological similarity with known anaerobic fungi. According to their ITS sequences, species were related to Neocallimastix and Piromyces. Rumen fungi tolerated exposure to an aerobic atmosphere for at least four days. Under anaerobic conditions, they could grow on both, defined or complex substrates. Growth in liquid media was monitored by the continuous measurement of metabolic gases (O2, CO2, H2, CO, H2S, CH4). Monitored metabolism was complex, showed that both CO2 and H2 were produced and subsequently consumed by yet unknown metabolic pathway(s). CO and H2S were evolved similarly, but not identically with the generation of CO2 and H2 suggesting their connection with energetic metabolism. Anaerobic fungi from snail faeces and compost produced concentrations of H2S, H2, CO near the lower limit of detection. The rumen isolates produced cellulases and xylanases with similar pH and temperature optima. Proteolytic enzymes were secreted as well. Activities of some enzymes of the main catabolic pathways were found in cell-free homogenates of mycelia. The results indicate the presence of the pentose cycle, the glyoxylate cycle and an incomplete citrate cycle in these fungi. Differences between isolates indicate phenotypic variability between anaerobic fungi.

Nutrient transport into germinating Trichoderma atroviride conidia and development of its driving force.

The exit from dormancy and the start of growth should be preceded or at least accompanied by the uptake of nutrients. In this work we studied changes in the transport of several nutrients into Trichoderma atroviride conidia. Germination started with a short period of isodiametric growth (conidial swelling), followed by polarized growth (germ tube formation) after about 8 h at 26 °C. The onset of isodiametric growth required the presence of external both phosphate and nitrate. At the same time, an increased uptake of precursors of macromolecules and phospholipids ((14)C- or (3)H-labelled valine, uracil, N-acetylglucosamine and choline) occurred. A low uptake of these precursors was observed also in non-germinating conidia. Concomitantly, this uptake developed an increased sensitivity to the uncoupler 3,3',4',5-tetrachlorosalicylanilide. Expression and activity of H(+)-ATPase started after completing isodiametric growth, suggesting that the proton-motive force (PMF) generated by H(+)-ATPase may be an accelerator of nutrient uptake and metabolism. (14)C-valine uptake was also measured into a mutant with disrupted pma1 gene. This mutant did not form conidia. The mutant also exhibited uncoupler sensitivity of (14)C-valine uptake. These observations showed that a PMF must have been generated by a mechanism(s) other than the H(+)-ATPase activity in the WT before H(+)-ATPase expression and in mycelia with disrupted H(+)-ATPase.

Changes in metabolome and in enzyme activities during germination of Trichoderma atroviride conidia.

The aim of this work was to study the metabolic changes during germination of Trichoderma atroviride conidia along with selected marker enzyme activities. The increase in proteinogenic amino acid concentrations together with the increase in glutamate dehydrogenase activity suggests a requirement for nitrogen metabolism. Even though the activities of tricarboxylic acid cycle enzymes also increased, detected organic acid pools did not change, which predisposes this pathway to energy production and supply of intermediates for further metabolism. The concentrations of many metabolites, including the main osmolytes mannitol and betaine, also increased during the formation of germ tubes. The activities of H(+)-ATPase and GDPase, the only marker enzymes that did not have detectable activity in non-germinated conidia, were shown with germ tubes.

Glutamic acid decarboxylase gene disruption reveals signalling pathway(s) governing complex morphogenic and metabolic events in Trichoderma atroviride.

Glutamate decarboxylase (GAD) catalyses decarboxylation of glutamate to gamma-aminobutyrate (GABA) in a metabolic pathway connected to citrate cycle and known as GABA shunt. The gene (gad) was disrupted in Trichoderma atroviride CCM F-534 and viable mutants were characterized. Two of them were found to arise by homologous recombination and were devoid of both GAD activity and GABA. Mutants grew slower as compared to the wild type (F534). In the submerged culture, mutants developed less CO2 and consumed less O2 than the F534 without changing their respiratory quotients. Hyphae of mutants were more ramified than those of F534. Their ramification, in contrast to F534, was not increased by cyclosporin A, a drug causing hyphae ramification of several fungi and which is a calcineurin/cyclophilin inhibitor, or by FK506. Rapamycin, which is a cyclophilin but not calcineurin inhibitor, had a different effect on hyphae ramification in F534 and mutants. To examine the presence of GABA receptors in the fungus the effect of mammalian GABA-receptor modulators, such as bicuculline, gabapentin or carbamazepine on fungal morphology were investigated. Conidia of mutants germinated in a multipolar manner more frequently (up to 80 %) than those of F534. This trait was modified with cyclosporine A, FK506 and GABA receptor modulators in a different manner. Transport of chlorides, an intimate feature of GABA-regulated receptors/channels in animal cells, was measured in vegetative mycelia by means (36)Cl(-) uptake. It was significantly reduced in gad mutants. The results suggest that T. atroviride possesses a signalling pathway that involves GABA, putative GABA receptor(s), calcineurin, target of rapamycin and chloride transporter(s) to regulate physiological functions.

Properties of secreted protease from vegetative Trichoderma atroviride mycelia cultivated with protein inducer reveal a complex protein-recognition mechanism.

In this study, the submerged cultivation of Trichoderma atroviride with protein substrate (bovine serum albumin, casein, ovalbumin, etc.) led to the secretion of protease activity (PA) into the medium. The secretion started within 30 h and rose to the maximum after 72-h cultivation. It continued upon the prolonged cultivation (up to 8 days) with lower secreted PA. Zymography of the secreted protease (SDS-PAGE with 0.2% gelatine), revealing high molecular weight (m.w.) protease(s) (~200 kDa) with high autolytic activity as the only secretory product. Enzymological characteristics of high m.w. proteases elicited by different inducer proteins were surprisingly different, suggesting that mycelia can recognize not only the presence of inducer proteins but also some qualitative characteristics. Low m.w. protease(s), as found from other studies was(were) seen after prolonged cultivation only, as a band with m.w. about 36 kDa. Expression of known Trichoderma spp. genes encoding secreted proteases, prb1 and proA, showed that only prb1 was expressed after 3-4 days of cultivation, i.e., after the early-secretion phase. The secretory activity of the earlier phase was impaired by tunicamycin and brefeldin A and was significantly stimulated by uncoupler. The existence of a biphasic fungal secretory response and the protein quality recognition represent previously unrecognized processes.

Transient excretion of succinate from Trichoderma atroviride submerged mycelia reveals the complex movements and metabolism of carboxylates.

Submerged growth of Trichoderma atroviride CCM F 534 on glucose-containing medium was accompanied by the excretion of organic acids (succinate, citrate, alpha-ketoglutarate, fumarate, aconitate). The excretion of succinate was transient. After 48-72 h cultivation, millimolar amounts of succinate disappeared from the medium. We studied the mechanism of the removal of succinate from the medium and demonstrated the activation of the inward transport of succinate by submerged mycelia. This transport was carrier-mediated, had a low solute specificity, and was driven by proton-motive force. The last aspect was provided by the activation of the H(+)-ATPase, as documented by measurements of ATPase activity and expression of the pma gene. The disruption of the pma gene abolished the capacity of the mycelia to re-uptake succinate but not its production. Results show that excreted carboxylates could serve as alternative nutrients in the late phase(s) of submerged growth, explain why inward transport system(s) for carboxylates are induced, and indicate that the inward-directed transport could interfere with the production of carboxylic acids by fungi.

Spontaneous and protein-induced secretion of proteinases from Saccharomyces cerevisiae.

Many fungi are capable of secreting the wide spectrum of hydrolytic enzymes. We characterized an inducible proteinase secretion in yeasts, Saccharomyces cerevisiae. The proteinase secretion by S. cerevisiae was induced in the presence of yeast extract, or of purified proteins, such as bovine serum albumin, casein, or ovalbumin, and some proteolytic activity was present also without protein inducer. We found that properties of proteinases induced under cultivation conditions were different in various aspects (temperature- and pH-dependencies, substrate specificities, sensitivities to proteinase inhibitors). Proteinase activities were also characterized by gelatin zymography. Multiple proteinase bands with wide-molecular weights (ranging from 45 to 240 kDa) were detected and patterns of proteinase bands were different. S. cerevisiae cells were able to retain the information about previous contacts with protein inducer resulting in faster and more intensive proteinase secretion response after repeated induction.

Ca2+-dependent induction of conidiation in submerged cultures of Trichoderma viride.

The presence of Ca2+ (up to 0.1 mol/L) in the cultivation media was found to induce the formation of conidia in submerged mycelia of Trichoderma viride in a concentration-dependent manner. Ca2+ dramatically stimulated conidiation after 70 h of cultivation. The effect was present in the dark, and illumination stimulated it only marginally. Low (less than 100 micromol/L) Ca2+ concentrations induced the formation of chlamydospores. Sr2+ could substitute Ca2+ in conidiogenesis with lower efficiency (almost 2 orders of magnitude), while the efficiency of Mg2+, Mn2+, or Ba2+ was lower by almost 3 orders of magnitude. Our results demonstrate that mycelial Ca2+ homeostasis has powerful effects on the conidiation and mycelial morphogenesis in T. viride, and they suggest that there is an additional mechanism of conidiation in addition to those induced by light and starvation.

Characterization and regulation of basal calcium influx in human peripheral blood lymphocytes.

The basal 45Ca2+ influx into resting human blood lymphocytes was measured. This process showed biphasic kinetics with first rapid phase followed by the second long-lasting and markedly slower phase. Further, it showed signs of saturability and reaches maximal values at 37 degrees C and extracellular pH 7.2. The basal 45Ca2+ influx was stimulated by addition of submicromolar concentrations of 4 beta-phorbol-12-myristate-13-acetate, and this effect was abolished by protein kinase C (PKC) inhibitor Ro-31-8220. In the regulation of basal 45Ca2+ influx is probably only partially involved adenylate cyclase pathway as show results with intracellular c-AMP elevating agents (dB-c-AMP, 3-isobutyl-1-metylxantine and forskolin). Uncoupler 3,3',4',5-tetrachloro-salicylanilide (TCS) in micromolar concentrations stimulated basal 45Ca2+ influx and its effect was more significant in media with high extracellular concentration of K+.

H+ -mediated coupling of transmembrane Ca2+ fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca2+ concentrations.

The adaptation to extreme concentrations of Ca(2+) and its consequence on the properties of the (45)Ca(2+) transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca(2+)](o) did not cause changes in kinetic parameters of the (45)Ca(2+) influx but the adaptation to high [Ca(2+)](o) increased the K(M(Ca2+)). The V(max) of the (45)Ca(2+) influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K(M(Ca2+)) these changes were prevented in mycelia adapted to high Ca(2+). High [Ca(2+)](o) decreased the stimulation by the uncoupler, 3, 3', 4', 5-tetrachloro salicylanilide (TCS) (30 muM), as compared to the control, whereas the Ca(2+) chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the (45)Ca(2+) influx faded away, in parallel with the activity of the H(+)-ATPase. The (45)Ca(2+) efflux from mycelia was affected by TCS in a similar way as the (45)Ca(2+) influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca(2+) homeostasis and ageing are in agreement with a notion that both Ca(2+)-influx and-efflux are coupled by the H(+)-homeostasis at the plasma membrane.

Changes in growth competence of aged Trichoderma viride vegetative mycelia.

Identical masses of submerged Trichoderma viride mycelia of various ages were used as inoculum for a second submerged cultivation lasting for 24 h. It was found that the growth yield of secondary culture was dependent on the age of inoculum. The growth yields increased when the age of primary culture was less than 3 d, and decreased down to zero when older mycelia were inoculated. The mycelia were living even after 1 month of submerged cultivation, as they formed conidia after inoculating onto solid medium. In order to elucidate underlying biochemical processes, developmental changes of specific activities of organellar marker enzymes were measured in the mitochondrial/vacuolar and microsomal fractions of mycelia. These activities changed during the growth of mycelia in a biphasic manner and their time courses were remarkably similar. Only the H(+)-ATPase activity decreased monophasically with the age of mycelia. Membrane-bound proteases of both membrane fractions changed differently upon ageing. These results could not be explained as a consequence of nutrient starvation and indicate that the prolonged submerged cultivation triggers coordinated series of biochemical events which leads to the loss of growth competence.

Light accelerates the splicing of srh1 homologue gene transcripts in aerial mycelia of Trichoderma viride.

The expression of the Tvsrh1 gene encoding conidial hydrophobin was investigated during the development of surface-cultivated Trichoderma viride mycelia under different illumination regimes. Three transcripts of the whole gene amplified from the total mRNA were found with lengths of 400, 323 and 272 bp. The 400-bp transcript was slowly converted to the shorter forms in the dark. Light-pulse dramatically increased the rate of conversion, and a permanent illumination of mycelia was most efficient in this process. The sequencing of transcripts revealed that the 400 bp transcript contains two introns, whereas the intermediate one contains only one intron located distally from the 5'-end. The shortest transcript was without introns. The sum of all transcripts remained almost unchanged in the dark and increased upon the light pulse but decreased during development under permanent illumination. The appearance of conidia coincided with the complete conversion of the transcripts. The results showed that the splicing of the two introns was not random but sequential, and that it did not follow the cotranscriptional mechanism. Furthermore, they suggested that mRNA processing could represent another regulation level of gene expression by light during the photo-induced conidiation in T. viride.

Study of Trichoderma viride metabolism under conditions of the restriction of oxidative processes.

Trichoderma viride was capable of growth and conidiation in the presence of high concentrations of the uncoupler 3,3',4',5-tetrachlorosalicylanilide (up to 100 micromol x L(-1) and of the respiration inhibitor mucidin (up to 100 micromol x L(-1) ) in both submerged and surface cultivation. When vegetative mycelia were cultivated on the solid Czapek-Dox medium with yeast autolysate under an anaerobic and CO2-containing atmosphere, the growth was observed only rarely but the microorganism survived as long as 3 months under these conditions. Major products of metabolism of both aerobic and anaerobic submerged mycelia were identified by means of 1H-NMR measurements. Major products excreted to the medium under aerobic conditions were succinic and citric acids. Major metabolites present in the submerged mycelia were gamma-aminobutyric (and glutamic) acid and alanine. Under anaerobic conditions, citric acid was not excreted into the medium but ethanol appeared. Its production could not be increased upon increasing the sugar concentration. The appearance of secondary metabolites was found to be modified by oxygen availability during the mycelial growth. Results suggest that the vegetative form of T. viride is capable of fermentative metabolism characterized by the production of ethanol and succinate and that the excretion of carboxylic acids is developmentally regulated and modified by oxygen availability.

Characterization of microorganisms isolated from lignite excavated from the Záhorie coal mine (southwestern Slovakia).

Microorganisms were isolated from lignite freshly excavated in the Záhorie coal mine (southwestern Slovakia) under conditions excluding contamination with either soil or air-borne microorganisms. The isolates represented both Prokarya and Eukarya (fungi). All were able to grow on standard media, although some microorganisms were unstable and became extinct during storage of coal samples. Bacteria belonged to the genera Bacillus, Staphylococcus, and Rhodococcus, according to both morphological criteria and ITS sequences. Several bacterial isolates were resistant to antibiotics. The presence of anaerobic bacteria was also documented, although they have not yet been identified. Fungal isolates were typified by using their ITS sequences. They belonged to the genera Trichoderma (Hypocrea), Penicillium, Epicoccum, Metarhizium (Cordyceps), and Cladosporium. Several fungi produced compounds with antibiotic action against standard bacterial strains. The evidence for the presence of microorganisms in native lignite was obtained by means of fluorescence microscopy, scanning electron microscopy, and electron microprobe analysis. Results demonstrated that microorganisms were able to survive in the low-rank coal over a long time period.

Developmental changes in Trichoderma viride enzymes abundant in conidia and the light-induced conidiation signalling pathway.

The expression of glutamic acid decarboxylase gene and the laccase activity were measured during the development of surface-cultivated Trichoderma viride mycelia in order to examine their up-regulation by light. The results show that the changes in activity of GAD induced by light observed previously are caused by transcriptional regulation of gad gene expression in both submerged mycelia and aerial mycelia after photoinduction. The expression of tga gene encoding a T. viride G(alpha) protein was found not to be up-regulated by light and was also present in the non-conidiating mutant of T. viride suggesting that this protein is not involved in the regulation of conidiation in this fungus, or that it plays a role is in later stages of conidia development. The activity of laccase was also not light-inducible and may be related to the maturation of conidia.