Duplications and losses of genes encoding known elements of the stress defence system of the Aspergilli contribute to the evolution of these filamentous fungi but do not directly influence their environmental stress tolerance.

The contribution of stress protein duplication and deletion events to the evolution of the Aspergilli was studied. We performed a large-scale homology analysis of stress proteins and generated and analysed three stress defence system models based on Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Although both yeast-based and A. nidulans-based models were suitable to trace evolutionary changes, the A. nidulans-based model performed better in mapping stress protein radiations. The strong Mantel correlation found between the positions of species in the phylogenetic tree on the one hand and either in the A. nidulans-based or S. cerevisiae-based models on the other hand demonstrated that stress protein expansions and reductions contributed significantly to the evolution of the Aspergilli. Interestingly, stress tolerance attributes correlated well with the number of orthologs only for a few stress proteins. Notable examples are Ftr1 iron permease and Fet3 ferro-O2-oxidoreductase, elements of the reductive iron assimilation pathway, in the S. cerevisiae-based model, as well as MpkC, a HogA-like mitogen activated protein kinase in the A. nidulans-based model. In the case of the iron assimilation proteins, the number of orthologs showed a positive correlation with H2O2-induced stress tolerance while the number of MpkC orthologs correlated positively with Congo Red induced cell wall stress, sorbitol induced osmotic stress and H2O2 induced oxidative stress tolerances. For most stress proteins, changes in the number of orthologs did not correlate well with any stress tolerance attributes. As a consequence, stress tolerance patterns of the studied Aspergilli did not correlate with either the sets of stress response proteins in general or with the phylogeny of the species studied. These observations suggest that stress protein duplication and deletion events significantly contributed to the evolution of stress tolerance attributes of Aspergilli. In contrast, there are other processes, which may counterbalance the effects of stress gene duplications or deletions including (i) alterations in the structures of stress proteins leading to changes in their biological activities, (ii) varying biosynthesis of stress proteins, (iii) rewiring stress response regulatory networks or even (iv) acquiring new stress response genes by horizontal gene transfer. All these multilevel changes are indispensable for the successful adaptation of filamentous fungi to altering environmental conditions, especially when these organisms are entering new ecological niches.

Interactions between naturally occurring antifungal agents.

Pairwise interactions between four antifungal compounds were studied. The ß-1,3-glucan synthase inhibitor echinocandin B (ECB) showed synergistic effect with the cell wall hydrolase ChiB chitinase and EngA ß-1,3-glucanase on Saccharomyces cerevisiae, Candida albicans, Aspergillus rugulosus and A. fumigatus. The antifungal protein of Penicillium chrysogenum (PAF) did not influence the antifungal activity of ChiB or EngA, but showed antagonistic effect with ECB on A. nidulans, A. rugulosus and A. fumigatus. PAF had no significant effect on the growth of the tested yeasts as it was expected and did not influence significantly the antifungal activity of ECB, ChiB or EngA against yeasts.

The extracellular ß-1,3-endoglucanase EngA is involved in autolysis of Aspergillus nidulans.

AIMS: To elucidate the roles of the ß-1,3-endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. METHODS AND RESULTS: A ß-1,3-endoglucanase was purified from carbon-starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene-expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. CONCLUSIONS: The ß-1,3-endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall-degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. SIGNIFICANCE AND IMPACT OF THE STUDY: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.

Optimization of coprogen production in Neurospora crassa.

Coprogen production of Neurospora crassa was dependent on glucose, aspartate and iron contents as well as on initial pH of the culture media. Surplus iron and acidic pH hindered the production of coprogen as well as the transcription of the sid1 gene (NCU07117) encoding putative L-ornithine-N5-monooxygenase, the first enzyme in the coprogen biosynthetic pathway. High glucose (40 g/l) and aspartate (21 g/l) concentrations were beneficial for coprogen synthesis, but neither glucose nor aspartate affected the sid1 transcription. Moreover, efficient coprogen production was observed after glucose had been consumed, which suggested that N. crassa accumulated iron even in non-growing, carbon-starving cultures.

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production.

AIMS: Elucidation of the regulation of ChiB production in Aspergillus nidulans. METHODS AND RESULTS: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. CONCLUSIONS: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.

PepJ is a new extracellular proteinase of Aspergillus nidulans.

Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 degrees C, respectively. The enzyme was stable at pH 3.5-10.5 and was still active at 95 degrees C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.

The glutathione response to salt stress in the thermophilic fungus, Thermomyces lanuginosus.

In order to investigate the role of glutathione in response to salt stress in the thermophilic fungus, Thermomyces lanuginosus, the biomass and the intracellular pool of protein and the glutathione + glutathione disulphid (GSH + GSSG) was measured for four days in a medium with NaCl or KCl added and in the basal medium. Due to the osmotic and ionic stress imposed by the salts, the growth of T. lanuginosus was delayed and the inhibitory effect of KCl exceeded that of NaCl. Glutathione seemed to be involved in the response of T. lanuginosus towards high concentrations of salt, as the level of stress was negatively correlated with the amount of total glutathione. Salt stress did not result in an increased intracellular protein production. GSH accumulated while nutrients were abundant and were subsequently degraded later, suggesting that nutrients stored in GSH are used when the medium is depleted.

Characterization and heterologous expression of an age-dependent fungal/bacterial type chitinase of Aspergillus nidulans.

Under carbon starvation, Aspergillus nidulans produced a fungal/bacterial type chitinase, ChiB. The chiB gene was cloned and subcloned into pJC40 expression vector containing a 10XHis fusion tag, and the ChiB protein was expressed heterologously in Escherichia coli. Recombinant and native ChiB enzymes shared the same optimal pH ranges and showed similar substrate specificities with endo-acting cleavage patterns.

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies.

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed.

Yeast-like cell formation and glutathione metabolism in autolysing cultures of Penicillium chrysogenum.

The bulk formation of yeast-like (arthrospore-like) cells were typical in carbon-depleted submerged cultures of the high beta-lactam producer Penicillium chrysogenum NCAIM 00237 strain independently of the nitrogen-content of the culture medium. This morphogenetic switch was still quite common in carbon-starving cultures of the low-penicillin-producer strain P. chrysogenum ATCC 28089 (Wis 54-1255) when the nitrogen-content of the medium was low but was a very rare event in wild-type P. chrysogenum cultures. The mycelium-->yeast-like cell transition correlated well with a relatively high glutathione concentration and a reductive glutathione/glutathione disulfite (GSH/GSSG) redox balance in autolysing cultures, which was a consequence of industrial strain development. Paradoxically, the development of high beta-lactam productivity resulted in a high intracellular GSH level and, concomitantly, in an increased y-glutamyltranspeptidase (i.e. GSH-decomposing) activity in the autolytic phase of growth of P. chrysogenum NCAIM 00237. The hypothesized causal connection between GSH metabolism and cell morphology, if verified, may help us in future metabolic engineering of industrially important filamentous fungi.

A novel aspect of NADPH production in ageing Penicillium chrysogenum.

NADPH is involved in many basically important anabolic processes. For a long time, pentose phosphate pathway (PPS) was regarded as the most important source of NADPH in fungi. Here we present evidence of a metabolic switch to an alternative NADPH-producing pathway in ageing Penicillium chrysogenum cultures, which involves NADP+ -specific isocitrate dehydrogenase (NADP+ -ID) rather than PPS enzymes. Considering the main biochemical functions of NADPH, we propose that NADP+ -ID could have deep impact on many physiological processes switched on glucose deprivation including proteinase production or penicillin biosynthesis. We also demonstrate that although the alternative pathway was inferior to PPS when the fungus was grown on well-utilisable carbon sources yet it could have an important role in fatty acid biosynthesis as well as in the maintenance of high intracellular NADPH/NADP+ ratios.

Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans.

N-Acetyl-D-glucosamine, chito-oligomers and carbon starvation regulated chiA, chiB, and nagA gene expressions in Aspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and the N-acetyl-beta-D-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-related chiA or the autolysis-coupled chiB genes although both of them were down-regulated under diamide stress. The transcription of the chiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration.

Penicillium chrysogenum glucose oxidase -- a study on its antifungal effects.

AIMS: Purification and characterization of the high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. METHODS AND RESULTS: The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (M(r)) = 155 000, subunit structure alpha(2) with M(r,alpha) = 76 000, isoelectric point (pI) = 5.4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1.0 mg ml(-1)) prevented oxidative cell injuries triggered by 0.004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX-catalase system was likely to replace the primary inhibition exerted by H(2)O(2). CONCLUSIONS: Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent.

Physiological and morphological changes in autolyzing Aspergillus nidulans cultures.

Physiological and morphological changes in carbon-limited autolyzing cultures of Aspergillus nidulans were described. The carbon starvation arrested conidiation while the formation of filamentous and "yeast-like" hyphal fragments with profoundly altered metabolism enabled the fungus to survive the nutritional stress. The morphological and physiological stress responses, which maintained the cellular integrity of surviving hyphal fragments at the expense of autolyzing cells, were highly concerted and regulated. Moreover, sublethal concentrations of the protein synthesis inhibitor cycloheximide or the mitochondrial uncoupler 2,4-dinitrophenol completely blocked the autolysis. In accordance with the propositions of the free-radical theory of ageing reactive oxygen species accumulated in the surviving fragments with a concomitant increase in the specific superoxide dismutase activity and a continuous decrease in cell viability. Glutathione was degraded extensively in carbon-starving cells due to the action of gamma-glutamyltranspeptidase, which resulted in a glutathione-glutathione disulfide redox imbalance during autolysis.

In situ localization of manganese peroxidase production in mycelial pellets of Phanerochaete chrysosporium.

The ultrastructure of Phanerochaete chrysosporium hyphae from pellets in submerged liquid cultures was investigated in order to learn more about the interrelation between fungal architecture and manganese peroxidase (MnP) production. At day 2 of cultivation, some subapical regions of hyphae in the outer and middle zones of the pellet initiated differentiation into intercalary thick-walled chlamydospore-like cells of about 10 micro m diameter. At the periphery of the cytoplasm of these cells, a large number of mitochondria and Golgi-like vesicles were observed. The sites of MnP production were localized at different stages of cultivation by an immunolabelling procedure. The immunomarker of MnP was mainly concentrated in the chlamydospore-like cells and principally distributed in Golgi-like vesicles located at the periphery of the cytoplasm. The apices of hyphae in the outer layer of the pellets were apparently minor sites of MnP production. Maximal MnP release into the culture supernatant coincided with apparent autolysis of the chlamydospore-like cells. Production of extracellular autolytic chitinase and protease coincided with the disappearance of these structures from the pellets. The chlamydospore-like cells observed in the mycelial pellets of P. chrysosporium could be metabolically active entities operating as an enzyme reservoir, delivering their content into the surrounding medium possibly by an enzyme-mediated autolytic process.

Glutathione metabolism of Acremonium chrysogenum in relation to cephalosporin C production: is gamma-glutamyltransferase in the center?

Methionine increased the intracellular glutathione (reduced) (GSH) pool and the specific gamma-glutamyltransferase (gamma-GT) activity in the cephalosporin C (CPC) producer Acremonium chrysogenum. The accelerated turnover of GSH might be indicative of the existence of a functioning gamma-glutamate cycle, and might supply the CPC biosynthetic machinery with L-cysteine. The gamma-GT was not subject to nitrogen metabolic repression but was more active in cells exposed to different oxidative-stress-generating agents. Exogenous cysteine hindered both the uptake of methionine and the induction of gamma-GT, and was not beneficial for CPC production. There was no causal relationship between the redox status of the cells and the observed cell morphology.

Autolysis and ageing of Penicillium chrysogenum under carbon starvation: respiration and glucose oxidase production.

During the exponential growth phase of Penicillium chrysogenum NCAIM 00237 the effective conversion of glucose and O2 to gluconate and H2O2 by glucose oxidase (GOX) was the most likely source of intracellular ROS measured. In glucose-supplemented autolysing cultures, the increased of intracellular ROS concentration was attributed to respiration in the absence of any significant GOX activity. The induction of GOX and catalase by glucose and H2O2 was clearly age-dependent in P. chrysogenum. In ageing cryptic growth phase cultures, superoxide dismutase and cyanide-resistant respiration were the major elements of antioxidative defence but these activities were insufficient to prevent the progressive accumulation of ROS and the concomitant decrease in cell vitality.

Distribution and susceptibility of Candida species isolated in the Medical University of Debrecen.

Data of Candida albicans and non-albicans Candida species isolated during the 1997-2000 period in the Medical and Health Science Center of the University of Debrecen are analysed. The number of yeast isolates increased from 408 to 1213 per year during this period. Dominance of C. albicans has been persistent, but a slight increase of C. glabrata and C. krusei could be observed. Distribution of different Candida species isolated from 16 body sites indicates that C. albicans seems to be still the most aggressive Candida species. Investigation of 244 urinary Candida isolates (parallel with bacterial cultures) suggests that tha aetiological role of Candida species in the pathogenesis of urinary tract infections can be hypothesized if colony forming unit (CFU) number of yeasts is higher than 10(4)/ml and bacteria are present in low CFU number or are absent. Antifungal susceptibility testing of C. albicans, C. glabrata, C. tropicalis and C. krusei against Flucytosine, Amphotericin-B, Miconazole, Ketoconazole and Fluconazole suggests that Amphotericin-B is still the most effective antifungal agent. Finally, the problems in judging the aetiological role of isolated Candida species in the pathogenesis of different types of diseases are critically discussed.

Preparation and chemical modification of poly-gamma-L-glutamic acid.

Poly-gamma-L-glutamic acid was synthesized in optimized fermentor cultivation of Bacillus licheniformis. The polypeptide obtained was esterified using benzyl- and butylbromides. Benzyl esters with 30, 60 and 100% degree of esterification were prepared. Solubility of the products in dimethyl sulfoxide, chloroform, methanol and in water was significantly different from these ones of original polymer.