Search

Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans. We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napAunder tert-butylhydroperoxide (tBOOH) elicited oxidative stress. Furthermore, gene expression of napAnegatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans. High rsmAexpression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmAand catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmAand napAexpression as well as 0.2 mM tBOOH treatment.

In the ever-changing fungal environment, fungi have to cope with a wide array of very different stresses. These stresses frequently act in combination rather than independently, i.e., they quickly follow one another or occur concomitantly. Combinatorial stress response studies revealed that the response of fungi to a stressor is highly dependent on the simultaneous action of other stressors or even on earlier stresses to which the fungi adapted. Several important phenomena were discovered, such as stress pathway interference, acquired stress tolerance, stress response memory or stress cross-protection/sensitization, which cannot be interpreted when we study the consequences of a single stressor alone. Due to the interactions between stressors and stress responses, a stress response that develops under a combined stress is not the simple summation of stress responses observed during single stress treatments. Based on the knowledge collected from single stress treatment experiments, we cannot predict how fungi will respond to a certain combination of stresses or even whether this combination will be more harmful than single stress treatments. This uncertainty warns us that if we want to understand how fungi adapt to a certain habitat (e.g., to the human body) to find a point of weakness in this adaptation, we must understand how the fungi cope with combinations of stresses, rather than with single stressors.

The fungal genus Aspergillusis of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. We have generated genome sequences for ten novel, highly diverse Aspergillusspecies and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Abstract: Carbon starvation induced autolysis is an active process of self-digestion and is under complex regulation in Aspergillus nidulans. In this study we investigated how autolysis depends on the composition of the culture medium, especially on the presence of yeast extract. We demonstrated that the rate of autolytic cell wall degradation as well as the extracellular chitinase and proteinase productions significantly decreased in the presence of this nutrient. The effect of yeast extract on carbon starved cultures was independent of loss-of-function mutations in the carbon and nitrogen regulatory genes creA and areA and in the heterotrimeric G protein signalling genes fadA and ganB. In contrast, the nitrogen regulating transcription factor MeaB was involved in the yeast-extract-mediated repression of autolysis. Reverse transcriptase — polymerase chain reaction (RT-PCR) experiments demonstrated that MeaB affects the FluG-BrlA sporulation regulatory pathway by affecting transcription of brlA, a gene also initiating the autolytic cell wall degradation in this fungus.

Physiological changes taking place in carbon-starved, autolysing cultures of Aspergillus (Emericella) nidulans strains with mutations in the GanB/RgsA heterotrimeric G protein signalling pathway were studied and compared. Deletion of the ganB, rgsA or both genes did not alter markedly either the autolytic loss of biomass or the extracellular chitinase production. However, they caused a significant decrease in the proteinase formation, which was detected by measuring both extracellular enzyme activity and the transcription of the prtA gene. The deletion mutants also showed significantly higher specific γ -glutamyltranspeptidase activities than the control strain. Deletion of the rgsA gene affected the glutathione peroxidase and catalase formation, as well as the peroxide content of the cells. The concomitant initiations of cell death and developmental genomic programmes may be interconnected via heterotrimeric G-protein signalling and subsequent changes in intracellular ROS levels in ageing A. nidulans. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

ABSTRACTThe small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the β-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulanshyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulansprotoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulansstrain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans ΔflbAand fadAG203Rstrains were studied. Loss-of-function mutation of the flbAgene resulted in an altered germination with unusually thick germination tubes, “fluffy” pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadAG203Rmutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the ΔflbAstrain, whereas the protease and ammonia production of the fadAG203Rmutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of γ-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.

The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulansFGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly, progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulansFGSC26 and a loss-of-function FlbAautolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative effect on the sporulation of strain FGSC26 and the FadAG203Rhypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross autolysis should be avoided.

The glutathione (GSH) S-conjugation of 1,2-epoxy-3-(4'-nitrophenoxy)propane was catalysed predominantly by microsomal glutathione S-transferase (mGST) in Penicillium chrysogenum. The specific mGST activity unlike the cytosolic GST (cGST) activity increased substantially when the penicillin side-chain precursor phenoxyacetic acid (POA) was included in the culture medium. Therefore, a microsomal monooxygenase (causing possible release of epoxide intermediates) and mGST-dependent detoxification pathway may exist for the side-chain precursors as an alternative to microsomal activation to acyl-CoA and subsequent transfer to β-lactam molecules. The P. chrysogenum pahA and Aspergillus nidulans phacA gene products, which are cytochrome P450 monooxygenases and are able to hydroxylate phenylacetic acid (PA) at position 2 on the aromatic ring, are unlikely to release toxic epoxide intermediates but epoxidation of PA and POA due to the action of other microsomal monooxygenases cannot be excluded. The GSH-dependent detoxification of POA was provoked by a well-controlled transient lowering of pH (down to 5.0) at the beginning of the production phase in a fed-batch fermentation system. Both the specific GST and γGT activities were increased but the intracellular GSH concentrations remained unaltered unless the pH of the feed was transiently lowered below 5.0. At pH 4.6, the GSH pool was depleted rapidly but no antibiotic production was observed. Although sucrose was taken up effectively by the cells, cell death and autolysis were progressing. Therefore, the industrial exploitation of the GSH-dependent detoxification of penicillin side-chain precursors to reduce intracellular GSH-levels in order to avoid the GSH inhibition of the β-lactam biosynthetic enzymes seems to be rather unlikely. P. chrysogenum mGST and cGST were separated using GSH-Sepharose 6B affinity chromatography. The purified cGST possessed a homodimer (α2) tertiary structure with Mr, α = 29500.

The stable Cr(VI)-tolerant chr1-66T mutant of Schizosaccharomyces pombe, which carries one simple gene mutation responsible for Cr(VI) tolerance, accumulated and reduced the chromate anion (CrO4^2–) significantly more slowly than did its parental strain 6chr⁺. The mutant chr1-66T proved to be sensitive to oxidative stressors such as H2O2, menadione, tert-butyl hydroperoxide and Cd²⁺. Both the Cr(VI) tolerance and the oxidative stress sensitivity were attributed to a decreased specific glutathione reductase activity. These effects were also enhanced with a decrease in the specific mitochondrial Mn-SOD activity.

The Cr(VI)-sensitive mutant chr-51S of the Schizosaccharomyces pombe accumulated chromate (CrO4^2–) and reduced Cr(V) to much greater extent, than did its parental strain 6 chr⁺. Sublethal doses of K2Cr2O7 did not induce any adaptive stress response, while H2O2 or menadione pretreatment proved protective against the cell injuries caused by Cr(VI). The intracellular GSH concentration in chr-51S cells was approximately half of that for the 6 chr⁺. Moreover, the glutathione disulfide reducing capacity of chr-51S was characterized by significantly increased glutathione reductase (GR) and glucose-6-phosphate dehydrogenase activities. These data strongly suggested that, instead of GSH, the NADPH/GR system was the major one-electron Cr(VI) reductant in vivo. The increased Cr(V) reduction in chr-51S mutant was accompanied by high intracellular superoxide and peroxide concentrations, required for formation of the hydroxyl radical (^•OH). The decreased intracellular GSH levels and the Cr(VI)-sensitive phenotype of the chr-51S cells indicates that GSH might act effectively against chromate by scavenging ^•OH.

In submerged carbon-limited autolysing cultures of Penicillium chrysogenum, round-ended yeast-like fragments became the dominant surviving morphological forms and a new equilibrium between cell lysis and growth was observed, termed as a phase of ‘cryptic growth’. The formation of yeast-like fragments and their cryptic growth represented the only way that enabled the fungus to cope with harsh environmental conditions and survive. The surviving morphological forms reverted to vegetative growth after carbon supplementation. The glutathione metabolism and formation of reactive oxygen species (ROS) were influenced by numerous endogenous and exogenous factors depending on the phase of growth. Consequently, no firm correlation between the redox status, ROS levels and the observed morphological and physiological characteristics of the cells was found. The Dioxygen Avoidance Theory of Cell Differentiation failed to explain the complex physiological changes observed prior to and during autolysis, and any possible intrinsic analogy between the autolysis of P. chrysogenum and the oxidative stress dependent apoptosis of higher eucaryotes was also questioned. Nevertheless, ageing yeast-like fragments accumulated ROS as a function of incubation time and seem to be promising objects for future apoptosis research in filamentous fungi.

In submerged carbon-limited autolysing cultures of Penicillium chrysogenum, round-ended yeast-like fragments became the dominant surviving morphological forms and a new equilibrium between cell lysis and growth was observed, termed as a phase of ‘cryptic growth’. The formation of yeast-like fragments and their cryptic growth represented the only way that enabled the fungus to cope with harsh environmental conditions and survive. The surviving morphological forms reverted to vegetative growth after carbon supplementation. The glutathione metabolism and formation of reactive oxygen species (ROS) were influenced by numerous endogenous and exogenous factors depending on the phase of growth. Consequently, no firm correlation between the redox status, ROS levels and the observed morphological and physiological characteristics of the cells was found. The Dioxygen Avoidance Theory of Cell Differentiation failed to explain the complex physiological changes observed prior to and during autolysis, and any possible intrinsic analogy between the autolysis of P. chrysogenumand the oxidative stress dependent apoptosis of higher eucaryotes was also questioned. Nevertheless, ageing yeast-like fragments accumulated ROS as a function of incubation time and seem to be promising objects for future apoptosis research in filamentous fungi.

Both toxicity and penicillin productivity of the hydroxylated derivatives of phenylacetic acid (PA) and phenoxyacetic acid (POA) were highly dependent on the position of hydroxylation on the aromatic ring in Penicillium chrysogenum. Hydroxylation at position 2 diminished penicillin production but the compounds retained most of their toxicity. On the other hand, hydroxylation at position 4 resulted in barely toxic derivatives with still significant penicillin productivity. 3-Hydroxy-PA was a weak side-chain precursor with considerably reduced toxicity. The activity of the glutathione-dependent detoxification pathway correlated well with the toxicity of the compounds but there was no correlation between acidity, toxicity and penicillin productivity.

Yeastmycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. On the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR),γ-glutamyltranspeptidase (γGT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific γGT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific γGT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Both toxicity and penicillin productivity of the hydroxylated derivatives of phenylacetic acid (PA) and phenoxyacetic acid (POA) were highly dependent on the position of hydroxylation on the aromatic ring in Penicillium chrysogenum. Hydroxylation at position 2 diminished penicillin production but the compounds retained most of their toxicity. On the other hand, hydroxylation at position 4 resulted in barely toxic derivatives with still significant penicillin productivity. 3-Hydroxy-PA was a weak side-chain precursor with considerably reduced toxicity. The activity of the glutathione-dependent detoxification pathway correlated well with the toxicity of the compounds but there was no correlation between acidity, toxicity and penicillin productivity.

Yeastmycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. On the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR),γ-glutamyltranspeptidase (γGT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific γGT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific γGT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Both toxicity and penicillin productivity of the hydroxylated derivatives of phenylacetic acid (PA) and phenoxyacetic acid (POA) were highly dependent on the position of hydroxylation on the aromatic ring in Penicillium chrysogenum. Hydroxylation at position 2 diminished penicillin production but the compounds retained most of their toxicity. On the other hand, hydroxylation at position 4 resulted in barely toxic derivatives with still significant penicillin productivity. 3-Hydroxy-PA was a weak side-chain precursor with considerably reduced toxicity. The activity of the glutathione-dependent detoxification pathway correlated well with the toxicity of the compounds but there was no correlation between acidity, toxicity and penicillin productivity.

Yeastmycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. On the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR),γ-glutamyltranspeptidase (γGT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific γGT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific γGT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Both toxicity and penicillin productivity of the hydroxylated derivatives of phenylacetic acid (PA) and phenoxyacetic acid (POA) were highly dependent on the position of hydroxylation on the aromatic ring in Penicillium chrysogenum. Hydroxylation at position 2 diminished penicillin production but the compounds retained most of their toxicity. On the other hand, hydroxylation at position 4 resulted in barely toxic derivatives with still significant penicillin productivity. 3-Hydroxy-PA was a weak side-chain precursor with considerably reduced toxicity. The activity of the glutathione-dependent detoxification pathway correlated well with the toxicity of the compounds but there was no correlation between acidity, toxicity and penicillin productivity.

Yeastmycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. On the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR),γ-glutamyltranspeptidase (γGT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific γGT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific γGT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Both toxicity and penicillin productivity of the hydroxylated derivatives of phenylacetic acid (PA) and phenoxyacetic acid (POA) were highly dependent on the position of hydroxylation on the aromatic ring in Penicillium chrysogenum. Hydroxylation at position 2 diminished penicillin production but the compounds retained most of their toxicity. On the other hand, hydroxylation at position 4 resulted in barely toxic derivatives with still significant penicillin productivity. 3-Hydroxy-PA was a weak side-chain precursor with considerably reduced toxicity. The activity of the glutathione-dependent detoxification pathway correlated well with the toxicity of the compounds but there was no correlation between acidity, toxicity and penicillin productivity.

Yeastmycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. On the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR),γ-glutamyltranspeptidase (γGT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific γGT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific γGT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

In the idiophase of penicillin V fermentations, the β-lactam biosynthetic machinery of Penicillium chrysogenum is subject to the inhibitory effect of glutathione (GSH) meanwhile the GSH-dependent activation of phenoxyacetic acid (POA) via the formation of (S-phenoxyacetyl)glutathione seems to be unlikely. Our experimental findings suggest that both the intracellular GSH concentrations and the redox capacity of the GSH/GSSG system overweigh the needs raised by the β-lactam production in well-controlled fed-batch penicillin V fermentations. That is, keeping the intracellular GSH levels low, e.g. by the construction of low GSH-producer P. chrysogenum strains, could be beneficial for the antibiotic production. Importantly, its exceptionally potent antioxidant defence system enables the micro-organism to prevent the intracellular accumulation of reactive oxygen species even at low intracellular GSH levels. As a protonophore which is transported across the plasma membrane solely by free diffusion, POA is toxic to P. chrysogenum and accumulates inside mycelia especially when the extracellular pH drops below 6.0. Nevertheless, P. chrysogenum cells eliminate POA quite efficiently most probably via a GSH-dependent pathway under these circumstances.

In the idiophase of penicillin V fermentations, the β-lactam biosynthetic machinery of Penicillium chrysogenum is subject to the inhibitory effect of glutathione (GSH) meanwhile the GSH-dependent activation of phenoxyacetic acid (POA) via the formation of (S-phenoxyacetyl)glutathione seems to be unlikely. Our experimental findings suggest that both the intracellular GSH concentrations and the redox capacity of the GSH/GSSG system overweigh the needs raised by the β-lactam production in well-controlled fed-batch penicillin V fermentations. That is, keeping the intracellular GSH levels low, e.g. by the construction of low GSH-producer P. chrysogenum strains, could be beneficial for the antibiotic production. Importantly, its exceptionally potent antioxidant defence system enables the micro-organism to prevent the intracellular accumulation of reactive oxygen species even at low intracellular GSH levels. As a protonophore which is transported across the plasma membrane solely by free diffusion, POA is toxic to P. chrysogenum and accumulates inside mycelia especially when the extracellular pH drops below 6.0. Nevertheless, P. chrysogenum cells eliminate POA quite efficiently most probably via a GSH-dependent pathway under these circumstances.

In the idiophase of penicillin V fermentations, the β-lactam biosynthetic machinery of Penicillium chrysogenum is subject to the inhibitory effect of glutathione (GSH) meanwhile the GSH-dependent activation of phenoxyacetic acid (POA) via the formation of (S-phenoxyacetyl)glutathione seems to be unlikely. Our experimental findings suggest that both the intracellular GSH concentrations and the redox capacity of the GSH/GSSG system overweigh the needs raised by the β-lactam production in well-controlled fed-batch penicillin V fermentations. That is, keeping the intracellular GSH levels low, e.g. by the construction of low GSH-producer P. chrysogenum strains, could be beneficial for the antibiotic production. Importantly, its exceptionally potent antioxidant defence system enables the micro-organism to prevent the intracellular accumulation of reactive oxygen species even at low intracellular GSH levels. As a protonophore which is transported across the plasma membrane solely by free diffusion, POA is toxic to P. chrysogenum and accumulates inside mycelia especially when the extracellular pH drops below 6.0. Nevertheless, P. chrysogenum cells eliminate POA quite efficiently most probably via a GSH-dependent pathway under these circumstances.

In the idiophase of penicillin V fermentations, the β-lactam biosynthetic machinery of Penicillium chrysogenum is subject to the inhibitory effect of glutathione (GSH) meanwhile the GSH-dependent activation of phenoxyacetic acid (POA) via the formation of (S-phenoxyacetyl)glutathione seems to be unlikely. Our experimental findings suggest that both the intracellular GSH concentrations and the redox capacity of the GSH/GSSG system overweigh the needs raised by the β-lactam production in well-controlled fed-batch penicillin V fermentations. That is, keeping the intracellular GSH levels low, e.g. by the construction of low GSH-producer P. chrysogenum strains, could be beneficial for the antibiotic production. Importantly, its exceptionally potent antioxidant defence system enables the micro-organism to prevent the intracellular accumulation of reactive oxygen species even at low intracellular GSH levels. As a protonophore which is transported across the plasma membrane solely by free diffusion, POA is toxic to P. chrysogenum and accumulates inside mycelia especially when the extracellular pH drops below 6.0. Nevertheless, P. chrysogenum cells eliminate POA quite efficiently most probably via a GSH-dependent pathway under these circumstances.

In the idiophase of penicillin V fermentations, the β-lactam biosynthetic machinery of Penicillium chrysogenum is subject to the inhibitory effect of glutathione (GSH) meanwhile the GSH-dependent activation of phenoxyacetic acid (POA) via the formation of (S-phenoxyacetyl)glutathione seems to be unlikely. Our experimental findings suggest that both the intracellular GSH concentrations and the redox capacity of the GSH/GSSG system overweigh the needs raised by the β-lactam production in well-controlled fed-batch penicillin V fermentations. That is, keeping the intracellular GSH levels low, e.g. by the construction of low GSH-producer P. chrysogenum strains, could be beneficial for the antibiotic production. Importantly, its exceptionally potent antioxidant defence system enables the micro-organism to prevent the intracellular accumulation of reactive oxygen species even at low intracellular GSH levels. As a protonophore which is transported across the plasma membrane solely by free diffusion, POA is toxic to P. chrysogenum and accumulates inside mycelia especially when the extracellular pH drops below 6.0. Nevertheless, P. chrysogenum cells eliminate POA quite efficiently most probably via a GSH-dependent pathway under these circumstances.

The intracellular superoxide and glutathione disulphide concentrations increased in Penicillium chrysogeum treated with 50,250 or 500 μM menadione (MQ). A significant increase in the intracellular peroxide concentration was also observed when mycelia were exposed to 250 or 500 μM MQ. The specific activity of Cu,Zn and Mn superoxide dismutases, glutathione reductase and glutathione S-transferase as well as the glutathione producing activity increased in the presence of MQ while glutathione peroxidase and γ-glutamyltranspeptidase were only induced by high intracellular peroxide levels. The glucose-6-phosphate dehydrogenase and catalase activities did not respond to the oxidative stress caused by MQ.

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3^— and NO2^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3^— and NO2^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3^— and NO2^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3^— and NO2^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3^— and NO2^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

Changes in the de novo glutathione (GSH) synthesis (GSH producing activity; GPA), in the γ-glutamyltranspeptidase (γGT) and GSH reductase (GR) activities as well as in the GSH pools were measured in a high β-lactam producing strain of Penicillium chrysogenum in the presence of different nitrogen, sulphur and carbon sources. When (NH4)2HPO4 was used as a nitrogen source both the specific GPA and the specific γGT activity were significantly lower than those observed with Na-Glu, meanwhile nitrogen starvation increased the γGT activity only. Although neither the addition of ammonium ions nor the withdrawal of nitrogen sources influenced the GSH pool during a 10 h cultivation the addition of the sulphur containing amino acids cysteine (Cys) or methionine (Met) at a concentration of 20 mM increased considerably both the specific GPA and the intracellular GSH levels. Nevertheless, GPA decreased significantly when either Cys or Met was added at a three times higher concentration. When glucose was replaced by lactose as a carbon source or during carbon starvation the GPA values and the GSH concentrations were higher while the GR activities were lower than those observed with glucose. Nevertheless, no changes in either the enzyme activities or the GSH levels were detected when glucose was replaced with 2-deoxy-D-glucose or glucose together with caffeine was added to the culture media.

Changes in the de novo glutathione (GSH) synthesis (GSH producing activity; GPA), in the γ-glutamyltranspeptidase (γGT) and GSH reductase (GR) activities as well as in the GSH pools were measured in a high β-lactam producing strain of Penicillium chrysogenum in the presence of different nitrogen, sulphur and carbon sources. When (NH4)2HPO4 was used as a nitrogen source both the specific GPA and the specific γGT activity were significantly lower than those observed with Na-Glu, meanwhile nitrogen starvation increased the γGT activity only. Although neither the addition of ammonium ions nor the withdrawal of nitrogen sources influenced the GSH pool during a 10 h cultivation the addition of the sulphur containing amino acids cysteine (Cys) or methionine (Met) at a concentration of 20 mM increased considerably both the specific GPA and the intracellular GSH levels. Nevertheless, GPA decreased significantly when either Cys or Met was added at a three times higher concentration. When glucose was replaced by lactose as a carbon source or during carbon starvation the GPA values and the GSH concentrations were higher while the GR activities were lower than those observed with glucose. Nevertheless, no changes in either the enzyme activities or the GSH levels were detected when glucose was replaced with 2-deoxy-D-glucose or glucose together with caffeine was added to the culture media.

Changes in the de novo glutathione (GSH) synthesis (GSH producing activity; GPA), in the γ-glutamyltranspeptidase (γGT) and GSH reductase (GR) activities as well as in the GSH pools were measured in a high β-lactam producing strain of Penicillium chrysogenum in the presence of different nitrogen, sulphur and carbon sources. When (NH4)2HPO4 was used as a nitrogen source both the specific GPA and the specific γGT activity were significantly lower than those observed with Na-Glu, meanwhile nitrogen starvation increased the γGT activity only. Although neither the addition of ammonium ions nor the withdrawal of nitrogen sources influenced the GSH pool during a 10 h cultivation the addition of the sulphur containing amino acids cysteine (Cys) or methionine (Met) at a concentration of 20 mM increased considerably both the specific GPA and the intracellular GSH levels. Nevertheless, GPA decreased significantly when either Cys or Met was added at a three times higher concentration. When glucose was replaced by lactose as a carbon source or during carbon starvation the GPA values and the GSH concentrations were higher while the GR activities were lower than those observed with glucose. Nevertheless, no changes in either the enzyme activities or the GSH levels were detected when glucose was replaced with 2-deoxy-D-glucose or glucose together with caffeine was added to the culture media.

Changes in the de novo glutathione (GSH) synthesis (GSH producing activity; GPA), in the γ-glutamyltranspeptidase (γGT) and GSH reductase (GR) activities as well as in the GSH pools were measured in a high β-lactam producing strain of Penicillium chrysogenum in the presence of different nitrogen, sulphur and carbon sources. When (NH4)2HPO4 was used as a nitrogen source both the specific GPA and the specific γGT activity were significantly lower than those observed with Na-Glu, meanwhile nitrogen starvation increased the γGT activity only. Although neither the addition of ammonium ions nor the withdrawal of nitrogen sources influenced the GSH pool during a 10 h cultivation the addition of the sulphur containing amino acids cysteine (Cys) or methionine (Met) at a concentration of 20 mM increased considerably both the specific GPA and the intracellular GSH levels. Nevertheless, GPA decreased significantly when either Cys or Met was added at a three times higher concentration. When glucose was replaced by lactose as a carbon source or during carbon starvation the GPA values and the GSH concentrations were higher while the GR activities were lower than those observed with glucose. Nevertheless, no changes in either the enzyme activities or the GSH levels were detected when glucose was replaced with 2-deoxy-D-glucose or glucose together with caffeine was added to the culture media.

Changes in the de novo glutathione (GSH) synthesis (GSH producing activity; GPA), in the γ-glutamyltranspeptidase (γGT) and GSH reductase (GR) activities as well as in the GSH pools were measured in a high β-lactam producing strain of Penicillium chrysogenum in the presence of different nitrogen, sulphur and carbon sources. When (NH4)2HPO4 was used as a nitrogen source both the specific GPA and the specific γGT activity were significantly lower than those observed with Na-Glu, meanwhile nitrogen starvation increased the γGT activity only. Although neither the addition of ammonium ions nor the withdrawal of nitrogen sources influenced the GSH pool during a 10 h cultivation the addition of the sulphur containing amino acids cysteine (Cys) or methionine (Met) at a concentration of 20 mM increased considerably both the specific GPA and the intracellular GSH levels. Nevertheless, GPA decreased significantly when either Cys or Met was added at a three times higher concentration. When glucose was replaced by lactose as a carbon source or during carbon starvation the GPA values and the GSH concentrations were higher while the GR activities were lower than those observed with glucose. Nevertheless, no changes in either the enzyme activities or the GSH levels were detected when glucose was replaced with 2-deoxy-D-glucose or glucose together with caffeine was added to the culture media.