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Grape production worldwide is increasingly threatened by grapevine trunk diseases (GTDs). No grapevine cultivar is known to be entirely resistant to GTDs, but susceptibility varies greatly. To quantify these differences, four Hungarian grape germplasm collections containing 305 different cultivars were surveyed to determine the ratios of GTDs based on symptom expression and the proportion of plant loss within all GTD symptoms. The cultivars of monophyletic Vitis vinifera L. origin were amongst the most sensitive ones, and their sensitivity was significantly (p < 0.01) higher than that of the interspecific (hybrid) cultivars assessed, which are defined by the presence of Vitis species other than V. vinifera (e.g., V. labrusca L., V. rupestris Scheele, and V. amurensis Rupr.) in their pedigree. We conclude that the ancestral diversity of grapes confers a higher degree of resilience against GTDs.

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species

Grape production worldwide is increasingly threatened by grapevine trunk diseases (GTDs). No grapevine cultivar is known to be entirely resistant to GTDs, but susceptibility varies greatly. To quantify these differences, four Hungarian grape germplasm collections containing 305 different cultivars were surveyed to determine the ratios of GTDs based on symptom expression and mortality rate. The cultivars of monophyletic Vitis vinifera L. origin were amongst the most sensitive ones, and their sensitivity was significantly (p

Walnut (Juglans regia L.) production is a developing sector in Hungarian horticulture, where preharvest fruit rot increasingly causes economic losses. Previously, fungi from the Diaporthaceae and Botryosphaeriaceae families were isolated with a high incidence from rotten fruits. Here, we identify these pathogens from different plant parts (twigs, buds, and shoots) in orchards in the north-east of Hungary, and test their pathogenicity on immature nuts. Diaporthe eres Nitschke and Diplodia seriata De Notaris were identified in isolates from different symptomatic and asymptomatic plant parts based on their ITS (nuclear ribosomal internal transcribed spacer) and tef1 (translation elongation factor EF-1-alpha gene) DNA sequences. Purified monocultures caused rot of immature nuts following in vitro inoculation. Our results suggest that D. eres Nitschke and D. seriata De Notaris lingering on buds and overwintering woody parts may affect the seasonal nuts through wound infection. Infection by Diaporthe and Botryosphaeriaceae species present on woody plant parts, as well as on/in buds has been reported in Mediterranean countries. This is the first report of such stepwise aetiology from a region with continental weather. Climate change, associated weather patterns and the limitations of fungicide use in the European Union, among other factors, could be responsible for the increasing number of infections and economic damage caused by these pathogens.

In the first part of this two-piece publication, the isolation, identification and in vitro characterization of ten endophytic Trichoderma isolates were reported. Here we report the ability of two different mixes of some of these isolates (Trichoderma simmonsii, Trichoderma orientale and Trichoderma gamsii as well as of Trichoderma afroharzianum and T. simmonsii) to colonize and stimulate the growth of grapevines. Two commercial vineyards about 400 km away from the site of isolation were used as experimental fields, from which the strains of three Trichoderma species were re-isolated up to four years after rootstock soaking treatment with conidiospores, performed before planting. The treatments decreased the overall percentage of lost plants of about 30%, although a low number of lost plants (about 5%) were observed also in the control plot. For all cultivars and clones, the Trichoderma treatments significantly increased both the bud burst ratio and bud burst vigor index. In addition, the grape must parameters such as the Brix degrees, as well as the extract, the D-glucose and the D-fructose concentrations all appeared to be improved, suggesting a potentially higher ethanol content of the produced wine. We conclude that grapevine-endophytic Trichoderma isolates promote plant growth, which could be a useful feature for sustainable agriculture in general and integrated plant production in particular.

High-yield citric acid production by the filamentous Ascomycete fungus

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s).

Organic acid accumulation is probably the best-known example of primary metabolic overflow. Both bacteria and fungi are capable of producing various organic acids in large amounts under certain conditions, but in terms of productivity-and consequently, of commercial importance-fungal platforms are unparalleled. For high product yield, chemical composition of the growth medium is crucial in providing the necessary conditions, of which the concentrations of four of the first-row transition metal elements, manganese (Mn2+), iron (Fe2+), copper (Cu2+) and zinc (Zn2+) stand out. In this paper we critically review the biological roles of these ions, the possible biochemical and physiological consequences of their influence on the accumulation of the most important mono-, di- and tricarboxylic as well as sugar acids by fungi, and the metal ion-related aspects of submerged organic acid fermentations, including the necessary instrumental analytics. Since producing conditions are associated with a cell physiology that differs strongly to what is observed under “standard” growth conditions, here we consider papers and patents only in which organic acid accumulation levels achieved at least 60% of the theoretical maximum yield, and the actual trace metal ion concentrations were verified.

The effects of the interplay of copper(II) and manganese(II) ions on growth, morphology and itaconic acid formation was investigated in a high-producing strain of Aspergillus terreus (NRRL1960), using carbon sources metabolized either mainly via glycolysis (D-glucose, D-fructose) or primarily via the pentose phosphate shunt (D-xylose, L-arabinose). Limiting Mn2+ concentration in the culture broth is indispensable to obtain high itaconic acid yields, while in the presence of higher Mn2+ concentrations yield decreases and biomass formation is favored. However, this low yield in the presence of high Mn2+ ion concentrations can be mitigated by increasing the Cu2+ concentration in the medium when D-glucose or D-fructose is the growth substrate, whereas this effect was at best modest during growth on D-xylose or L-arabinose. A. terreus displays a high tolerance to Cu2+ which decreased when Mn2+ availability became increasingly limiting. Under such conditions biomass formation on D-glucose or D-fructose could be sustained at concentrations up to 250 mg L–1 Cu2+, while on D-xylose- or L-arabinose biomass formation was completely inhibited at 100 mg L–1. High (>75%) specific molar itaconic acid yields always coincided with an “overflow-associated” morphology, characterized by small compact pellets (

This paper reports on the identification and in vitro characterization of several Trichoderma strains isolated from the Tokaj Wine Region in North-East Hungary. Ten isolates were analyzed and found to consist of six individual species—T. gamsii, T. orientale, T. simmonsii, T. afroharzianum, T. atrobrunneum and T. harzianum sensu stricto. The growth potential of the strains was assessed at a range of temperatures. We also report here on the in vitro biocontrol properties and fungicide tolerance of the most promising strains.

Itaconic acid is used as a bio-based, renewable building block in the polymer industry. It is produced by submerged fermentations of the filamentous fungus Aspergillus terreus from molasses or starch, but research over the efficient utilization of non-food, lignocellulosic plant biomass is soaring. The objective of this study was to test whether the application of two key cultivation parameters for obtaining itaconic acid from D-glucose in high yields – Mn2+ ion deficiency and high concentration of the carbon source – would also occur on D-xylose, the principal monomer of lignocellulose. To this end, a carbon and energy balance for itaconic acid formation was established, which is 0.83 moles/mole D-xylose. The effect of Mn2+ ions on itaconic acid formation was indeed similar to that on D-glucose and maximal yields were obtained below 3 μg L–1 Mn2+ ions, which were, however, only 0.63 moles of itaconic acid per mole D-xylose. In contrast to the case on D-glucose, increasing D-xylose concentration over 50 g L–1 did not change the above yield. By-products such as xylitol and α-ketoglutarate were found, but in total they remained below 2% of the concentration of D-xylose. Mass balance of the fermentation with 110 g L–1 D-xylose revealed that >95% of the carbon from D-xylose was accounted as biomass, itaconic acid, and the carbon dioxide released in the last step of itaconic acid biosynthesis. Our data show that the efficiency of biomass formation is the critical parameter for itaconic acid yield from D-xylose under otherwise optimal conditions.

In this study, we analyzed the expression of the structural genes encoding the five enzymes comprising the Leloir pathway of D-galactose catabolism in the industrial cell factory Penicillium chrysogenum on various carbon sources. The genome of P. chrysogenum contains a putative galactokinase gene at the annotated locus Pc13g10140, the product of which shows strong structural similarity to yeast galactokinase that was expressed on lactose and D-galactose only. The expression profile of the galactose-1-phosphate uridylyl transferase gene at annotated locus Pc15g00140 was essentially similar to that of galactokinase. This is in contrast to the results from other fungi such as Aspergillus nidulans, Trichoderma reesei and A. niger, where the ortholog galactokinase and galactose-1-phosphate uridylyl transferase genes were constitutively expressed. As for the UDP-galactose-4-epimerase encoding gene, five candidates were identified. We could not detect Pc16g12790, Pc21g12170 and Pc20g06140 expression on any of the carbon sources tested, while for the other two loci (Pc21g10370 and Pc18g01080) transcripts were clearly observed under all tested conditions. Like the 4-epimerase specified at locus Pc21g10370, the other two structural Leloir pathway genes - UDP-glucose pyrophosphorylase (Pc21g12790) and phosphoglucomutase (Pc18g01390) - were expressed constitutively at high levels as can be expected from their indispensable function in fungal cell wall formation.

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light.

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light.

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light.

Itaconic acid is a five-carbon dicarboxylic acid with an unsaturated alkene bond, frequently used as a building block for the industrial production of a variety of synthetic polymers. It is also one of the major products of fungal "overflow metabolism" which can be produced in submerged fermentations of the filamentous fungus Aspergillus terreus. At the present, molar yields of itaconate are lower than those obtained in citric acid production in Aspergillus niger. Here, we have studied the possibility that the yield may be limited by the oxygen supply during fermentation and hence tested the effect of the dissolved oxygen concentration on the itaconic acid formation rate and yield in lab-scale bioreactors. The data show that a dissolved oxygen concentration of 2% saturation was sufficient for maximal biomass formation. Raising it to 30% saturation had no effect on biomass formation or the growth rate, but the itaconate yield augmented substantially from 0.53 to 0.85 mol itaconate/mol glucose. Furthermore, the volumetric and specific rates of itaconic acid formation ameliorated by as much as 150% concurrent with faster glucose consumption, shortening the fermentation time by 48 h. Further increasing the dissolved oxygen concentration over 30% saturation had no effect. Moreover, we show that this increase in itaconic acid production coincides with an increase in alternative respiration, circumventing the formation of surplus ATP by the cytochrome electron transport chain, as well as with increased levels of alternative oxidase transcript. We conclude that high(er) itaconic acid accumulation requires a dissolved oxygen concentration that is much higher than that needed for maximal biomass formation, and postulate that the induction of alternative respiration allows the necessary NADH reoxidation ratio without surplus ATP production to increase the glucose consumption and the flux through overflow metabolism.

Aldose 1-epimerases or mutarotases (EC 5.1.3.3) are catalyzing the interconversion of α- and β-anomers of hemiacetals of aldose sugars such as D-glucose and D-galactose, and are presumed to play an auxiliary role in carbohydrate metabolism as mutarotation occurs spontaneously in watery solutions. The first step in the Leloir pathway of D-galactose breakdown is preceded by accelerated conversion of β-D-galactopyranose into the α-anomer, the substrate of the anomer-specific D-galactose 1-kinase. Here, we identified two putative aldose-1-epimerase genes (galmA and galmB) in the model organism Aspergillus nidulans, and characterized them upon generation of single- and double deletion mutant strains, as well as overexpressing mutants carrying multiple copies of either. Assaying cell-free extracts from the galmB single- and galm double mutants, we observed that the mutarotation hardly exceeded spontaneous anomer conversion, while galmB multicopy strains displayed higher activities than the wild type, increasing with the copy number. When grown on D-galactose in submerged cultures, biomass formation and D-galactose uptake rates in mutants lacking galmB were considerably reduced. None such effects were observed studying galmA deletion mutants, which consistently behave like the wild type. We conclude that GalmB is the physiologically relevant mutarotase for the utilization of D-galactose in A. nidulans.

[Background] The fungal genus Aspergillus is 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., [Results] We have generated genome sequences for ten novel, highly diverse Aspergillus species 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., [Conclusions] 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., The sugar transporter analysis was supported by grants AGL2011-29925 and AGL2015-66131- AGL2015-66131-C2-2-R (MINECO/FEDER).

Spliceosomal introns can occupy nearby rather than identical positions in orthologous genes (intron sliding or shifting). Stwintrons are complex intervening sequences, where an ‘internal’ intron interrupts one of the sequences essential for splicing, generating after its excision, a newly formed canonical intron defined as ‘external’. In one experimentally demonstrated configuration, two alternatively excised internal introns, overlapping by one G, disrupt respectively the donor and the acceptor sequence of an external intron, leading to mRNAs encoding identical proteins. In a gene encoding a DHA1 antiporter in Pezizomycotina, we find a variety of predicted intron configurations interrupting the DNA stretch encoding a conserved peptidic sequence. Some sport a stwintron where the internal intron interrupts the donor of the external intron (experimentally confirmed for Aspergillus nidulans). In others, we found and demonstrate (for Trichoderma reesei) alternative, overlapping internal introns. Discordant canonical introns, one nt apart, are present in yet other species, exactly as predicted by the alternative loss of either of the internal introns at the DNA level from an alternatively spliced stwintron. An evolutionary pathway of 1 nt intron shift, involving an alternatively spliced stwintron intermediate is proposed on the basis of the experimental and genomic data presented.

Background In the primary transcript of nuclear genes, coding sequences—exons—usually alternate with non-coding sequences—introns. In the evolution of spliceosomal intron–exon structure, extant intron positions can be abandoned and new intron positions can be occupied. Spliceosomal twin introns (“stwintrons”) are unconventional intervening sequences where a standard “internal” intron interrupts a canonical splicing motif of a second, “external” intron. The availability of genome sequences of more than a thousand species of fungi provides a unique opportunity to study spliceosomal intron evolution throughout a whole kingdom by means of molecular phylogenetics. Results A new stwintron was encountered in Aspergillus nidulans and Aspergillus niger. It is present across three classes of Leotiomyceta in the transcript of a well-conserved gene encoding a putative lipase (lipS). It occupies the same position as a standard intron in the orthologue gene in species of the early divergent classes of the Pezizomycetes and the Orbiliomycetes, suggesting that an internal intron has appeared within a pre-extant intron. On the other hand, the stwintron has been lost from certain taxa in Leotiomycetes and Eurotiomycetes at several occasions, most likely by a mechanism involving reverse transcription and homologous recombination. Another ancient stwintron present across whole Pezizomycotina orders—in the transcript of the bifunctional biotin biosynthesis gene bioDA—occurs at the same position as a standard intron in many species of non-Dikarya. Nevertheless, also the bioDA stwintron has disappeared from certain lineages within the taxa where it occurs, i.e., Sordariomycetes and Botryosphaeriales. Intriguingly, only the internal intron was lost from the Sordariomycetes bioDA stwintron at all but one occasion, leaving a standard intron in the same position, while where the putative lipase stwintron was lost, no intronic sequences remain. Conclusions Molecular phylogeny of the peptide product was used to monitor the existence and fate of a stwintron in the transcripts of two neatly defined fungal genes, encoding well conserved proteins. Both defining events—stwintron emergence and loss—can be explained with extant models for intron insertion and loss. We thus demonstrate that stwintrons can serve as model systems to study spliceosomal intron evolution. Electronic supplementary material The online version of this article (doi:10.1186/s40694-017-0037-y) contains supplementary material, which is available to authorized users.

In this study, we analyze the expression of the Aspergillus nidulans bgaD-lacpA gene couple (encoding an intracellular beta-galactosidase and a lactose permease) in the presence of d-galactose. This monosaccharide can be catabolized via alternative, independent pathways in this model organism. The inductive capabilities of intermediates of the two alternative routes of d-galactose utilization were addressed in loss-of-function mutants defective in a defined step in one of the two pathways. In a galactokinase ( galE9 ) mutant, the cluster is strongly induced by d-galactose, suggesting that formation of Leloir pathway intermediates is not required. The expression profiles of bgaD and lacpA were similar in wild type, l-arabinitol dehydrogenase ( araA1 ), and hexokinase ( hxkA1 ) negative backgrounds, indicating that intermediates of the oxido-reductive pathway downstream of galactitol are not necessary either. Furthermore, bgaD-lacpA transcription was not induced in any of the tested strains when galactitol was provided as the growth substrate. An hxkA1/galE9 double mutant cannot grow on d-galactose at all, but still produced bgaD and lacpA transcripts upon transfer to d-galactose. We therefore concluded that the physiological inducer of the bgaD-lacpA gene cluster upon growth on d-galactose is the nonmetabolized sugar itself.

Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis.

In Aspergillus nidulans, uptake rather than hydrolysis is the rate-limiting step of lactose catabolism. Deletion of the lactose permease A-encoding gene (lacpA) reduces the growth rate on lactose, while its overexpression enables faster growth than wild-type strains are capable of. We have identified a second physiologically relevant lactose transporter, LacpB. Glycerol-grown mycelia from mutants deleted for lacpB appear to take up only minute amounts of lactose during the first 60 h after a medium transfer, while mycelia of double lacpA/lacpB-deletant strains are unable to produce new biomass from lactose. Although transcription of both lacp genes was strongly induced by lactose, their inducer profiles differ markedly. lacpA but not lacpB expression was high in d-galactose cultures. However, lacpB responded strongly also to β-linked glucopyranose dimers cellobiose and sophorose, while these inducers of the cellulolytic system did not provoke any lacpA response. Nevertheless, lacpB transcript was induced to higher levels on cellobiose in strains that lack the lacpA gene than in a wild-type background. Indeed, cellobiose uptake was faster and biomass formation accelerated in lacpA deletants. In contrast, in lacpB knockout strains, growth rate and cellobiose uptake were considerably reduced relative to wild-type, indicating that the cellulose and lactose catabolic systems employ common elements. Nevertheless, our permease mutants still grew on cellobiose, which suggests that its uptake in A. nidulans prominently involves hitherto unknown transport systems.

Nitrosomonas europaea is a chemolithoautotrophic nitrifier, a gram-negative bacterium that can obtain all energy required for growth from the oxidation of ammonia to nitrite, and this may be beneficial for various biotechnological and environmental applications. However, compared to other bacteria, growth of ammonia oxidizing bacteria is very slow. A prerequisite to produce high cell density N. europaea cultures is to minimize the concentrations of inhibitory metabolic by-products. During growth on ammonia nitrite accumulates, as a consequence, N. europaea cannot grow to high cell concentrations under conventional batch conditions. Here, we show that single-vessel dialysis membrane bioreactors can be used to obtain substantially increased N. europaea biomasses and substantially reduced nitrite levels in media initially containing high amounts of the substrate. Dialysis membrane bioreactor fermentations were run in batch as well as in continuous mode. Growth was monitored with cell concentration determinations, by assessing dry cell mass and by monitoring ammonium consumption as well as nitrite formation. In addition, metabolic activity was probed with in vivo acridine orange staining. Under continuous substrate feed, the maximal cell concentration (2.79 × 10(12)/L) and maximal dry cell mass (0.895 g/L) achieved more than doubled the highest values reported for N. europaea cultivations to date.

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.

International audience; Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

Botrytis cinerea has been reported as a species complex containing two cryptic species, groups I (Botrytis pseudocinerea) and II (B. cinerea sensu stricto). In order to compare the pathogenicity of group I and group II of B. cinerea, we have selected 4 strains of group I and 4 strains of group II. The results demonstrated that competitive infection of group II was more on grape, cucumber and paprika leaves, than group I. However the results on bean leaves did not correlate the applied B. cinerea group.

The genes being in the mitochondrial DNA primarily encode the enzymes of cellular respiration. Fungicides belonging to the family of quinol oxidase inhibitors (QoIs) play on important role in the protection against several plant diseases caused by fungi. These fungicides bind to the cytochrome bc1 complex so they block electron transport between cytochrome b and cytochrome c1. This way these fungicides inhibit the ATP synthesis consequently they inhibit the mitochondrial respiration. The QoI resistance has two mechanisms. One of them is the point mutation of the cytochrome b gene (CYTB), e.g. the substitution of a single glycine by alanine at position 143 results in high-resistance. The other is the cyanide-resistant alternative respiration sustained by the alternative oxidase.In a cell there are several mitochondria. The phenomenon when the genomes of all mitochondria in the cell are identical is called homoplazmy. If in the cell there is wild and mutant mitochondrial DNA this is called heteroplasmy. Whether the mutation in the mitochondria causes fenotypical diversity or does not depend on the dose, i.e. it depends on the percentage of the changed mitochondrials. During our work we investigated Botrytis cinerea single spore isolates which have been collected in 2008-2009 on different host plants. Our goal was to decide whether heteroplasmy influences the level of resistance. We managed to detect the change of the level of heteroplasmy, so the change the level of the resistance due to the treatment with fungicide.

In the mitochondrion of eukaryotes, cytochrome b is a component of respiratory chain complex III. Cytochrome b is encoded by thecytochrome b (CYTB) gene located in the mitochondrial genome. The fungicidal activity of QoIs relies on their ability to inhibit mitochondrial respiration by binding at the so-called Qo site (the outer quinol-oxidation site) of the complex III. Since their introduction, QoIs (like azoxystrobin) have become essential components of plant disease control programs because of their wide-ranging efficacy against many agriculturally important fungal diseases like grey mould on various crops. QoI resistance primarily arises from a target-site-based mechanism involving mutations in the mitochondrial CYTB. As the management of grey mould is often dependent on chemicals, the rational design of control programs requires the information about the diversity of genes connected with resistance in field populations of the pathogen.Monospore B. cinerea field isolates has been collected during 2008-2009 from different hosts in Hungary. PCR fragment length analysisindicated the high frequency presence of type large intron in the isolates while in a few strains G143A substitution could also be detected.These results indicated the heterogeneity of CYTB in the Hungarian B. cinerea populations, which possibly involve the heteroplasmy of thismitochondrial gene, moreover indicates the existence op azoxystrobin resistant populations in Hungary.This work was supported by NKFP-A2-2006/0017 grant. Erzsébet Fekete is a grantee of the János Bolyai Scholarship (BO/00519/09/8).

Botrytis cinerea (anamorph of Botryotinia fuckeliana) causes gray mold on a high number of crop plants including grapes. In this study, we investigated the genetic properties of a grape pathogenic population of B. cinerea in the area of Eger, Hungary. A total of 109 isolates from 12 areas were sampled. Based on the sequence of the β-tubulin (tub1) locus, they all belong to group II, a phylogenetic species within B. cinerea. Seventy-four isolates were classified as transposa, with both the Flipper and Boty transposons, and 10 were classified as vacuma, lacking both transposons. The remaining isolates contained either only Flipper (13) or Boty (12). Multilocus analysis of sequences from tub1 and two other loci (elongation factor 1-alpha, tef1, and a minisatellite from the intron of an ATPase, MSB1) led to poor phylogenetic resolution of strains in individual clades. Analysis of five microsatellites (Bc2, Bc3, Bc5, Bc6, and Bc10) resulted in 55 microsatellite haplotypes within the 109 strains. No correlation was detected among individual haplotypes and the presence/absence of Flipper and/or Boty, the geographic origin, or the year of isolation. Application of the index of association, the chi-square test, and the phi test consistently indicated that the population of Hungarian isolates of B. cinerea undergoes sexual reproduction. However, the index of association test suggested the presence of some clonality, and the fixation index showed a low or occasionally moderate level of fixation in the Flipper populations. We conclude that the B. cinerea populations in Hungary consist of a strongly recombining group II phylogenetic species.

The disaccharide lactose is a byproduct of cheese production accumulating to amounts of 800,000 tons per year worldwide, of which 15% is used as a carbon source for various microbial fermentations. Nevertheless, little is known about the regulation of its metabolism in filamentous fungi. Lactose is metabolized slowly, and some important fungi such as A. niger cannot use it at all. A more detailed knowledge on the rate-limiting steps would be helpful to improve its industrial application. We have chosen A. nidulans as an object for investigating how lactose and galactose metabolism are regulated because it has long become a model system for biochemical and genetic research on fungi, and mutants in the lactose-metabolizing pathway of A. nidulans are available. In this paper, we will review the contributions of our research group achieved on this field.

The heterodisaccharide lactose (1,4-O-β- d -galactopyranosyl- d -glucose) induces cellulase formation in the ascomycete Hypocrea jecorina (= Trichoderma reesei ). Lactose assimilation is slow, and the assimilation of its β- d -galactose moiety depends mainly on the operation of a recently described reductive pathway and depends less on the Leloir pathway, which accepts only α- d -galactose. We therefore reasoned whether galactomutarotase [aldose 1-epimerase (AEP)] activity might limit lactose assimilation and thus influence cellulase formation. We identified three putative AEP-encoding genes ( aep1 , aep2 , aep3 ) in H. jecorina , of which two encoded intracellular protein (AEP1 and AEP2) and one encoded an extracellular protein (AEP3). Although all three were transcribed, only the aep3 transcript was detected on lactose. However, no mutarotase activity was detected in the mycelia, their cell walls, or the extracellular medium during growth on lactose. Therefore, the effect of galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal galactose mutarotase part of the Saccharomyces cerevisiae Gal10. These strains showed increased growth on lactose in a gene copy number-dependent manner, although their formation of extracellular β-galactosidase activity and transcription of the genes encoding the first steps in the Leloir and the reductive pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the β-anomer of d -galactose and reveal the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.

The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 beta-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and d-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (=specific growth) rates. The data showed that bga1 expression by lactose, d-galactose and galactitol positively correlated with the dilution rate, and that galactitol and d-galactose induced the highest activities of beta-galactosidase at comparable growth rates. To know more about the actual inducer for beta-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two d-galactose-degrading pathways was compared. Induction by d-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir pathway of d-galactose catabolism. However, in a strain deleted in the aldose/d-xylose reductase gene xyl1, which performs the reduction of d-galactose to galactitol in a recently identified second pathway, induction by d-galactose, but not by galactitol, was impaired. On the other hand, induction by d-galactose and galactitol was not affected in an l-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on d-galactose in H. jecorina.

Itaconic acid (IA), an unsaturated dicarboxylic acid with a high potential as a platform for chemicals derived from sugars, is industrially produced by large-scale submerged fermentation by Aspergillus terreus. Although the biochemical pathway and the physiology leading to IA is almost the same as that leading to citric acid production in Aspergillus niger, published data for the volumetric (g L(-1)) and the specific yield (mol/mol carbon source) of IA are significantly lower than for citric acid. Citric acid is known to accumulate to high levels only when a number of nutritional parameters are carefully adjusted, of which the concentration of the carbon source and that of manganese ions in the medium are particularly important. We have therefore investigated whether a variation in these two parameters may enhance IA production and yield by A. terreus. We show that manganese ion concentrations 3 ppb are necessary to obtain highest yields. Highest yields were also dependent on the concentration of the carbon source (D-glucose), and highest yields (0.9) were only obtained at concentrations of 12-20 % (w/v), thus allowing the accumulation of up to 130 g L(-1) IA. These findings perfectly mirror those obtained when these parameters are varied in citric acid production by A. niger, thus showing that the physiology of both processes is widely identical. Consequently, applying the fermentation technology established for citric acid production by A. niger citric acid production to A. terreus should lead to high yields of IA, too.

Carbon catabolite repression by the CreA-transcriptional repressor is widespread in filamentous fungi, but the mechanism by which glucose triggers carbon catabolite repression is still poorly understood. We investigated the hypothesis that the growth rate on glucose may control CreA-dependent carbon catabolite repression by using glucose-limited chemostat cultures and the intracellular beta-galactosidase activity of Aspergillus nidulans, which is repressed by glucose, as a model system. Chemostat cultures at four different dilution rates (D = 0.095, 0.068, 0.045 and 0.015 h-1) showed that formation of beta-galactosidase activity is repressed at the two highest Ds, but increasingly derepressed at the lower Ds, the activity at 0.015 h-1 equalling that in derepressed batch cultures. Chemostat cultures with the carbon catabolite derepressed A. nidulans mutant strain creADelta4 revealed a dilution-rate independent constant beta-galactosidase activity of the same range as that found in the wild-type strain at D = 0.015 h-1. Two other enzymes--isocitrate lyase, which is almost absent on glucose due to a CreA-independent mechanism; and galactokinase, which is formed constitutively and independent of CreA--were measured as controls. They were formed at constant activity at each dilution rate, both in the wild-type strain as well as in the carbon catabolite derepressed mutant strain. We conclude that the growth rate on glucose is a determinant of carbon catabolite repression in A. nidulans, and that below a certain growth rate carbon catabolite derepression occurs.

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.

The relationship between oxygen input and activity of the cyanide-resistant alternative respiration of submerged cultures of Acremonium crysogenum was investigated. The volumetric oxygen transfer coefficient of the respective cultures correlated positively within almost two ranges of magnitude with the size of the intracellular peroxide pool, which in turn, correlated with the activity of the cyanide-resistant alternative respiratory pathway. Increased aeration also stimulated the glucose uptake rate but had no effect on the total respiration rate or the growth rate. Addition of the lipid peroxyl radical scavenger DL-α-tocopherol to A. chrysogenum cultures decreased the rate of intracellular peroxide production as well as glucose uptake. An increase in the cyanide-resistant fraction of total respiration was observed, while growth and the total respiratory activity remained unchanged. We conclude that intracellular peroxides may stimulate the alternative respiration in A. chrysogenum.Key words: Acremonium chrysogenum, alternative respiration, oxygen, peroxide, Kla.

Background The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi. In Trichoderma reesei, its ortholog LAE1 appears to predominantly regulate genes involved in increasing competitive fitness in its environment, including expression of cellulases and polysaccharide hydrolases. A drawback in all studies related to LaeA/LAE1 function so far, however, is that the respective loss-of-function and overexpressing mutants display different growth rates. Thus some of the properties attributed to LaeA/LAE1 could be simply due to changes of the growth rate. Results We cultivated T. reesei, a Δlae1 mutant and a lae1-overexpressing strain in chemostats on glucose at two different growth rates (0.075 and 0.020 h-1) which resemble growth rates at repressing and derepressing conditions, respectively. Under these conditions, the effect of modulating LAE1 expression was mainly visible in the Δlae1 mutant, whereas the overexpressing strain showed little differences to the parent strain. The effect on the expression of some gene categories identified earlier (polyketide synthases, heterokaryon incompatibility proteins, PTH11-receptors) was confirmed, but in addition GCN5-N-acetyltransferases, amino acid permeases and flavin monooxygenases were identified as so far unknown major targets of LAE1 action. LAE1 was also shown to interfere with the regulation of expression of several genes by the growth rate. About a tenth of the genes differentially expressed in the Δlae1 mutant under either growth condition were found to be clustered in the genome, but no specific gene group was associated with this phenomenon. Conclusions Our data show that – using T. reesei LAE1 as a model - the investigation of transcriptome in regulatory mutants at constant growth rates leads to new insights into the physiological roles of the respective regulator. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-447) contains supplementary material, which is available to authorized users.

Fungi, in particular Aspergilli, are well known for their potential to overproduce a variety of organic acids. These microorganisms have an intrinsic ability to accumulate these substances and it is generally believed that this provides the fungi with an ecological advantage, since they grow rather well at pH 3 to 5, while some species even tolerate pH values as low as 1.5. Organic acid production can be stimulated and in a number of cases conditions have been found that result in almost quantitative conversion of carbon substrate into acid. This is exploited in large-scale production of a number of organic acids like citric-, gluconic- and itaconic acid. Both in production volume as well as in knowledge available, citrate is by far the major organic acid. Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is a true bulk product with an estimated global production of over 900 thousand tons in the year 2000. Till the beginning of the 20th century, it was exclusively extracted from lemons. Since the global market was dominated by an Italian cartel, other means of production were sought. Chemical synthesis was possible, but not suitable due to expensive raw materials and a complicated process with low yield. The discovery of citrate accumulation by Aspergillus niger led to a rapid development of a fermentation process, which only a decade later accounted for a large part of the global production. The application of citric acid is based on three of its properties: (1) acidity and buffer capacity, (2) taste and flavour, and (3) chelation of metal ions. Because of its three acid groups with pKa values of 3.1, 4.7 and 6.4, citrate is able to produce a very low pH in solution, but is also useful as a buffer over a broad range of pH values (2 to 7). Citric acid has a pleasant acid taste which leaves little aftertaste. It sometimes enhances flavour, but is also able to mask sweetness, such as the aspartame taste in diet beverages. Chelation of metal ions is a very important property that has led to applications such as antioxidant and preservative. Moreover, it is a "natural" substance and fully biodegradable.

International audience; The spliceosome is an RNA/protein complex, responsible for intron excision from eukaryotic nuclear transcripts. In bacteria, mitochondria and plastids, intron excision does not involve the spliceosome, but occurs through mechanisms dependent on intron RNA secondary and tertiary structure. For group II/III chloroplast introns, "twintrons" (introns within introns) have been described. The excision of the external intron, and thus proper RNA maturation, necessitates prior removal of the internal intron, which interrupts crucial sequences of the former. We have here predicted analogous instances of spliceosomal twintrons ("stwintrons") in filamentous fungi. In two specific cases, where the internal intron interrupts the donor of the external intron after the first or after the second nucleotide, respectively, we show that intermediates with the sequence predicted by the "stwintron" hypothesis, are produced in the splicing process. This implies that two successive rounds of RNA scanning by the spliceosome are necessary to produce the mature mRNA. The phylogenetic distributions of the stwintrons we have identified suggest that they derive from "late" events, subsequent to the appearance of the host intron. They may well not be limited to fungal nuclear transcripts, and their generation and eventual disappearance in the evolutionary process are relevant to hypotheses of intron origin and alternative splicing.

Lactose (1,4-0-β-d-galactopyranosyl-d-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for - among other purposes - use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography-mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-β-1,6-Gal-β-1,4-Glc/Fru and Gal-β-1,4-Gal-β-1,4-Glc/ Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-β-1,3/1,4/1,6-Gal; Gal-β-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-β-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-β-1,4-Gal-β-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation. © 2013 Springer-Verlag Berlin Heidelberg.

International audience; The necrotrophic fungus Botrytis cinerea is reported to infect more than 220 host plants worldwide. In phylogenetical-taxonomical terms, the pathogen is considered a complex of two cryptic species, group I and group II. We sampled populations of B. cinerea on sympatric strawberry and raspberry cultivars in the North-East of Hungary for three years during flowering and the harvest period. Four hundred and ninety group II B. cinerea isolates were analyzed for the current study. Three different data sets were generated: (i) PCR-RFLP patterns of the ADP-ATP translocase and nitrate reductase genes, (ii) MSB1 minisatellite sequence data, and (iii) the fragment sizes of five microsatellite loci. The structures of the different populations were similar as indicated by Nei's gene diversity and haplotype diversity. The F statistics (Fst, Gst), and the gene flow indicated ongoing differentiation within sympatric populations. The population genetic parameters were influenced by polymorphisms within the three data sets as assessed using Bayesian algorithms. Data Mining analysis pointed towards the five microsatellite loci as the most defining markers to study differentiation in the 490 isolates. The results suggest the occurrence of host-specific, sympatric divergence of generalist phytoparasites in perennial hosts.

Lactose is intracellularly hydrolysed by Aspergillus nidulans. Classical mutation mapping data and the physical characteristics of the previously purified glycosyl hydrolase facilitated identification of the clustered, divergently transcribed intracellular β-galactosidase (bgaD) and lactose permease (lacpA) genes. At the transcript level, bgaD and lacpA were coordinately expressed in response to d-galactose, lactose or l-arabinose, while no transcription was detectable in the additional presence of glucose. In contrast, creA loss-of-function mutants derepressed for both genes to a considerable extent (even) under non-inducing or repressing growth conditions. Lactose- and d-galactose induction nevertheless occurred only in the absence of glucose, indicating a regulatory role for CreA-independent repression. Remarkably, bgaD deletion mutants grew normal on lactose. In contrast, lacpA deletants grew at a much slower rate in lactose liquid medium than wild-type while strains that carried more than one copy of lacpA grew faster, showing that transport is the limiting step in lactose catabolism. The effect of lacpA gene deletion on lactose uptake was exacerbated at lower substrate concentrations, evidence for the existence of a second transport system with a lower affinity for this disaccharide in A. nidulans.

Background The identification and characterization of the transcriptional regulatory networks governing the physiology and adaptation of microbial cells is a key step in understanding their behaviour. One such wide-domain regulatory circuit, essential to all cells, is carbon catabolite repression (CCR): it allows the cell to prefer some carbon sources, whose assimilation is of high nutritional value, over less profitable ones. In lower multicellular fungi, the C2H2 zinc finger CreA/CRE1 protein has been shown to act as the transcriptional repressor in this process. However, the complete list of its gene targets is not known. Results Here, we deciphered the CRE1 regulatory range in the model cellulose and hemicellulose-degrading fungus Trichoderma reesei (anamorph of Hypocrea jecorina) by profiling transcription in a wild-type and a delta-cre1 mutant strain on glucose at constant growth rates known to repress and de-repress CCR-affected genes. Analysis of genome-wide microarrays reveals 2.8% of transcripts whose expression was regulated in at least one of the four experimental conditions: 47.3% of which were repressed by CRE1, whereas 29.0% were actually induced by CRE1, and 17.2% only affected by the growth rate but CRE1 independent. Among CRE1 repressed transcripts, genes encoding unknown proteins and transport proteins were overrepresented. In addition, we found CRE1-repression of nitrogenous substances uptake, components of chromatin remodeling and the transcriptional mediator complex, as well as developmental processes. Conclusions Our study provides the first global insight into the molecular physiological response of a multicellular fungus to carbon catabolite regulation and identifies several not yet known targets in a growth-controlled environment.

International audience; Some of the Aspergilli are reputed for their versatile and efficient catabolism of soluble carbon sources and related metabolites as well as raw polymeric materials. Here, we present a detailed investigation of the genomic and evolutionary basis for this versatility, using seven Aspergillus and one Neosartorya genome sequences. We manually annotated about 155 genes per genome covering glycolysis, the pentose phosphate cycle, alternative routes of D-glucose metabolism, catabolism of D-galactose and pentoses, and the TCA cycle, as well as the utilization of acetate and ethanol, propionate metabolism, and gluconeogenesis.The annotation reveals that the Aspergilli have re-enforced several areas of their primary metabolism(notably glycolysis, TCA cycle, ethanol utilization, and pentose and polyol metabolism) by gene duplications,horizontal gene transfer or gene clustering. Results from the phylogenetic analysis of several enzymes encoded by duplicated genes also suggests that some gene products may have acquired new(physiological) functions, that render primary carbon metabolism of the Aspergilli more complex than previously thought.

Lactose (1,4-O-β-d-galactopyranosyl-d-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on d-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that d-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with d-galactose, lactose, galactitol and d-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that d-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0·015 h−1) but not at higher dilution rates. At the same dilution rate, growth on d-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on d-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0·015 h−1 than d-galactose and induced cellulases even at growth rates up to 0·042 h−1. Results of chemostats with an equimolar mixture of d-galactose and d-glucose essentially mimicked the behaviour on d-galactose alone, whereas an equimolar mixture of d-galactose and galactitol, the first intermediate of a recently described second pathway of d-galactose catabolism, led to cellulase induction at D=0·030 h−1. It is concluded that d-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase ( galE) can still grow on d-galactose in the presence of ammonium-but not nitrate-ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme ( araA1) did not show NAD(+)-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase ( frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD(+)-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.

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.