Advanced mycotoxin control and decontamination techniques in view of an increased aflatoxin risk in Europe due to climate change.

Aflatoxins are toxic secondary metabolites produced by Aspergillus spp. found in staple food and feed commodities worldwide. Aflatoxins are carcinogenic, teratogenic, and mutagenic, and pose a serious threat to the health of both humans and animals. The global economy and trade are significantly affected as well. Various models and datasets related to aflatoxins in maize have been developed and used but have not yet been linked. The prevention of crop loss due to aflatoxin contamination is complex and challenging. Hence, the set-up of advanced decontamination is crucial to cope with the challenge of climate change, growing population, unstable political scenarios, and food security problems also in European countries. After harvest, decontamination methods can be applied during transport, storage, or processing, but their application for aflatoxin reduction is still limited. Therefore, this review aims to investigate the effects of environmental factors on aflatoxin production because of climate change and to critically discuss the present-day and novel decontamination techniques to unravel gaps and limitations to propose them as a tool to tackle an increased aflatoxin risk in Europe.

The impact of bZIP Atf1ortholog global regulators in fungi.

Regulation of signal transduction pathways is crucial for the maintenance of cellular homeostasis and organismal development in fungi. Transcription factors are key elements of this regulatory network. The basic-region leucine zipper (bZIP) domain of the bZIP-type transcription factors is responsible for DNA binding while their leucine zipper structural motifs are suitable for dimerization with each other facilitiating the formation of homodimeric or heterodimeric bZIP proteins. This review highlights recent knowledge on the function of fungal orthologs of the Schizosaccharomyces pombe Atf1, Aspergillus nidulans AtfA, and Fusarium verticillioides FvAtfA, bZIP-type transcription factors with a special focus on pathogenic species. We demonstrate that fungal Atf1-AtfA-FvAtfA orthologs play an important role in vegetative growth, sexual and asexual development, stress response, secondary metabolite production, and virulence both in human pathogens, including Aspergillus fumigatus, Mucor circinelloides, Penicillium marneffei, and Cryptococcus neoformans and plant pathogens, like Fusarium ssp., Magnaporthe oryzae, Claviceps purpurea, Botrytis cinerea, and Verticillium dahliae. KEY POINTS: • Atf1 orthologs play crucial role in the growth and development of fungi. • Atf1 orthologs orchestrate environmental stress response of fungi. • Secondary metabolite production and virulence are coordinated by Atf1 orthologs.

FvmnSOD is involved in oxidative stress defence, mitochondrial stability and apoptosis prevention in Fusarium verticillioides.

Superoxide dismutases are key enzymes in elimination of the superoxide anion radical (O2 •- ) generated intracellularly or by exogenous oxidative stress eliciting agents, like menadione. In this study, we investigated the physiological role of the manganese superoxide dismutase-encoding gene in Fusarium verticillioides via the construction of a gene deletion mutant, ΔFvmnSOD and comparing its phenotype with that of the wild-type parental strain and a ΔFvmnSOD' C strain, complemented with the functional manganese superoxide dismutase gene. Deletion of FvmnSOD had no effect on the relative intracellular superoxide ratio but increased the sensitivity of the fungus to menadione sodium bisulphite on Czapek-Dox stress agar plates. The lack of FvmnSOD caused changes in mitochondrial morphology and physiology: The volumetric ratio of these cell organelles in the second hyphal segment, as well as the total, the KCN-sensitive cytochrome c-dependent and the KCN+SHAM (salicylhidroxamic acid)-resistant residual respiration rates, were higher in the mutant as compared to the wild-type and the complemented strains. Nevertheless, changes in the respiration rates were attributable to the higher volumetric ratio of mitochondria found in the gene deletion mutant. Changes in the mitochondrial functions also brought about higher sensitivity to apoptotic cell death elicited by the Penicillium chrysogenum antifungal protein. The gene deletion mutant developed significantly thinner hyphae in comparison to the wild-type strain. Deletion of FvmnSOD had no effect on fumonisin B1 and B2 production of the fungus grown in Myro medium as a static culture.

FvatfA regulates growth, stress tolerance as well as mycotoxin and pigment productions in Fusarium verticillioides.

FvatfA from the maize pathogen Fusarium verticillioides putatively encodes the Aspergillus nidulans AtfA and Schizasaccharomyces pombe Atf1 orthologous bZIP-type transcription factor, FvAtfA. In this study, a ΔFvatfA deletion mutant was constructed and then genetically complemented with the fully functional FvatfA gene. Comparing phenotypic features of the wild-type parental, the deletion mutant and the restored strains shed light on the versatile regulatory functions played by FvAtfA in (i) the maintenance of vegetative growth on Czapek-Dox and Potato Dextrose agars and invasive growth on unwounded tomato fruits, (ii) the preservation of conidiospore yield and size, (iii) the orchestration of oxidative (H2O2, menadione sodium bisulphite) and cell wall integrity (Congo Red) stress defences and (iv) the regulation of mycotoxin (fumonisins) and pigment (bikaverin, carotenoid) productions. Expression of selected biosynthetic genes both in the fumonisin (fum1, fum8) and the carotenoid (carRA, carB) pathways were down-regulated in the ΔFvatfA strain resulting in defected fumonisin production and considerably decreased carotenoid yields. The expression of bik1, encoding the polyketide synthase needed in bikaverin biosynthesis, was not up-regulated by the deletion of FvatfA meanwhile the ΔFvatfA strain produced approximately ten times more bikaverin than the wild-type or the genetically complemented strains. The abolishment of fumonisin production of the ΔFvatfA strain may lead to the development of new-type, biology-based mycotoxin control strategies. The novel information gained on the regulation of pigment production by this fungus can be interesting for experts working on new, Fusarium-based biomass and pigment production technologies. Key points • FvatfA regulates vegetative and invasive growths of F. verticillioides. • FvatfA also orchestrates oxidative and cell wall integrity stress defenses. • The ΔFvatfA mutant was deficient in fumonisin production. • FvatfA deletion resulted in decreased carotenoid and increased bikaverin yields.

Toxicity of abiotic stressors to Fusarium species: differences in hydrogen peroxide and fungicide tolerance.

Stress sensitivity of three related phytopathogenic Fusarium species (Fusarium graminearum, Fusarium oxysporum and Fusarium verticillioides) to different oxidative, osmotic, cell wall, membrane, fungicide stressors and an antifungal protein (PAF) were studied in vitro. The most prominent and significant differences were found in oxidative stress tolerance: all the three F. graminearum strains showed much higher sensitivity to hydrogen peroxide and, to a lesser extent, to menadione than the other two species. High sensitivity of F. verticillioides strains was also detectable to an azole drug, Ketoconazole. Surprisingly, no or limited differences were observed in response to other oxidative, osmotic and cell wall stressors. These results indicate that fungal oxidative stress response and especially the response to hydrogen peroxide (this compound is involved in a wide range of plant-fungus interactions) might be modified on niche-specific manner in these phylogenetically related Fusarium species depending on their pathogenic strategy. Supporting the increased hydrogen peroxide sensitivity of F. graminearum, genome-wide analysis of stress signal transduction pathways revealed the absence one CatC-type catalase gene in F. graminearum in comparison to the other two species.

FSRD: fungal stress response database.

Adaptation to different types of environmental stress is a common part of life for today's fungi. A deeper understanding of the organization, regulation and evolution of fungal stress response systems may lead to the development of novel antifungal drugs and technologies or the engineering of industrial strains with elevated stress tolerance. Here we present the Fungal Stress Response Database (http://internal.med.unideb.hu/fsrd) aimed to stimulate further research on stress biology of fungi. The database incorporates 1985 fungal stress response proteins with verified physiological function(s) and their orthologs identified and annotated in 28 species including human and plant pathogens, as well as important industrial fungi. The database will be extended continuously to cover other fully sequenced fungal species. Our database, as a starting point for future stress research, facilitates the analysis of literature data on stress and the identification of ortholog groups of stress response proteins in newly sequenced fungal genomes. Database URL: http://internal.med.unideb.hu/fsrd

Regulation of patulin-induced oxidative stress processes in the fission yeast Schizosaccharomyces pombe.

Patulin (PAT), is one of the most widely disseminated mycotoxins found in agricultural products. In this study the PAT-induced accumulation of reactive oxygen species (ROS) and the regulation of the specific activities of antioxidant enzymes were investigated in the single cell eukaryotic organism Schizosaccharomyces pombe. In comparison with the untreated cells, 500 µM PAT treatment caused a 43% decrease in the concentration of the main intracellular antioxidant, glutathione (GSH); this depletion of GSH initiated a 2.44- and a 2.6-fold accumulation of superoxide anion and hydrogen peroxide, respectively, but did not increase the concentration of hydroxyl radicals; the reduction of ROS-induced adaptation processes via the activation of Pap1 transcription factor resulted in significantly increased specific activities of Cu/Zn superoxide dismutase, catalase and glutathione S-transferase to protect the cells against the ROS-induced unbalanced redox state. However, no change was measured in the activities of glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. It seems reasonable to assume that the temporary PAT-induced ROS accumulation plays a crucial role in adaptation processes. The adverse effects of PAT may be exerted mainly through the destruction of cellular membranes and protein/enzyme functions.

The MAT1-2-1 mating-type gene upregulates photo-inducible carotenoid biosynthesis in Fusarium verticillioides.

Filamentous ascomycetes, including mitotic holomorphs, have constitutively transcribed MAT (mating type) genes. These genes encode transcription factors considered to be the major regulators of sexual communication. The proven targets of the MAT transcription factors are pheromone precursor and pheromone receptor genes. However, recent studies demonstrated that MAT proteins may also affect other genes not involved directly in the mating process. When grown in the light, Fusarium verticillioides produces the acidic xanthophyll neurosporoxanthin and lower amounts of nonpolar precursor carotenes, such as phytoene, torulene, ß-carotene, and γ-carotene. Depending on the illumination conditions, a drastic decrease or the absence of light-inducible carotenoid accumulation was detected in three independent ΔFvMAT1-2-1 knockout mutants of F. verticillioides as compared with the parental wild-type strain. Transcript levels of the carB, carRA, and carT genes, encoding key enzymes of the carotenoid biosynthetic pathway, were also significantly reduced in the mutants. The downregulation of these genes in the ΔFvMAT1-2-1 mutant indicates that MAT genes play a role in the control of carotenogenesis in Fusarium. The finding that mating-type genes regulate important processes unrelated to sex helps to understand the presence of functional MAT genes in asexually reproducing fungus populations.

Time-lapse analysis of cell death in mammalian and fungal cells.

Time-lapse video microscopy was designed to follow the movement of single cells for an unlimited period of time under physiological conditions. The system is based on two inverted microscopes located in a CO(2) incubator and equipped with charge-coupled device cameras connected to the computer. Frames were recorded every minute and the subsequent video sequence was converted to database form. The system was applied to describe the movements of normal HaCaT cells and Pb-treated cells causing the so-called apoptotic dance during cell death. The apoptotic movement was also followed in high-osmolarity glycerol-type mitogen-activated protein kinase (MAPK) null mutant of Fusarium proliferatum, a filamentous fungus, during osmotic stress. The shortest (20 min) and most vigorous death movements were observed in apoptotic fungal cells subjected to salt stress. The necrotic process at higher Pb concentration (50 microM) took 2-3 h, whereas the apoptotic process at lower Pb concentrations lasted from minutes to days.

Adenylyl cyclase regulates heavy metal sensitivity, bikaverin production and plant tissue colonization in Fusarium proliferatum.

A homologue of the adenylyl cyclase (AC) gene of Neurospora crassa, named Fpacy1 was cloned from the genomic library of Fusarium proliferatum ITEM 2287 by screening the library with a DNA fragment amplified by using PCR primers designed from conserved sequences of the catalytic domain of AC genes from other fungi. The deduced FPACY1 protein had 53-77% identity with the AC proteins of other fungi. DeltaFpacy1 mutants obtained by targeted gene disruption showed retarded vegetative growth, increased conidiation and delayed conidial germination. Colonization capability of the mutants, assessed on maize seedlings and tomato fruits also was adversely affected. In sexual crosses the AC mutants retained full male fertility, but their female fertility decreased significantly. Disruption of Fpacy1 abolished vegetative self-incompatibility, suggesting that the AC gene is involved in multiple developmental processes related to vegetative growth, as well as sexual and parasexual events. The elevated thermo- and H(2)O(2)-tolerance of the DeltaFpacy1 mutants was coupled to an increased sensitivity towards Cd and Cu, indicating that the cAMP signaling pathway may have both negative and positive regulatory roles on the stress response mechanisms of fungal cells. When grown under nitrogen limitation conditions, the DeltaFpacy1 mutants produced an average of approximately 274 microg g(-1) bikaverin, whereas only traces of this metabolite was detected in the wild type. This finding provides further evidence of the role of the cAMP-PKA pathway in regulating bikaverin production.

N-starvation stress induced FUM gene expression and fumonisin production is mediated via the HOG-type MAPK pathway in Fusarium proliferatum.

During cultivation of a wild type strain of Fusarium proliferatum on ammonium dihydrogen phosphate containing defined medium, expression levels of FUM1 and FUM8, members of the fumonisin biosynthesis gene cluster significantly increased when ammonium ion concentration of the culture medium decreased below 10 mM, indicating that N-depletion triggers the fumonisin biosynthesis genes. Deletion of Fphog1, a HOG-type MAP kinase gene resulted in further increases in FUM1 and FUM8 expression under nitrogen starvation (absence of any N-source) conditions. Fumonisin B1 (FB1) production paralleled with increased FUM gene expression: significant amounts of FB1 were measured in culture filtrates of the DeltaFphog1 deleted mutant after five days culturing, whereas only traces of FB1 could be detected in filtrates of the wild type and the restored strain (R1) complemented with the wild-type Fphog1-24 gene. N-starvation strongly retarded the growth of the DeltaFphog1 mutant in comparison to wild type. The up-regulation of fumonisin biosynthesis genes in the DeltaFphog1 mutant could be explained by the increased sensitivity of these strains to N-starvation stress that appears in the absence of an intact HOG-type MAPK pathway.

Fphog1, a HOG-type MAP kinase gene, is involved in multistress response in Fusarium proliferatum.

Delta Fphog1 mutants of Fusarium proliferatum obtained by targeted gene disruption of Fphog1, an orthologue of the Saccharomyces cerevisiae hog1 MAPK gene showed increased sensitivity towards different abiotic stressors including UV-irradiation, heat, salt, osmotic and hydrogen peroxide treatments. Incubation of the Delta Fphog1 mutants under hyperosmotic conditions was accompanied with prolonged growth arrest, inhibition of conidial germination, morphological abnormalities and time-dependent increase of the cell death rate. The wild type Fphog1 gene, under the control of its own promoter, was able to rescue the multistress sensitivity of the mutant strain. Real time qPCR data demonstrated that under salt and sorbitol stress conditions the Fphog1 gene is not subject of transcriptional regulation. Levels of reactive oxygen species (ROS), mitochondrial membrane permeability transition, nuclear disintegration and DNA fragmentation, indicators of programmed cell death (PCD) all showed significant increases under osmotic stress conditions in the Delta Fphog1 mutant in comparison to the wild type strain. These results suggest that an important function of Fphog1 is attenuating apoptotic phenotypes under salt and sorbitol stressors.

Characterization of a new mitochondrial plasmid from Fusarium proliferatum.

A 10.3kb linear mitochondrial DNA plasmid designated pFP1 was isolated from Fusarium proliferatum. The DNA sequence of the plasmid consists of 10,336bp with perfect terminal inverted repeats of 400bp. Two major, non-overlapping ORFs were identified on opposite strands, encoding a phage-type RNA polymerase and a family B type DNA polymerase, respectively. One additional minor ORF encoding a putative highly basic protein was also identified. The copy number of pFP1, as determined by RT-PCR, ranged between 1.8 and 3.1 per mtDNA copies depending on the host strain. Real-time PCR analysis of a total of 400 cultures surviving ethidium bromide curing indicated that no plasmid-free strains could be obtained by this treatment. Further single spore selections of the survivors with reduced plasmid content were needed to obtain plasmid-free clones. No phenotypic differences were found between the wild-type strains and their plasmid-free progenies.

Genetic factors affecting sexual reproduction in toxigenic Fusarium species.

Mycotoxin producing capability greatly varies within species. In theory, the major source of this variability is meiotic recombination. However, a number of important toxigenic species have no known sexual stage and, therefore, the origin of the intraspecific diversity in these fungi is poorly understood. Mating in sexually reproducing Ascomycetes is controlled by MAT genes, but fungi with no known sexual stage also may have fully functional, constitutively transcribed mating type genes. The MAT genes, MAT1-1-1 and MAT1-2-1 encode putative transcription factors which, besides regulating pheromone and pheromone receptor genes, may affect other genes not involved directly in the mating process. By comparing the transcript profiles of a DeltaMAT1-2-1 knock-out mutant and the wild type of Fusarium verticillioides, more than 200 ESTs, either down- or up-regulated in the mutant, were identified. Sequences encoding proteins involved in protein synthesis and metabolism occurred more frequently among ESTs up-regulated in the mutant, while sequences involved in cell signaling and communication were more frequent in the down-regulated subset of ESTs. The lack of fertility in fungi with no known sexual stage and the limited fertility of local populations of sexually reproducing fungi are probably due to changes in one or more of numerous genes that cause female sterility. A number of gene disruption mutants of Fusarium proliferatum were assessed for their mating capabilities. Fphch (a Het-C homologue), Fpmtr (an amino acid transporter gene), and Fpnitr1 (a putative nitrilase encoding gene) encode proteins in seemingly unrelated pathways, but mutations at any of these loci can reduce female fertility. Thus, a number of genes, with functions not related directly to mating, can influence the frequency of sexual reproduction indicating that this process requires the concerted operation of many factors not obviously connected to female fertility/sterility.

Cloning and characterization of Fpmtr1, an amino acid transporter gene of Fusarium proliferatum (Gibberella intermedia).

Fpmtr1, an amino acid transporter gene from Fusarium proliferatum was strongly expressed during conidial germination and repressed in late stationary phase. To identify the specific function of this gene, DeltaFpmtr1 knock-out mutants were generated by gene replacement. Vegetative growth of the DeltaFpmtr1 mutants was normal both in liquid and on solid media, but conidial germination was delayed. The DeltaFpmtr1 mutants and the wild type were equally fertile when used as males in sexual crosses, however if the mutants were used as the female parent then the fertility of the cross decreased dramatically. Inactivation of Fpmtr1 abolished vegetative self-incompatibility in strain ITEM 2287 of F. proliferatum, but the DeltaFpmtr1 mutants were still vegetatively incompatible with the other strains of the fungus. Endophytic colonization capability of the mutants, assessed on maize seedlings also was adversely affected. These data suggest that Fpmtr1 is involved in multiple developmental processes related to both sexual and parasexual events in F. proliferatum. Furthermore, the fungus might have problems in adapting to a less than optimal environment if this otherwise dispensable transporter has been inactivated.

Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A).

Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes and therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, DeltaMAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down-regulated in the DeltaMAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the Gibberella zeae and Gibberella moniliformis genomes. Fifty-five percent of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 45% were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences down-regulated in the mutant.

The homologue of het-c of Neurospora crassa lacks vegetative compatibility function in Fusarium proliferatum.

For two fungal strains to be vegetatively compatible and capable of forming a stable vegetative heterokaryon they must carry matching alleles at a series of loci variously termed het or vic genes. Cloned het/vic genes from Neurospora crassa and Podospora anserina have no obvious functional similarity and have various cellular functions. Our objective was to identify the homologue of the Neurospora het-c gene in Fusarium proliferatum and to determine if this gene has a vegetative compatibility function in this economically important and widely dispersed fungal pathogen. In F. proliferatum and five other closely related Fusarium species we found a few differences in the DNA sequence, but the changes were silent and did not alter the amino acid sequence of the resulting protein. Deleting the gene altered sexual fertility as the female parent, but it did not alter male fertility or existing vegetative compatibility interactions. Replacement of the allele-specific portion of the coding sequence with the sequence of an alternate allele in N. crassa did not result in a vegetative incompatibility response in transformed strains of F. proliferatum. Thus, the fphch gene in Fusarium appears unlikely to have the vegetative compatibility function associated with its homologue in N. crassa. These results suggest that the vegetative compatibility phenotype may result from convergent evolution. Thus, the genes involved in this process may need to be identified at the species level or at the level of a group of species and could prove to be attractive targets for the development of antifungal agents.

Cloning, characterization and phylogenetic relationships of cel5B, a new endoglucanase encoding gene from Thermobifida fusca.

Thermobifida fusca, a thermophilic, aerobic, cellulolytic bacterium has a highly complex cellulase system comprising three endoglucanases, two exoglucanases and one processive endoglucanase. Zymogram analysis indicated that additional cellulases may exist in T. fusca strain TM51, therefore a TM51 expression library was prepared in Streptomyces lividans TK24 and screened for hydrolases. A new endoglucanase gene, named Tf cel5B, was identified. Heterologous Cel5B, produced in S. lividans, had temperature and pH optima of 77 degrees C and 8.2, respectively and retained more than 60% of its activity after 24 h incubation at 60 degrees C. Domain analysis revealed an N-terminal catalytic domain with homology to known endoglucanases in family GH5 and a C-terminal cellulose binding module III domain (CBD). Comparing the domain structures of all seven known T. fusca cellulases showed, that the cellulase system of this organism consists of pairs of enzymes from the same GH family, including Cel5A--Cel5B, Cel6A--Cel6B and Cel9A--Cel9B plus a single family GH48 enzyme (Cel48A). Furthermore, the catalytic and substrate binding domains of enzymes, belonging to the same GH family were arranged in opposite orientations. Phylogenetic comparisons of the catalytic domain sequences of the T. fusca cellulases to other family GH5, GH6, GH9 and GH48 cellulases of bacterial origin revealed that the enzyme pairs in the same GH family are not closely related to each other, instead they showed significant similarities to various cellulase enzymes from taxonomically distinct organisms. Therefore, the complex and highly efficient cellulase system of T. fusca seems to be evolved as a result of horizontal gene transfers rather than gene duplication events.

Mating type sequences in asexually reproducing Fusarium species.

To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative alpha and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The alpha box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria.

Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51.

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.