Cocoa-specific flavor components and their peptide precursors.

Essential precursors of the cocoa-specific roasting-flavor notes were formed during proteolysis of the cocoa vicilin-class(7S) globulin by a mixture of cocoa aspartic protease and carboxypeptidase. These could be partially purified by ligand-exchange chromatography. Many constituents of this peptide fraction were destroyed by post-treatment with pepsin, but the cocoa-specific flavor-precursor peptides were largely resistant against pepsin treatment. However, these peptides were not generated when the cocoa vicilin-class(7S) globulin was digested with a mixture of pepsin and carboxypeptidase. By nano-liquid chromatography mass spectrometry, the peptide composition of these peptide fractions were compared in order to identify the putative precursors of the cocoa-specific flavor components. These peptides were assigned to five regions of the cocoa vicilin-class(7S) globulin. Analyzing the roasting products of the different protein fractions by headspace solid-phase microextraction, followed by gas chromatography mass spectrometry, eight volatile compounds were detected, whose occurrence correlated with the sensory detection of cocoa-specific flavor notes.

pH-Dependency of the proteolytic formation of cocoa- and nutty-specific aroma precursors.

Sensory evaluation of roasted cocoa not only revealed cocoa-specific but also more or less pronounced nutty-specific aroma notes. Essential precursors of the corresponding volatile compounds could be generated in vitro by proteolysis of the cocoa vicilin-class(7S) globulin using a mixture of cocoa aspartic protease and cocoa carboxypeptidase. Since both proteases have rather different pH optima (pH 3.5 and 5.5-6.0, respectively), we have investigated the pH-dependency of proteolysis of this protein substrate in the presence of both proteases, the liberation of free amino acids and the generated aroma potential. Our findings revealed that the precursors of the nutty aroma notes were generated at higher pH-values (pH 4.8-5.6) than the cocoa-specific precursor peptides (pH 4.4-5.2). Longer peptide fragments of the cocoa vicilin were formed by proteolysis at pH 5.2 than at pH 4.8. Furthermore, our findings indicated that cocoa-vicilin derived peptides are essential precursors of both the cocoa- and the nutty-specific aroma components.

Bacterial Surface Traits Influence Digestion by Tetrahymena pyriformis and Alter Opportunity to Escape from Food Vacuoles.

Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide allows any substance carrying amino- or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm.

The fate of mitochondria after infection of the Mucoralean fungus Absidia glauca by the fusion parasite Parasitella parasitica: comparison of mitochondrial genomes in zygomycetes.

Absidia glauca and Parasitella parasitica constitute a versatile experimental system for studying horizontal gene transfer between a mucoralean host and its fusion parasite. The A. glauca chondriome has a length of approximately 63 kb and a GC content of 28%. The chondriome of P. parasitica is larger, 83 kb, and contains 31% GC base pairs. These mtDNAs contain the standard fungal mitochondrial gene set, small and large subunit rRNAs, plus ribonuclease P RNA. Comparing zygomycete chondriomes reveals an unusually high number of homing endonuclease genes in P. parasitica, substantiating the mobility of intron elements independent of host-parasite interactions.

The Cytotoxic Effects of Camptothecin and Mastoparan on the Unicellular Green Alga Chlamydomonas reinhardtii.

We have recently reported that protease inhibitors affecting the activity of the proteasome cause necrotic cell death in Chlamydomonas reinhardtii instead of inducing apoptosis as shown for some mammalian cell lines. Therefore, we have studied other well-known inducers of apoptosis in mammalian cells for their effects on C. reinhardtii cells. Mastoparan caused rapid cell death without a prominent lag-phase under all growth conditions, whereas the cytotoxic effect of the topoisomerase I inhibitor camptothecin exclusively occurred during the cell-division phase. Essentially no differences between wall-deficient and wild-type cells were observed with respect to dose-response and time-course of camptothecin and mastoparan. In cultures of the wall-deficient strain, cell death was accompanied by swelling and subsequent disruption of the cells, established markers of necrosis. In case of the wild-type strain, camptothecin and mastoparan caused accumulation of apparently intact, but dead cells instead of cell debris due to the presence of the wall. Both in cultures of the wall-deficient and the wild-type strains, cell death was accompanied by an increase of the protein concentration in the culture medium indicating a lytic process like necrosis. Taking together, we have severe doubts on the existence of an apoptotic program in case of C. reinhardtii.

Post-translational regulation by structural changes of 4-dihydromethyltrisporate dehydrogenase, a key enzyme in sexual and parasitic communication mediated by the trisporic acid pheromone system, of the fungal fusion parasite Parasitella parasitica.

Sexual communication between complementary mating partners in the fungal group of zygomycetes is mediated by the trisporoid pheromone system. A key enzyme towards biosynthesis of hormonally active trisporoids is 4-dihydromethyltrisporate dehydrogenase (TSP1), an enzyme occurring in all zygomycetous fungi. Trisporic acid and some of its precursor molecules serve as pheromones for recognizing complementary mating partners and for induction of the differentiation program towards sexual spore formation. In the parasitic zygomycete Parasitella parasitica, a biotrophic fusion parasite infecting many other zygomycetes, these substances have an additional function: They are also responsible for host-parasite recognition and the formation of the characteristic infection structures. Parasitic interactions are mating type dependent as well. In the Mucor-related mycoparasite P. parasitica we can study both types of communication in parallel. We were interested in protein structures of TSP1 from P. parasitica, the genome of which was recently sequenced by us, and especially in the mechanisms involved in the switch from sexual to parasitic communication. P. parasitica contains at least six genes coding for TSP1-like proteins. We created models of tertiary structures and performed protein-protein docking with the resulting protein structures to simulate dimerization and to provide support for probable regulatory mechanisms at the protein level. The resulting structure models show differences in putative activity and binding preferences between the different TSP1-like proteins. Two of them seem to be able to form solid binding pockets for substrate and cosubstrate after dimerization. The other four TSP1-like proteins are more likely to represent regulating subunits for the two active isoforms. The ability to form homodimers with enzymatic activity could be the crucial difference between sexual and parasitic communication pathways. TSP1 PARPA_07791 forms enzymatically inactive homodimers. The second TSP1, PARPA_04105, forms active homodimers and could be responsible for the parasitic pathway of communication. Both TSP1 proteins can form more or less active heterodimers with the additional TSP1-like proteins. TSP1 PARPA_07791 mediates the sexual pathway probably as in other zygomycetous fungi like Mucor mucedo. High sequence identities between this TSP1 isomer and TSP1 proteins from other zygomycetes substantiate its function. This bioinformatic study supports previous experimental findings of post-translational regulation of 4-dihydromethyltrisporate dehydrogenases in zygomycetes and, for the first time, provides a substantiated hypothesis of the underlying mechanism.

Dataset of cocoa aspartic protease cleavage sites.

The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans.

The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme.

Complete Mitochondrial DNA Sequence of the Mucoralean Fungus Absidia glauca, a Model for Studying Host-Parasite Interactions.

The mitochondrial DNA (mtDNA) ofAbsidia glaucahas been completely sequenced. It is 63,080 bp long, has a G+C content of 28%, and contains the standard fungal gene set.A. glaucais the recipient in a laboratory model for horizontal gene transfer withParasitella parasiticaas a donor of nuclei and mitochondria.

Partial purification and characterisation of the peptide precursors of the cocoa-specific aroma components.

Essential precursors of the cocoa-specific aroma notes are formed during fermentation of the cocoa beans by acid-induced proteolysis. It has been shown that, in addition to free amino acids, hydrophilic peptides derived from the vicilin-class(7S) globular storage protein are required for the generation of the cocoa-specific aroma notes during the roasting process. To identify those peptides responsible for the generation of the cocoa-specific aroma components, we have developed a procedure for the fractionation of the aroma precursor extract from well-fermented cocoa beans by ligand-exchange and subsequent Sephadex-LH20 chromatography. The cocoa-specific aroma precursor fractions were characterised by matrix-assisted laser-desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and the determination of their amino acid sequences by electrospray ionisation mass spectrometry (ESI-MS/MS).

Ingestion and digestion studies in Tetrahymena pyriformis based on chemically modified microparticles.

Recognition of food and, in consequence, ingestion of digestible particles is a prerequisite for energy metabolism in Tetrahymena pyriformis. Understanding why some particles are ingested and digested, whereas others are not, is important for many fields of research, e.g. survival of pathogens in single-celled organisms or establishment of endosymbiotic relationships. We offered T. pyriformis synthetical bovine-serum-albumin (BSA)-methacrylate microparticles of approximately 5.5 µm diameter and studied the ciliates' ingestion and digestion behaviour. Different staining techniques as well as co-feeding with a transformant strain of Escherichia coli revealed that T. pyriformis considers these particles as natural food source and shows no feeding preference. Further, they are ingested at normal rates and may serve as sole food source. A pivotal advantage of these particles is the convenient modification of their surface by binding different ligands resulting in defined surface properties. Ingestion rate of modified microparticles either increased (additional BSA, enzymes) or decreased (amino acids). Furthermore, we investigated glycosylation patterns by lectin binding. By binding different substances to the surface in combination with various staining techniques, we provide a versatile experimental tool for elucidating details on food recognition and digestion that may allow to study evading digestion by pathogens or potential endosymbionts, too.

Complete Mitochondrial DNA Sequence of the Mucoralean Fusion Parasite Parasitella parasitica.

The complete mitochondrial DNA sequence of the Mucor-related fungus Parasitella parasitica has been sequenced. It has a G+C content of 30% and a total length of 83,361 bp. All protein-coding genes normally found in fungi are present in the sequence. A special feature is the remarkably high number of 27 homing endonucleases.

Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP) kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG) transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT) and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

A model for endosymbiosis: interaction between Tetrahymena pyriformis and Escherichia coli.

Endosymbiosis in ciliates is a common and highly diverse phenomenon in nature, but its development at the mechanistic level and the origins are not easy to understand, since these associations may have arisen at any time during evolution. Therefore a laboratory model is helpful. It could be provided by the interaction of Tetrahymena pyriformis and Escherichia coli. Microscopic analyses with a genetically manipulated fluorescent strain of E. coli show single bacteria leaving food vacuoles and escaping digestion, an important prerequisite for further experiments. Under selective conditions, beneficial for T. pyriformis, the ciliate was shown to internalize E. coli cells. After feeding, bacteria, transformed with the plasmids pBS-neoTet or pNeo4, provide T. pyriformis with the ability to handle toxic conditions, caused by the aminoglykoside antibiotic paromomycin. Axenic cultures or cocultures with untransformed bacteria show lower cell numbers and survival rates compared to cocultures with transformed bacteria after transfer to paromomycin containing media. PCR detects bacterial DNA inside T. pyriformis cells. Additionally, microscopical analysis of selectively grown cocultures reveals fluorescing particles in the cytoplasm of T. pyriformis containing DNA and lipids, corresponding in size to E. coli. This system could be a reasonable model for understanding mechanisms of endosymbiosis establishment in ciliates.

Complementation of a stable Met2-1 mutant of the zygomycete Absidia glauca by the corresponding wild-type allele of the mycoparasite Parasitella parasitica, transferred during infection.

Compared with prokaryotes, where horizontal gene transfer events are frequently found and can be studied in the laboratory at the mechanistic level, few systems are known that allow direct experimental access to parasexual phenomena in eukaryotes. In zygomycetes, a basal lineage of fungi, several mycoparasitic fungi are known that inevitably form a cytoplasmic continuum with their hosts during infection. We provide evidence that, corresponding to the expectation suggested by the morphology of the infection process, gene transfer occurs from the parasite to the host. For analysing this parasexual system at the DNA level, we characterized interspecific recombinants obtained by infecting a stable methionine-auxotrophic Absidia glauca mutant with heavy rearrangements at the Met2-1 locus, which encodes homoserine acetyltransferase. Recipients were shown to be complemented by part of the corresponding gene from Parasitella parasitica. This foreign DNA is neither integrated at the putative Met2-2 locus in the recipient strain nor integrated at Met2-1, a locus encoding a hypothetical protein with amino acid similarity but with unknown function. Based on hybridization studies and on the phenotype of recipients that bear some mitotic instability of the acquired prototrophy, we propose that P. parasitica DNA is established in A. glauca recipients as extrachromosomally located replicons.

Correlation between sequence, structure and function for trisporoid processing proteins in the model zygomycete Mucor mucedo.

Terpenoids, steroids, carotenoids, phytoenes and other chemically related substance groups fulfill multiple functions in all realms of the organismic world. This analysis focuses on trisporoids that operate as pheromones in the phylogenetically ancient fungal group of mucoralean zygomycetes. Trisporoids serve as pheromones for recognizing complementary mating partners and for inducing the differentiation program towards sexual spore formation. Trisporoids are synthesized by oxidative degradation of ß-carotene. Structurally, they are related to retinoids in mammals and abscisic acid in vascular plants. In order to evaluate evolutionary relationships between proteins involved in trisporoid binding and also for checking possibilities to recognize functionally related proteins by sequence and structure comparisons, we compared representative proteins of different origins. Towards this goal, we calculated three-dimensional structures for 4-dihydromethyltrisporate dehydrogenase (TSP1) and 4-dihydrotrisporin dehydrogenase (TSP2), the two proteins involved in trisporic acid synthesis that have unequivocally been correlated with their catalytic function for the model zygomycete Mucor mucedo. TSP1 is an aldo-keto reductase with a TIM-barrel structure, TSP2 belongs to short-chain dehydrogenases, characterized by a Rossmann fold. Evidently, functional conservation, even implying very similar substrates and identical cosubstrates of enzymes in a single organism, turns out to be essentially independent of basic protein structure. The binding sites for NADP and trisporoid ligands in the proteins were determined by docking studies, revealing those regions affecting substrate specificity. Despite the pronounced differences in amino acid sequence and tertiary structure, the surfaces around the active sites are comparable between TSP1 and TSP2. Two binding regions were identified, one sterically open and a second closed one. In contrast to TSP1, all docking models for TSP2 place the trisporoid into the second, channel-like region.

The mating-related loci sexM and sexP of the zygomycetous fungus Mucor mucedo and their transcriptional regulation by trisporoid pheromones.

The putative mating type locus of mucoralean fungi consists of a single high mobility group (HMG)-domain transcription factor gene, sexM or sexP, flanked by genes for an RNA helicase and a triosephosphate transporter. We used degenerate primers derived from the amino acid sequence of the RNA helicase to sequence a fragment of this gene from Mucor mucedo. This fragment was extended by inverse PCR to obtain the complete sequences of the sex loci from both mating types of M. mucedo. The sex loci in M. mucedo reflect the general picture obtained previously for Phycomyces blakesleeanus, presenting a single HMG-domain transcription factor gene, sexM and sexP in the minus and plus mating types, respectively. These are located next to a gene for RNA helicase. Transcriptional analysis by quantitative real-time PCR showed that only transcription of sexM is considerably stimulated by adding trisporoid pheromones, thus mimicking sexual stimulation, whereas sexP is only slightly affected. These differences in regulation between sexM and sexP are supported by the observation that the promoter sequences controlling these genes show no similarities. The protein structures themselves are considerably different. The SexM, but not the SexP protein harbours a nuclear localization sequence. The SexM protein is indeed transported to nuclei. This was shown by means of a GFP fusion construct that was used to study the localization of SexM in the yeast Saccharomyces cerevisiae. The fusion protein is highly enriched in nuclei.

Transformation of the fungus Absidia glauca by complementation of a methionine-auxotrophic strain affected in the homoserine-acetyltransferase gene.

Transformation of fungi by complementation of auxotrophs is generally much more reliable than usage of antibiotic resistance markers. In order to establish such a system for the model zygomycete Absidia glauca, a stable methionine auxotrophic mutant was isolated after X-ray mutagenesis of the minus mating type and characterized at the molecular level. The mutant is disrupted in the coding region of the Met2-1 gene, encoding homoserine O-acetyltransferase. The corresponding wild type gene was cloned, sequenced and inserted into appropriate vector plasmids. Transformants are prototrophs and show restored methionine-independent growth, based on complementation by the autonomously replicating plasmids.

Targeted gene deletion and in vivo analysis of putative virulence gene function in the pathogenic dermatophyte Arthroderma benhamiae.

Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ΔacuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ΔacuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.

Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi.

BACKGROUND: Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans. RESULTS: 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species. CONCLUSIONS: Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens.