Virtual Reality has many more applications next to gaming, to which it is currently associated mostly. Ordina SMART Technologies was looking for a way to combine virtual reality with shopping, to create a Virtual Shopping Platform where users could shop and be immersed using virtual reality. An application had to be designed and implemented from scratch, combining elements from both shopping and virtual reality. An application like that has a huge potential scope, which is why the focus of the project was put on maintainability and extendability. The application supports multiple shops which reside on a server, and interaction with products. The groundworks of a Virtual Shopping Platform were created, intended to be expanded upon by Ordina developers.

A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homologue, sharing 30% identity and 50% similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70% of known serpins, and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.

The carbon disulfide (CS2)-oxidizing bacterium Thiomonas sp. WZW was enriched and isolated using activated sewage sludge as inoculum. Growth of Thiomonas sp. WZW was observed on CS2, thiosulfate, dimethylsulfide (DMS), dimethyldisulfide (DMDS), and H2S. No growth occurred on dimethylsulfoxide, methanol, acetate, and on complex media with glucose, yeast extract, or tryptone. DMDS-grown cells respired CS2, DMS, and DMDS, while thiosulfate-grown cells did not respire CS2. Chemostat cultures growing on thiosulfate could be rapidly adapted to growth on CS2. Growth was observed between pH 6 and 8. The K s values for CS2, thiosulfate, and sulfide of CS2-grown cells were between 5 and 10 μM. CS2 was inhibitory above 0.3 mM. A lab-scale biotrickling filter with lava stone as carrier material for treatment of CS2-polluted air was inoculated with Thiomonas sp. WZW. A rapid start up (95% removal in 1 week) was obtained at an inlet CS2 concentration of 2 cmol l−1 and an initial space velocity (SV) of 54 h−1. Subsequent thiosulfate addition for a week during start up increased the removal to 99%. The step-wise increase of SV to 130 h−1 and a CS2 concentration to 3 μmol l−1 resulted in a stable performance with a removal efficiency of 95%. Feeding mixtures of volatile sulfur compounds showed simultaneous conversion of H2S, CS2, dimethyldisulfide (DMDS), and DMS, with a preference in this order.

The complete oat gene and cDNA from the commercial mushroom, Agaricus bisporus, encoding ornithine aminotransferase (OAT) was characterized. The gene encodes a 466 amino acid protein and provides the first fully reported homobasidiomycete OAT protein sequence. The gene is interrupted by ten introns, and no mitochondrial targeting motif was present pointing to a cytoplasmic localization. The function of the gene was demonstrated by complementation of a Saccharomyces cerevisiae mutant unable to utilize ornithine as a sole source of nitrogen with an A. bisporus oat cDNA construct. Northern analysis of the oat gene together with the pruA gene (encoding Delta(1)-pyrroline-5-carboxylate dehydrogenase) showed that transcripts of both genes were lower during the first stages of fruiting body development. The higher expression of the oat gene in later stages of development, suggests the importance of ornithine metabolism for the redistribution of metabolites in the developing mushroom. Hplc analysis of all amino acids revealed that ornithine levels increased during fruiting body development whereas proline levels fell.

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. in this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61-63% and 51-55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59-60% identity. Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology. (C) 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

nammox in the genes Ten years ago a fortuitous discovery led to the identification of oceanic bacteria capable of anaerobic ammonium oxidation (anammox). It was soon recognized that the anammox reaction has great ecological significance, as it is responsible for removing up to 50% of fixed nitrogen from the oceans. The genome of the anammox bacterium Kuenenia stuttgartiensis has now been sequenced in a remarkable feat of what is called environmental genomics. Anammox bacteria grow very slowly and are not available in pure culture. For genome analysis an inoculum of wastewater sludge was grown in a bioreactor for one year, clocking up 10–15 generations. The DNA of the whole microbial community was sequenced and the genome of this one anammox bacterium was deduced from the results. With the genome sequence known, it will be possible to gain insight into the metabolism and evolution of these important bacteria.

Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.

An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies. An important role of the ornithine cycle enzymes in urea accumulation has been proposed. In this work, we present the cloning and sequencing of the arginase gene and its promoter region from A. bisporus. A PCR-probe based on fungal arginase was used to identify the A. bisporus arginase gene from a cDNA library. The arginase cDNA encodes a 311-aa protein which is most likely expressed in the cytosol. Expression of the cDNA in Escherichia coli was established as a His-tagged fusion protein. The arginase gene was used as a molecular marker to study expression and regulation during sporophore formation and postharvest development. The expression of the arginase gene was significantly up-regulated from developmental stage 3 onwards for all the tissues studied. A maximum of expression was reached at stage 6 for both stipe and cap tissue. In postharvest stages 5, 6 and 7 the level of expression observed was similar to normal growth stages 5, 6 and 7. A good correlation was found between arginase expression and urea content of stipe, velum, gills, cap and peel tissue. For all tissues the urea content decreased over the first four stages of development. From stage 4 onwards urea accumulated again except for stipe tissue where no significant changes were observed. The same trend was also observed for postharvest development, but the observed increase of urea in postharvest tissues was much higher.

Bacillus fastidiosus was cultivated in batch and continuous culture on various carbon and nitrogen sources. The enzymes involved in allantoin degradation (allantoinase, urease, carboligase) of B. fastidiosus were hardly affected by either carbon or nitrogen source. In contrast, the enzymes involved in glycerol utilization (glycerol kinase, glycerol 3-phosphate dehydrogenase) were induced during growth on glycerol, but were not affected by the amount of allantoin present.

Intestinal microorganisms play an important role in plant fiber degradation by larvae of the rose chafer Pachnoda marginata. In the hindgut of the larvae 2.5 to 7.4 × 108 bacteria per ml of gut content with xylanase or endoglucanase activity were found. Bacteria in the midgut were not (hemi)cellulolytic, but the alkaline environment in this part of the intestinal tract functions as a precellulolytic phase, solubilizing part of the lignocellulosic material. Accordingly, the degradation of lignocellulose-rich material in Pachnoda marginata larvae appeared to be a combination of a physico-chemical and microbiological process. A number of different facultative anaerobic and strictly anaerobic bacteria with (hemi)cellulolytic activity were isolated from the hindgut. A dominant (hemi)cellulolytic species was a Gram positive, irregular shaped, facultative aerobic bacterium. Further physiological identification placed the isolate in the genus Promicromonospora. Comparative 16S rDNA analysis and phenotypic features revealed that the isolate represented a new species for which the name Promicromonospora pachnodae is proposed. P. pachnodae produced xylanases and endoglucanases on several plant derived polymers, both under aerobic and anaerobic conditions.

Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (beta-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54-75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min−1mg protein-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.

The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular beta-glucosidase activity. To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737 bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp. strain E2 cellulosome.

Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.

Anaerobic fungi have very high cellulolytic activities and thus degrade cellulose very efficiently. In cellulose hydrolysis, beta-glucosidases play an important role in prevention of product inhibition because they convert oligosaccharides to glucose. A beta-glucosidase gene (cel1A) was isolated from a cDNA library of the anaerobic fungus Piromyces sp. E2. Sequence analysis revealed that the gene encodes a modular protein with a calculated mass of 75800 Da and a pI of 5.05. A secretion signal was followed by a negatively charged domain with unknown function. This domain was coupled with a short linker to a catalytic domain that showed high homology with glycosyl hydrolases belonging to family 1. Southern blot analysis revealed the multiplicity of the gene in the genome. Northern analysis showed that growth on fructose resulted in a high expression of cel1A. The cel1A gene was successfully expressed in Pichia pastoris. The purified heterologously expressed protein was shown to be encoded by the cel1A gene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of a tryptic digest. Purified heterologous Cel1A was active towards several artificial and natural substrates with beta-1-4 linked glucose molecules with a remarkably high activity on cellodextrins. The enzyme was strongly inhibited by D-glucono-1,5-delta-lactone (K(i)=22 microM), but inhibition by glucose was much less (K(i)=9.5 mM). pH and temperature optimum were 6 and 39 degrees C, respectively. The enzyme was fairly stable, retaining more than 75% of its activity when incubated at 37 degrees C for 5 weeks. Transglycosylation activity could be demonstrated by MALDI-TOF MS analysis of products formed during degradation of cellopentaose.

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts from fruit bodies.

The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components. To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

Microbial cycling of volatile organic sulfur compounds (VOSCs), especially dimethyl sulfide (DMS) and methanethiol (MT), is intensively studied because these compounds play an important role in the processes of global warming, acid precipitation, and the global sulfur cycle. VOSC concentrations in freshwater sediments are low due to the balance between the formation and degradation of these compounds. These reactions occur for the greater part at the oxic/anoxic interphase of sediment and water column. In contrast to marine ecosystems, where dimethylsulfoniopropionate is the main precursor of MT and DMS, in freshwater ecosystems, VOSCs are formed mainly by methylation of sulfide and to a lesser extent from the degradation of S-containing amino acids. One of the major routes for DMS and MT formation through sulfide methylation is anaerobic O-demethylation of methoxylated aromatic compounds. Inhibition studies have revealed that the major part of the endogenously produced MT and DMS is degraded anaerobically by methanogens. The major bacterial groups involved in formation and consumption of VOSCs are described.

Methanethiol (MT) and dimethyl sulfide (DMS) have been shown to be the dominant volatile organic sulfur compounds in freshwater sediments. Previous research demonstrated that in these habitats MT and DMS are derived mainly from the methylation of sulfide. In order to identify the microorganisms that are responsible for this type of MT and DMS formation, several sulfide-rich freshwater sediments were amended with two potential methyl group-donating compounds, syringate and 3,4,5-trimethoxybenzoate (0.5 mM). The addition of these methoxylated aromatic compounds resulted in excess accumulation of MT and DMS in all sediment slurries even though methanogenic consumption of MT and DMS occurred. From one of the sediment slurries tested, a novel anaerobic bacterium was isolated with syringate as the sole carbon source. The strain, designated Parasporobacterium paucivorans, produced MT and DMS from the methoxy groups of syringate. The hydroxylated aromatic residue (gallate) was converted to acetate and butyrate. Like Sporobacterium olearium, another methoxylated aromatic compound-degrading bacterium, the isolate is a member of the XIVa cluster of the low-GC-content Clostridiales group. However, the new isolate differs from all other known methoxylated aromatic compound-degrading bacteria because it was able to degrade syringate in significant amounts only in the presence of sulfide.

ABSTRACTAlthough several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related toMethanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene fromM. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogenM. hollandicain freshwater sediments.