Biodegradation of polyester polyurethane during commercial composting and analysis of associated fungal communities.

In this study the biodegradation of polyurethane (PU) during the maturation stage of a commercial composting process was investigated. PU coupons were buried in the centre and at the surface of a 10 m high compost pile. Fungal communities colonising polyester PU coupons were compared with the native compost communities using culture based and molecular techniques. Putative polyester PU degrading fungi were ubiquitous in compost and rapidly colonised the surface of polyester PU coupons with significant deterioration. As the temperature decreased, fungal diversity in the compost and on the surface of the polyester PU coupons increased and selection of fungal community on the polyester PU coupons occurs that is different from the surrounding compost.

Effect of phosphatidylcholine on the level expression of plc genes of Aspergillus fumigatus by real time PCR method and investigation of these genes using bioinformatics analysis.

BACKGROUND AND OBJECTIVES: Phosphlipases are a group of enzymes that breakdown phosphatidylcholine (phospholipids) molecules producing second products. These produced products have a divers role in the cell like signal transduction and digestion in humans. In this research the effect of phosphatidylcholine on the expression of plc genes of A. fumigatus was studied. The plc genes of this fungus were also interrogated using bioinformatics studies. MATERIALS AND METHODS: Real-time PCR was performed to study the expression of plc genes and these genes were interrogated using bioinformatics studies. RESULTS: There was more significant expression for all three plc genes when A. fumigatus was grown on the presence of phosphatidylcholine in the medium. The sequence of plc genes of A. fumigatus was also interrogated using bioinformatics analysis and their relationship with the other microorganisms was investigated. CONCLUSION: Real-time PCR revealed that afplc1, afplc2 and afplc3 were up-regulated in the presence of phosphatidylcholine. In this study we suggest either the plc's of A. fumigatus were present in an ancestral genome and have become lost in some lineages, or that they have been acquired from other organisms by horizontal gene transfer. We also found that plc's of this fungus appeared to be more closely related to the plant plc's than the bacterial plc's.

Occurrence of azole-resistant species of Aspergillus in the UK environment.

The aim of this study was to survey environmental isolates of Aspergillus resistant to azoles in azole-treated and naïve areas to determine whether resistance could be related to azole treatment history. Aspergillus fumigatus was sampled from the centre of a large city and from fields with known azole history. Azole resistance was determined and sequencing was performed to identify strains and mutations in the cyp51A gene. Azole resistance was detected in azole-treated field isolates but not in urban isolates (P=0.038). In addition, an azole-resistant isolate of Neosartorya fischeri was isolated. These results support the hypothesis that agricultural azole use may lead to resistance in environmental fungi of clinical importance. We report the first environmental UK TR34/L98H isolate of A. fumigatus.

Isolation and molecular characterization of polyvinyl chloride (PVC) plastic degrading fungal isolates.

The recalcitrant nature of polyvinyl chloride creates serious environmental concerns during manufacturing and waste disposal. The present study was aimed to isolate and screen different soil fungi having potential to biodegrade PVC films. After 10 months of soil burial experiment, it was observed that a number of fungal strains were flourishing on PVC films. On morphological as well as on 18rRNA gene sequence and phylogenetic basis they were identified as Phanerochaete chrysosporium PV1, Lentinus tigrinus PV2, Aspergillus niger PV3, and Aspergillus sydowii PV4. The biodegradation ability of these fungal isolates was further checked in shake flask experiments by taking thin films of PVC (C source) in mineral salt medium. A significant change in color and surface deterioration of PVC films was confirmed through visual observation and Scanning electron microscopy. During shake flask experiments, P. chrysosporium PV1 produced maximum biomass of about 2.57 mg ml(-1) followed by A. niger PV3. P. chrysosporium PV1 showed significant reduction (178,292 Da(-1)) in Molecular weight of the PVC film than control (200,000 Da(-1)) by gel permeation chromatography. Furthermore more Fourier transform infrared spectroscopy and nuclear magnetic resonance also revealed structural changes in the PVC. It was concluded that isolated fungal strains have significant potential for biodegradation of PVC plastics.

An investigation of anthraquinone dye biodegradation by immobilized Aspergillus flavus in fluidized bed bioreactor.

PURPOSE: Biodegradation and biodecolorization of Drimarene blue K(2)RL (anthraquinone) dye by a fungal isolate Aspergillus flavus SA2 was studied in lab-scale immobilized fluidized bed bioreactor (FBR) system. METHOD: Fungus was immobilized on 0.2-mm sand particles. The reactor operation was carried out at room temperature and pH 5.0 in continuous flow mode with increasing concentrations (50, 100, 150, 200, 300, 500 mg l(-1)) of dye in simulated textile effluent on the 1st, 2nd, 5th, 8th, 11th, and 14th days. The reactors were run on fill, react, settle, and draw mode, with hydraulic retention time (HRT) of 24-72 h. Total run time for reactor operation was 17 days. RESULTS: The average overall biological oxygen demand (BOD), chemical oxygen demand (COD), and color removal in the FBR system were up to 85.57%, 84.70%, and 71.3%, respectively, with 50-mg l(-1) initial dye concentration and HRT of 24 h. Reductions in BOD and COD levels along with color removal proved that the mechanism of biodecolorization and biodegradation occurred simultaneously. HPLC and LC-MS analysis identified phthalic acid, benzoic acid, 1, 4-dihydroxyanthraquinone, 2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione, and catechol as degradation products of Drimarene blue K(2)RL dye. Phytotoxicity analysis of bioreactor treatments provided evidence for the production of less toxic metabolites in comparison to the parent dye. CONCLUSION: The present fluidized bed bioreactor setup with indigenously isolated fungal strain in its immobilized form is efficiently able to convert the parent toxic dye into less toxic by-products.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

An HPLC method development for the assessment of degradation products of anthraquinone dye.

This paper describes the development of a simple and sensitive method with reduced run time for the estimation of biodegradation product of an anthraquinone dye, Drimarene blue K(2)RL. The chromatographic analysis was performed using a reversed-phase high performance liquid chromatography (HPLC) with a Lichrospher® RP-18 column, 5 µm particle size, 25 cm × 4.6 mm internal diameter using a 70:20:10 (v/v) mixture of acetonitrile-ammonium acetate buffer (0.02 M) with 0.8% Trifluoroacetic acid (pH 2.5) and methanol as eluent. Flow rate was adjusted to 1.2 mL min(-1). The metabolites (phthalic acid, benzoic acid, 1, 4-dihydroxyanthraquinone, and 2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione) were identified by running HPLC grade standards in defined concentrations. The retention time of the compounds were 2.0, 2.5, 5.2, and 7.2 min for phthalic acid, benzoic acid, 1, 4-dihydroxyanthraquinone, and 2,3-dihydro- 9,10-dihydroxy-1,4-anthracenedione, respectively. The reliability, sensitivity, and validation of the method were checked by calculating recoveries of the individual compounds in the acetonitrile and dye degradation media. The lower limits of detection for anthraquinone metabolites and the separation of acid and anthraquinone metabolites in short time were achieved.

Characterisation of Aft1 a Fot1/Pogo type transposon of Aspergillus fumigatus.

In recent years the filamentous fungus Aspergillus fumigatus has become a significant cause of infection in man and as such has become the focus of much study. It is thought to be the leading mould pathogen in leukaemia and transplant patients and is responsible for mortality in a large number of individuals with immunological disorders. In an attempt to develop molecular mutagenesis tools for assessment of this organism, the genome of A. fumigatus was analysed to identify possible functional transposable elements. An apparently intact Fot1/Pogo type transposon with 65% identity to the active Tan1 element of Aspergillus niger was identified and designated Aft1. Aft1 is a 1.9kb element present in multiple (>20) highly conserved copies. It encodes a 332 amino acid transposase which contains all the functional motifs required for transposition. In addition, the transposase was expressed in cultures grown at 37 degrees C in all three strains assessed and excision analysis suggests Aft1 may be active and of use in transposon tagging experiments. Southern hybridisation patterns indicate that Aft1 is widely distributed amongst clinical isolates of A. fumigatus with considerable variation in genomic localisation. A comprehensive analysis of the genomic localisation of Aft1 in the sequenced strain AF293 show that one insertion is 30 bases upstream of a predicted gene encoding a G-protein coupled receptor. Expression analysis indicates that this gene has been inactivated by the insertion.

Fungal communities associated with degradation of polyester polyurethane in soil.

Soil fungal communities involved in the biodegradation of polyester polyurethane (PU) were investigated. PU coupons were buried in two sandy loam soils with different levels of organic carbon: one was acidic (pH 5.5), and the other was more neutral (pH 6.7). After 5 months of burial, the fungal communities on the surface of the PU were compared with the native soil communities using culture-based and molecular techniques. Putative PU-degrading fungi were common in both soils, as <45% of the fungal colonies cleared the colloidal PU dispersion Impranil on solid medium. Denaturing gradient gel electrophoresis showed that fungal communities on the PU were less diverse than in the soil, and only a few species in the PU communities were detectable in the soil, indicating that only a small subset of the soil fungal communities colonized the PU. Soil type influenced the composition of the PU fungal communities. Geomyces pannorum and a Phoma sp. were the dominant species recovered by culturing from the PU buried in the acidic and neutral soils, respectively. Both fungi degraded Impranil and represented >80% of cultivable colonies from each plastic. However, PU was highly susceptible to degradation in both soils, losing up to 95% of its tensile strength. Therefore, different fungi are associated with PU degradation in different soils but the physical process is independent of soil type.

Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa.

Following exposure to light and attainment of steady-state in the chemostat, Neurospora was grown in constant conditions of darkness at 25 degrees C for 6 days. Biomass samples were taken every 4h for the extraction of RNA and protein, and the state of the circadian clock was assessed by assaying the levels of three rhythmically expressed mRNAs; frequency (frq), antisense frq (qrf) and clock-controlled gene-14 (ccg-14), and by monitoring the clock-controlled rhythm of sporulation. Our results indicate that the Neurospora clock continued to run in the chemostat. This is the longest reported time that Neurospora has been grown in a chemostat in filamentous form and opens up the possibility of studying the response of Neurospora to a range of stimuli in the absence of confounding effects due to; alterations in growth rate, aging, and changing conditions of the growth medium.

Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae.

BACKGROUND: Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR) pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. RESULTS: Chemostat cultures of T. reesei expressing human tissue plasminogen activator (tPA) and batch bioreactor cultures treated with dithiothreitol (DTT) to prevent correct protein folding were analysed with cDNA subtraction and cDNA-amplified fragment length polymorphism (AFLP) experiments. ESTs corresponding to 457 unique genes putatively induced under secretion stress were isolated and the expression pattern of 60 genes was confirmed by Northern analysis. Expression of these genes was also studied in a strain over-expressing inositol-requiring enzyme 1 (IREI) protein, a sensor for the UPR pathway. To compare the data with that of S. cerevisiae, published transcriptome profiling data on various stress responses in S. cerevisiae was reanalysed. The genes up-regulated in response to secretion stress included a large number of secretion related genes in both organisms. In addition, analysis of T. reesei revealed up regulation of the cpc1 transcription factor gene and nucleosomal genes. The induction of the cpcA and histone gene H4 were shown to be induced also in cultures of Aspergillus nidulans treated with DTT. CONCLUSION: Analysis of the genes induced under secretion stress has revealed novel features in the stress response in T. reesei and in filamentous fungi. We have demonstrated that in addition to the previously rather well characterised induction of genes for many ER proteins or secretion related proteins also other types of responses exist.

Comparative analysis of programmed cell death pathways in filamentous fungi.

BACKGROUND: Fungi can undergo autophagic- or apoptotic-type programmed cell death (PCD) on exposure to antifungal agents, developmental signals, and stress factors. Filamentous fungi can also exhibit a form of cell death called heterokaryon incompatibility (HI) triggered by fusion between two genetically incompatible individuals. With the availability of recently sequenced genomes of Aspergillus fumigatus and several related species, we were able to define putative components of fungi-specific death pathways and the ancestral core apoptotic machinery shared by all fungi and metazoa. RESULTS: Phylogenetic profiling of HI-associated proteins from four Aspergilli and seven other fungal species revealed lineage-specific protein families, orphan genes, and core genes conserved across all fungi and metazoa. The Aspergilli-specific domain architectures include NACHT family NTPases, which may function as key integrators of stress and nutrient availability signals. They are often found fused to putative effector domains such as Pfs, SesB/LipA, and a newly identified domain, HET-s/LopB. Many putative HI inducers and mediators are specific to filamentous fungi and not found in unicellular yeasts. In addition to their role in HI, several of them appear to be involved in regulation of cell cycle, development and sexual differentiation. Finally, the Aspergilli possess many putative downstream components of the mammalian apoptotic machinery including several proteins not found in the model yeast, Saccharomyces cerevisiae. CONCLUSION: Our analysis identified more than 100 putative PCD associated genes in the Aspergilli, which may help expand the range of currently available treatments for aspergillosis and other invasive fungal diseases. The list includes species-specific protein families as well as conserved core components of the ancestral PCD machinery shared by fungi and metazoa.

Role of the bga1-encoded extracellular {beta}-galactosidase of Hypocrea jecorina in cellulase induction by lactose.

Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major beta-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular beta-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Deltabga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular beta-galactosidase activity that was not encoded by bga1 but no intracellular beta-galactosidase activity. In conclusion, cellulase production on lactose occurs when beta-galactosidase activity levels are low but decreases as the beta-galactosidase activities increase. The data indicate that bga1-encoded beta-galactosidase activity is a critical factor for cellulase production on lactose.

Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus.

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.