Geoff Robson

Fungal siderophores: structures, functions and applications

Siderophores are low molecular weight, iron-chelating ligands produced by nearly all microorganisms. Fungi synthesize a wide range of hydroxamate siderophores. This review considers the chemical and biological aspects of these siderophores, their distribution amongst fungal genera and their possible applications. Siderophores function primarily as iron transport compounds. Expression of siderophore biosynthesis and the uptake systems is regulated by internal iron concentrations. Transport of siderophores is an energy-dependent process and is stereoselective, depending on recognition of the metal ion coordination geometry. In addition to transporting iron, siderophores have other functions and effects, including enhancing pathogenicity, acting as intracellular iron storage compounds and suppressing growth of other microorganisms. Siderophores can complex other metals apart from iron, in particular the actinides. Because of their metal-binding ability there are potential applications for siderophores in medicine, reprocessing of nuclear fuel, remediation of metal-contaminated sites and the treatment of industrial waste.

CADRE: the Central Aspergillus Data REpository 2012

The Central Aspergillus Data REpository (CADRE; http://www.cadre-genomes.org.uk) is a public resource for genomic data extracted from species of Aspergillus. It provides an array of online tools for searching and visualising features of this significant fungal genus. CADRE arose from a need within the medical community to understand the human pathogen Aspergillus fumigatus. Due to the paucity of Aspergillus genomic resources 10 years ago, the long-term goal of this project was to collate and maintain Aspergillus genomes as they became available. Since our first release in 2004, the resource has expanded to encompass annotated sequence for eight other Aspergilli and provides much needed support to the international Aspergillus research community. Recent developments, however, in sequencing technology are creating a vast amount of genomic data and, as a result, we shortly expect a tidal wave of Aspergillus data. In preparation for this, we have upgraded the database and software suite. This not only enables better management of more complex data sets, but also improves annotation by providing access to genome comparison data and the integration of high-throughput data.

Comparative analysis of programmed cell death pathways in filamentous fungi

BackgroundFungi 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.ResultsPhylogenetic 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.ConclusionOur 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.

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

BackgroundSecretion 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.ResultsChemostat 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.ConclusionAnalysis 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.

Fungal Communities Associated with Degradation of Polyester Polyurethane in Soil

ABSTRACT 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.

Role of the bga1-Encoded Extracellular β-Galactosidase of Hypocrea jecorina in Cellulase Induction by Lactose

ABSTRACT 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 β-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 β-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. Δbga1 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 β-galactosidase activity that was not encoded by bga1 but no intracellular β-galactosidase activity. In conclusion, cellulase production on lactose occurs when β-galactosidase activity levels are low but decreases as the β-galactosidase activities increase. The data indicate that bga1-encoded β-galactosidase activity is a critical factor for cellulase production on lactose.

Biofilms in water distribution systems.

This chapter describes the biofilm formation and investigates the relationship between the amount of biofilm and the composition of the pipe material. Biofilm formation at the interface between a solid substratum and a liquid is a common phenomenon in natural, medical and industrial environments. The walls of the pipes in the water distribution systems provide ideal surfaces for the microbial colonization, and the biofilms formed cause a number of problems for the water companies. The attached cells represent the major fraction of biomass in a distribution system and contribute to the continuous contamination of the water phase because bacteria are sheared from the surfaces of the pipes. Several factors influence the biofilm development. Some of these factors are temperature, nutrients, disinfectant residuals, hydraulic regime, and the characteristics of the substratum. The microbial growth on materials gives rise to three categories of problems: those of public health significance, aesthetic problems and those causing detrimental effects on the water distribution network at a major cost to water companies. Bacterial growth in biofilms is a potential threat to health as it links to the increased bacterial counts in treated water and to gastrointestinal illnesses.

Effect of pH on hen egg white lysozyme production and evolution of a recombinant strain of Aspergillus niger.

An Aspergillus niger strain (B1) transformed to produce mature hen egg white lysozyme (HEWL) from a glucoamylase fusion protein under control of the A. niger glucoamylase promoter was grown in glucose-limited chemostat culture at a dilution rate of 0.07 h-1 at various pH values. Maximum HEWL production (9.3 mg g-1; specific production rate = 0.65 mg g-1 per h) was obtained at pH 4.5. However, in chemostat culture, HEWL production was not stable at any pH tested. After 240 h in steady state, specific production decreased to only 0.03 +/- 0.01 and 0.24 +/- 0.02 mg g-1 per h at pH 6.5 and 4.5, respectively. Some isolates removed from the chemostat cultures had lost copies of the HEWL gene and when grown in shake flask cultures all of the isolates produced less HEWL than the parental strain. Morphological mutants with similar phenotypes were isolated at all pHs, but their rate of increase in the population was pH dependent, with cultures at low pH (< 4.5) being more morphologically stable than cultures at high (> 4.5) pH. The selective advantage of these mutants was also generally dependent on pH. Both yellow pigment producing mutants and brown sporulation mutants had higher selective advantages over the parental strain at high than at low pH, regardless of the pH at which they were isolated. However, the selective advantage of densely sporulating mutants was independent of pH.

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.

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.