Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag.

Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Entomopathogens for control of Hypsipyla spp

Published information on the incidence of pathogens in the field and laboratory infections of Hypsipyla spp. with entomopathogens is reviewed. In addition, some preliminary results of field collections from Ghana and Costa Rica are presented. Fungal pathogens from the Deuteromycetes have been isolated from both H. robusta Moore and H. grandella Zeller. Mermithid nematodes, Hexamermis spp., have been frequently isolated from larvae in the field and incidence of infection with these pathogens can reach significant levels. Microsporidia have been found in cadavers of larvae collected in the field but none have been identified so far. A number of pathogens of other Lepidoptera have been shown to be infectious to H. grandella , including Bacillus thuringiensis , Deuteromycete fungi and a nucleopolyhedrovirus (NPV) from Autographa californica . Hypsipyla spp. are difficult targets for microbial control, since the larvae are cryptic, occur at low density and occur sporadically. In addition, there is a low damage threshold, the plant is susceptible for a number of years and the susceptible part of the plant will rapidly outgrow any surface application. Key features of the biology of entomopathogens with relevance to the control of low density and cryptic pests are discussed. In the light of this experience, we discuss strategies to improve the possibilities of microbial control of this pest and suggest areas for research.

DNA adduction by the potent carcinogen aflatoxin B1 : mechanistic studies

Aflatoxin B1, a potently carcinogenic fungal metabolite, is converted to the biologically active form by chemical oxidation using dimethyldioxirane and enzymatically by cytochrome P450 mixed-function oxidases. Both processes give rise to mixtures of the exo- and endo-8,9-epoxides. Methanolysis studies reveal exclusive trans opening of both epoxides under neutral conditions in CH3OH and CH3OH/H2O mixtures; an SN2 mechanism is postulated. Under acidic conditions, the exo isomer gives mixtures of trans and cis solvolysis products, suggesting that the reaction is, at least in part, SN1; the endo isomer gives only the trans product. The exo isomer reacts with DNA by attack of the nitrogen atom at the 7 position of guanine on C8 of the epoxide to give the trans adduct; the endo epoxide fails to form an adduct at this or any other site in DNA. The exo isomer is strongly mutagenic in a base-pair reversion assay employing Salmonella typhimurium; the endo isomer is essentially nonmutagenic. Aflatoxin B1 and its derivatives intercalate in DNA. These results are consistent with a mechanism in which intercalation of the exo epoxide optimally orients the epoxide for an SN2 reaction with guanine but intercalation of the endo isomer places the epoxide in an orientation which precludes reaction. Thus, while the exo epoxide is a potent mutagen, the endo epoxide fails to react with DNA.

Incontinence-associated dermatitis: A cross-sectional prevalence study in the Australian acute care hospital setting

The purpose of this cross-sectional study was to identify the prevalence of incontinence and incontinence-associated dermatitis (IAD) in Australian acute care patients and to describe the products worn to manage incontinence, and those provided at the bedside for perineal skin care. Data on 376 inpatients were collected over 2 days at a major Australian teaching hospital. The mean age of the sample group was 62 years and 52% of the patients were male. The prevalence rate of incontinence was 24% (91/376). Urinary incontinence was significantly more prevalent in females (10%) than males (6%) (χ2  = 4·458, df = 1, P = 0·035). IAD occurred in 10% (38/376) of the sample group, with 42% (38/91) of incontinent patients having IAD. Semi-formed and liquid stool were associated with IAD (χ2  = 5·520, df = 1, P = 0·027). Clinical indication of fungal infection was present in 32% (12/38) of patients with IAD. Absorbent disposable briefs were the most common incontinence aids used (80%, 70/91), with soap/water and disposable washcloths being the clean-up products most commonly available (60%, 55/91) at the bedside. Further data are needed to validate this high prevalence. Studies that address prevention of IAD and the effectiveness of management strategies are also needed.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

Plant diversity predicts beta but not alpha diversity of soil microbes across grasslands worldwide

Aboveground–belowground interactions exert critical controls on the composition and function of terrestrial ecosystems, yet the fundamental relationships between plant diversity and soil microbial diversity remain elusive. Theory predicts predominantly positive associations but tests within single sites have shown variable relationships, and associations between plant and microbial diversity across broad spatial scales remain largely unexplored. We compared the diversity of plant, bacterial, archaeal and fungal communities in one hundred and forty-five 1 m2 plots across 25 temperate grassland sites from four continents. Across sites, the plant alpha diversity patterns were poorly related to those observed for any soil microbial group. However, plant beta diversity (compositional dissimilarity between sites) was significantly correlated with the beta diversity of bacterial and fungal communities, even after controlling for environmental factors. Thus, across a global range of temperate grasslands, plant diversity can predict patterns in the composition of soil microbial communities, but not patterns in alpha diversity.

Airborne viable fungi in school environments in different climatic regions – A review

Elevated levels of fungi in indoor environments have been linked with mould/moisture damage in building structures. However, there is a lack of information about “normal” concentrations and flora as well as guidelines of viable fungi in the school environment in different climatic conditions. We have reviewed existing guidelines for indoor fungi and the current knowledge of the concentrations and flora of viable fungi in different climatic areas, the impact of the local factors on concentrations and flora of viable fungi in school environments. Meta-regression was performed to estimate the average behaviour for each analysis of interest, showing wide variation in the mean concentrations in outdoor and indoor school environments (range: 101-103 cfu/m3). These concentrations were significantly higher for both outdoors and indoors in the moderate than in the continental climatic area, showing that the climatic condition was a determinant for the concentrations of airborne viable fungi. The most common fungal species both in the moderate and continental area were Cladosporium spp. and Penicillium spp. The suggested few quantitative guidelines for indoor air viable fungi for school buildings are much lower than for residential areas. This review provides a synthesis, which can be used to guide the interpretation of the fungi measurements results and help to find indications of mould/moisture in school building structures.

Design and synthesis of a screening library using the natural product scaffold 3-chloro-4-hydroxyphenylacetic acid

The fungal metabolite 3-chloro-4-hydroxyphenylacetic acid (1) was utilized in the generation of a unique drug-like screening library using parallel solution-phase synthesis. A 20-membered amide library (3–22) was generated by first converting 1 to methyl (3-chloro-4-hydroxyphenyl)acetate (2), then reacting this scaffold with a diverse series of primary amines via a solvent-free aminolysis procedure. The structures of the synthetic analogues (3–22) were elucidated by spectroscopic data analysis. The structures of compounds 8, 12, and 22 were confirmed by single X-ray crystallographic analysis. All compounds were evaluated for cytotoxicity against a human prostate cancer cell line (LNCaP) and for antiparasitic activity toward Trypanosoma brucei brucei and Plasmodium falciparum and showed no significant activity at 10 μM. The library was also tested for effects on the lipid content of LNCaP and PC-3 prostate cancer cells, and it was demonstrated that the fluorobenzyl analogues (12–14) significantly reduced cellular phospholipid and neutral lipid levels.

Infectious complications during therapy of acute leukaemia in Saudi Arabia

In 40 febrile neutropenic episodes during the induction and consolidation chemotherapy of acute leukaemia in Riyadh, 51% of organisms causing septicaemia were gram-negative, 26% gram-positive, 8% anaerobes and 15% fungi. In 21 (52%) febrile episodes there were pulmonary infiltrates; of the 12 where aetiology was known, six were due to fungi. Pulmonary infiltrates progressed to adult respiratory distress syndrome and death in nine instances. There was no significant occurrence of parasitic and tropical infections. The results show that the pattern of infections, during therapy of acute leukaemia in developing countries, may have important differences when compared with western centres. Empiric amphotericin B may need to be introduced at an earlier stage in patients with persistent fever or progressive pulmonary infiltrates.

Exposure to bioaerosols in the school environment in Brisbane, Australia

Introduction: Exposure to bioaerosols in indoor environments has been linked to various adverse health effects, such as airway disorders and upper respiratory tract symptoms. The aim of this study was to assess exposure to bioaerosols in the school environment in Brisbane, Australia. Methods: Culturable fungi and endotoxin measurements were conducted in six schools between October 2010 and May 2011. Culturable fungi (2 indoor air and 1-2 outdoor air samples per school) were assessed using a Biotest RCS High Flow Air Sampler, with a flow rate of either 50L/min or 20L/min. A rose pengar agar was used for recovery, which was incubated prior to counting and partial identification. Endotoxins were sampled (8h, 2L/min) using SKC glass fibre filters (4 indoor air samples per school) and analysed using an endpoint chromogenic LAL assay. Results: The arithmetic mean for fungi concentration in indoor and outdoor air was 710 cfu/m3(125- 1900 cfu/m3) and 524 cfu/m3 (140-1250 cfu/m3), respectively. The most frequently isolated fungal genus from the outdoor air was Cladosporium (over 40 %), followed by isolated Penicillium (21%) and Aspergillus (12%). The percent of Penicillium, Cladosporium and Aspergillus in indoor air samples was 32%, 32% and 8%, respectively. The aritmetic mean of endotoxin concentration was 0.59 EU/m3 (0-2,2 EU/m3). Discussion: The results of the current study are in agreement with previously reported studies, in that airborne fungi and endotoxin concentrations varied extensively, and were mostly dependent on climatic conditions. In addition, the indoor air mycoflora largely reflected the fungal flora present in the outdoor air, with Cladosporium being the most common in both outdoor and indoor (with Penicillium) air. In indoor air, unusually high endotoxin levels, over 1 EU/m3, were detected at 2 schools. Although these schools were not affected by the recent Brisbane floods, persistent rain prior to and during the study perios could explain the results.

Airborne culturable fungi in naturally ventilated primary school environments in a subtropical climate

There is currently a lack of reference values for indoor air fungal concentrations to allow for the interpretation of measurement results in subtropical school settings. Analysis of the results of this work established that, in the majority of properly maintained subtropical school buildings, without any major affecting events such as floods or visible mould or moisture contamination, indoor culturable fungi levels were driven by outdoor concentration. The results also allowed us to benchmark the “baseline range” concentrations for total culturable fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings. The measured concentration of total culturable fungi and three individual fungal genera were estimated using Bayesian hierarchical modelling. Pooling of these estimates provided a predictive distribution for concentrations at an unobserved school. The results indicated that “baseline” indoor concentration levels for indoor total fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings were generally ≤ 1450, ≤ 680, ≤ 480 and ≤ 90 cfu/m3, respectively, and elevated levels would indicate mould damage in building structures. The indoor/outdoor ratio for most classrooms had 95% credible intervals containing 1, indicating that fungi concentrations are generally the same indoors and outdoors at each school. Bayesian fixed effects regression modeling showed that increasing both temperature and humidity resulted in higher levels of fungi concentration.

Consistent responses of soil microbial communities to elevated nutrient inputs in grasslands across the globe

Soil microorganisms are critical to ecosystem functioning and the maintenance of soil fertility. However, despite global increases in the inputs of nitrogen (N) and phosphorus (P) to ecosystems due to human activities, we lack a predictive understanding of how microbial communities respond to elevated nutrient inputs across environmental gradients. Here we used high-throughput sequencing of marker genes to elucidate the responses of soil fungal, archaeal, and bacterial communities using an N and P addition experiment replicated at 25 globally distributed grassland sites. We also sequenced metagenomes from a subset of the sites to determine how the functional attributes of bacterial communities change in response to elevated nutrients. Despite strong compositional differences across sites, microbial communities shifted in a consistent manner with N or P additions, and the magnitude of these shifts was related to the magnitude of plant community responses to nutrient inputs. Mycorrhizal fungi and methanogenic archaea decreased in relative abundance with nutrient additions, as did the relative abundances of oligotrophic bacterial taxa. The metagenomic data provided additional evidence for this shift in bacterial life history strategies because nutrient additions decreased the average genome sizes of the bacterial community members and elicited changes in the relative abundances of representative functional genes. Our results suggest that elevated N and P inputs lead to predictable shifts in the taxonomic and functional traits of soil microbial communities, including increases in the relative abundances of faster-growing, copiotrophic bacterial taxa, with these shifts likely to impact belowground ecosystems worldwide.

Microbial oil production from palm oil empty fruit bunch hydrolysates

This study explores the potential use of empty fruit bunch (EFB) residues from palm oil processing residues, as an alternative feedstock for microbial oil production. EFB is a readily available, lignocellulosic biomass that provides cheaper substrates for oil production in comparison to the use of pure sugars. In this study, potential oleaginous microorganisms were selected based on a multi-criteria analysis (MCA) framework which utilised Analytical Hierarchy Process (AHP) with Preference Ranking Organization Method for Enrichment Evaluation (PROMETHEE) aided by Geometrical Analysis for Interactive Aid (GAIA). The MCA framework was used to evaluate several strains of microalgae (Chlorella protothecoides and Chlorella zofingiensis), yeasts (Cryptococcus albidus and Rhodotorula mucilaginosa) and fungi (Aspergillus oryzae and Mucor plumbeus) on glucose, xylose and glycerol. Based on the results of PROMETHEE rankings and GAIA plane, fungal strains A. oryzae and M. plumbeus and yeast strain R. mucilaginosa showed great promise for oil production from lignocellulosic hydrolysates. The study further cultivated A. oryzae, M. plumbeus and R. mucilaginosa on EFB hydrolysates for oil production. EFB was pretreated with dilute sulfuric acid, followed by enzymatic saccharification of solid residue. Hydrolysates tested in this study are detoxified liquid hydrolysates (LH) and enzymatic hydrolysate (EH).