Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli–maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP₁) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.     

Removal of malachite green from aqueous solution by immobilized Pseudomonas sp. DY1 with Aspergillus oryzae

Triphenylmethane dyes are considered to be one of the most recalcitrant pollutants in the environment. Malachite Green (MG) was successfully removed from aqueous solution by Pseudomonas sp. DY1 immobilization with Aspergillus oryzae. Inhibition test in the presence of sodium azide and nystatin indicated that A. oryzae was a natural immobilization reagent, and removal of MG by the immobilized cell pellets was attributed to the biodegradation by Pseudomonas sp. DY1. Optimum conditions of immobilization for maximum biodegradation were obtained using Taguchi design at 37 °C, inoculation size of Pseudomonas sp. DY1 (dry cell mass) 0.01 g, of A. oryzae (spore number) 1.0 × 10⁹, initial pH 6.5. Decolorization and biodegradation of MG by immobilized pellets under optimum conditions were 99.5% and 93.3%, respectively. Immobilized pellets exhibited more than 96% decolorization after 16 days in batch condition, indicating it had stable and high biodegradation capabilities when immobilized for long-term operation.     

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91 725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100 000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5–5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.     

Aspernolides A and B, butenolides from a marine-derived fungus Aspergillus terreus

Two aromatic butenolides, aspernolides A and B along with the known metabolites, butyrolactone I, terrein and physcion were isolated from the fermentation broth of a soft coral derived fungus Aspergillus terreus. The structures of these metabolites were assigned on the basis of detailed spectroscopic analysis. The absolute stereochemistry of aspernolides A (1) and B (2) was established by their preparation from the known butyrolactone I. Biogenetically aspernolides A and B must be derived from butyrolactone I, a well known specific inhibitor of cyclin dependent kinase (cdk) from A. terreus. When tested, aspernolide A exhibited mild cytotoxicity against cancer cell lines.     

Targeted gene deletion in Aspergillus fumigatus using microbial machinery and a recyclable marker

The emerging invasive fungal pathogen Aspergillus fumigatus causes very serious infections among immunocompromised patient populations. While the genome of this pathogen has been sequenced, a major barrier to better understanding the complex biology of this eukaryotic organism is a lack of tools for efficient genetic manipulation. To improve upon this, we have generated a new gene deletion system for A. fumigatus using yeast recombinational cloning and Agrobacterium tumefaciens mediated transformation (ATMT) employing a recyclable marker system. This system reduced the time for generating a gene deletion strain in our hands by two-thirds (12 weeks to 3 weeks) using minimal human labor, and we demonstrate that it can be used to efficiently generate multiple gene deletions within a single strain.     

Extension of the breeding season and its effects on fertilization and development in two species of lugworm (Arenicola marina and A. defodiens)

In the autumn/winter breeding polychaetes, Arenicola marina and A. defodiens, spawning can be advanced or delayed by a number of months through temperature manipulation of the adults. However, this manipulation may have significant consequences for fertilization rates and embryo developmental success and so in vitro fertilizations were performed to assess the impact of manipulation. Firstly, we used oocytes and sperm obtained from advanced or delayed individuals. For both species, using gametes from 4 weeks advanced individuals did not result in a significant reduction in development, however, gametes from individuals advanced (A. marina only) or delayed by 8 weeks resulted in significantly fewer embryos developing normally. Reciprocal crosses of temperature-manipulated A. marina gametes (from 4 weeks advanced and 4 weeks delayed individuals) with those at the natural spawning time confirmed that the reduction in developmental success in both was attributable to the oocytes. After 5 h post-fertilization, the majority of oocytes from advanced individuals had fertilized, but by 24 h most were abnormal. For fertilizations with gametes from delayed individuals, nearly 100% of the embryos were developing normally after 24 h, but after 144 h significantly more were abnormal in crosses involving oocytes from delayed females. Although both species have reproductive plasticity to extend their breeding season, the significant reduction in the numbers of competent larvae produced as the spawning is delayed or advanced further may be a significant bottleneck in aquaculture and it may also have considerable implications for the long-term reproductive success of a population in response to environmental change.Sympatric populations of the species exist in many locations and the inherent variability in the breeding seasons could allow spawning times to overlap. Artificially delaying A. marina individuals enabled fertilizations to be performed with A. defodiens at the natural spawning time in the laboratory. Both conspecific fertilizations produced 100% trochophore larvae after 120 h, but A. defodiens oocytes failed to fertilize after incubation with A. marina sperm, in comparison to the A. marina oocytes incubated with A. defodiens sperm where 40% developed to the trochophore stage. This asymmetric gamete incompatibility may be one of a suite of mechanisms to minimise hybridisation.     

Genetic diversity and inter-specific relations of western Mediterranean relic Abies taxa as compared to the Iberian A. alba

Several Abies species are currently present in the Mediterranean region and most of them are endemic taxa and tertiary relicts. Using six nuclear microsatellites, we studied the genetic structure and inter-specific relationships among West Mediterranean firs, A. pinsapo (Spain), A. maroccana and A. tazaotana (Morocco). Based on the hypothesis that A. pinsapo could historically exchange genes with A. alba growing in the Pyrenees via secondary contact, we investigated the level of genetic admixture between these species using a Bayesian approach. The studied populations showed moderate genetic diversity (mean H_E = 0.598) and a high level of genetic differentiation (F_ST = 0.225) that was especially pronounced between A. alba and the African firs. All populations experienced a strong bottleneck effect that was likely induced by climatic changes occurring in the West Mediterranean during the last glacial cycle and the Holocene. According to Bayesian clustering, both African taxa grouped together in a single cluster, the two A. pinsapo populations formed a second cluster, and two additional clusters were detected within A. alba. Our results indicate that A. tazaotana is genetically very close to A. maroccana, and hence these two taxa should probably not be considered as separate species. We found no genetic admixture between A. pinsapo and A. alba and only minor between A. pinsapo and the African fir populations suggesting an isolation effect of the Gibraltar Strait. Current limited distributions of firs in the Mediterranean region together with changing climate may lead to further deterioration of the genetic diversity levels. Hence, future efforts should focus on monitoring the demography and genetic threats to existing populations.     

Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation

This study aimed to assess physicochemical and microbiological properties of fresh spent mushroom substrates (SMSs) – without post-crop heat treatment – from Agaricus subrufescens and Lentinula edodes production to optimize the use of these residues in the soil enrichment for lettuce growth promotion and soil remediation. Organic matter and C content of both SMSs were high. Fresh A. subrufescens SMS was a good source of N, P and K. On the other hand, L. edodes SMS presented a lower concentration of these nutrients and a high level of immaturity. Both SMSs presented high electric conductivity values (2.5–3.4 mS/cm). Microbiological analysis, based upon enumeration of culturable bacteria (thermophilic and mesophilic) and fungi, and also evolution of CO₂, showed that SMSs played higher microbial diversity than soil control. Laccase activity from A. subrufescens SMS tended to remain constant during a 2-month period, while L. edodes SMS presented low laccase activity throughout the same period. Agaricus subrufescens and L. edodes were able to grow on a PDA (Potato Dextrose Agar) media supplemented with different concentrations of atrazine (1–50 μg/ml), degraded the herbicide, attaining rates of 35% and 26%, respectively. On experiments of lettuce growth promotion using a soil-based potting media with different SMS rates, 5% and 10% (dw) rates of A. subrufescens SMS resulted in higher lettuce aerial dry weights than the rates of 25% and 40%, the chemical fertilization (NPK) and the control (soil). At 10% supplementation, lettuce aerial dry weight increased 2.2 and 1.3 times compared to the control and the NPK treatment, respectively. Protein content increased along with SMS rates. Fresh A. subrufescens SMS was an excellent supplement for lettuce growth promotion and showed potential for remediation of biocides possibly due to improved microbial diversity and enzymatic activity. Fresh L. edodes SMS was not a good fertilizer, at least under the conditions tested. However, microbiological analysis showed that promising results may be achieved when using fresh L. edodes SMS for soil remediation.     

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger

Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL “Amano” produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 °C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by ¹H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.     

Prenylated acetophenones from Melicope obscura and Melicope obtusifolia ssp. obtusifolia var. arborea and their distribution in Rutaceae

Four prenylated acetophenones 2,6-dihydroxy-4-geranyloxyacetophenone (1), 4-geranyloxy-2,6,β-trihydroxyacetophenone (2), 2,6-dihydroxy-4-geranyloxy-3-prenylacetophenone (3), and 4-geranyloxy-3-prenyl-2,6,β-trihydroxyacetophenone (4) have for the first time been isolated from Melicope obscura (1 and 2) and Melicope obtusifolia ssp. obtusifolia var. arborea (3 and 4). The distribution of prenylated acetophenones in Rutaceae is reviewed and the results, including the new records, indicate that prenylated acetophenones are valuable as chemotaxonomic markers for the subfamily Rutoideae, tribe Xanthoxyleae sensu Engler.     

Comparative bioluminescence dynamics among multiple Armillaria gallica, A. mellea, and A. tabescens genets

Bioluminescence is well known among white-spored species of Basidiomycota including several species of the white-rot wood decay genus Armillaria. Previous work demonstrated consistent differences among A. gallica, A. mellea, and A. tabescens in luminescence magnitude and in luminescence expression relative to environmental stimuli. In the present studies, temporal fluctuations in mycelial luminescence were quantitatively characterized using genets matched for geographical location. All genets derived from rhizomorphs or basdiomata were constitutively luminescent while six of 13 genets originating from mycelial fans were inconsistently luminescent. Using time series of 1000 consecutive measurements over 800 ms intervals, fluctuation patterns had significantly quantifiable structure and were not simply ‘white noise’. Fluctuation patterns were qualitatively similar with alternating periods of rapid fluctuation and relative stability, regardless of luminescence magnitude. Anomalous spikes or shifts in luminescence were recorded for several genets suggesting further work to identify the transient stimuli which elicited these altered luminescence patterns.     

Mycoplasma feriruminatoris sp. nov., a fast growing Mycoplasma species isolated from wild Caprinae

Five Mycoplasma strains from wild Caprinae were analyzed: four from Alpine ibex (Capra ibex) which died at the Berlin Zoo between 1993 and 1994, one from a Rocky Mountain goat collected in the USA prior to 1987. These five strains represented a population different from the populations belonging to the ‘Mycoplasma mycoides cluster’ as tested using multi locus sequence typing, Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and DNA–DNA hybridization. Analysis of the 16S rRNA gene (rrs), genomic sequence based in silico as well as laboratory DNA–DNA hybridization, and the analysis of phenotypic traits in particular their exceptionally rapid growth all confirmed that they do not belong to any Mycoplasma species described to date. We therefore suggest these strains represent a novel species, for which we propose the name Mycoplasma feriruminatoris sp. nov. The type strain is G5847^T (= DSM 26019^T = NCTC 1362^T).     

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E₂ 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E₂ titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E₂ or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E₂ raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.     

Secondary metabolites from Centaurea ensiformis P.H. Davis

Centaurea ensiformis P.H. Davis was evaluated for its secondary metabolites. 20 different compounds have been isolated and identified; four phenolic compounds, one aminoacid, two acetophenone glycosides, three phenylpropanoide glycosides, one coumarin glucoside, four flavon glycosides, two neolignan glycosides, two megastigmane glycosides and schikimic acid methyl ester.     

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.     

Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli

NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitrogen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity using novel reactive dye-affinity resins. The pure enzyme was extensively characterized for its biochemical and kinetic properties and compared with its well studied Aspergillus niger counterpart. The A. terreus NADP-GDH was more stable and showed non-competitive ammonium inhibition with respect to glutamate. It exhibited hyperbolic 2-oxoglutarate saturation albeit with a weak substrate inhibition. This is in contrast to the allosteric nature of the enzyme from other Aspergilli. Differential susceptibility to chymotrypsin is also consistent with the absence of substrate cooperativity and conformational changes associated with A. terreus NADP-GDH. The non-allosteric nature of A. terreus NADP-GDH provides a unique opportunity to assess the contribution of allostery in metabolic regulation.     

Characterization of two related exoantigens from the biodeteriogenic fungus Aspergillus versicolor

Aspergillus versicolor is one of the most common fungi in damp buildings in U. K., various European and Scandinavian countries as well as the United States and Canada. It is a proxy for species that occur at similar material water activities. Based on studies from Finland, Norway and Germany, it is among the common species resulting in an IgE reaction. Using pre-screened human sera with antibodies to various fungi, two related proteins were discovered with molecular weights 43 and 41 kDa based on SDS electrophoresis. Both proteins were excreted on the surfaces of spores and into culture media. The 41 kDa protein has a pI of 4.5. Based on a partial sequence, it is a serine protease. There are a number of Aspergillus proteases with overlapping sequences but these have different molecular weights and pI values. Polyclonal and monoclonal antibodies were developed that were specific compared to a diverse taxonomic array of related and unrelated fungi that commonly occur in the built environment. Initially this was done to ensure the specificity of the target protein. The measurement of other allergens and antigens associated with the built environment has a number of uses. Most importantly, these can be used to assess reliably biodeterioration and contribute to improved exposure assessments for population health studies.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.