Transcriptional Response to Hypoxia in the Aquatic Fungus Blastocladiella emersonii

Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly down-regulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe(2+) ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1 alpha, caused a significant decrease in the levels of certain upregulated hypoxic genes.

Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.

PREDICTION OF METAL CATION TOXICITY TO THE BIOLUMINESCENT FUNGUS GERRONEMA VIRIDILUCENS

A correlation between the physicochemical properties of mono- [Li(I), K(I), Na(I)] and divalent [Cd(II), Cu(II), Mn(II), Ni(II), Co(II), Zn(II), Mg(II), Ca(II)] metal cations and their toxicity (evaluated by the free ion median effective concentration. EC50(F)) to the naturally bioluminescent fungus Gerronema viridilucens has been studied using the quantitative ion character activity relationship (QICAR) approach. Among the 11 ionic parameters used in the current study, a univariate model based on the covalent index (X(m)(2)r) proved to be the most adequate for prediction of fungal metal toxicity evaluated by the logarithm of free ion median effective concentration (log EC50(F)): log EC50(F) = 4.243 (+/-0.243) -1.268 (+/-0.125).X(m)(2)r (adj-R(2) = 0.9113, Alkaike information criterion [AIC] = 60.42). Additional two- and three-variable models were also tested and proved less suitable to fit the experimental data. These results indicate that covalent bonding is a good indicator of metal inherent toxicity to bioluminescent fungi. Furthermore, the toxicity of additional metal ions [Ag(I), Cs(I), Sr(II), Ba(II), Fe(II), Hg(II), and Pb(II)] to G. viridilucens was predicted, and Pb was found to be the most toxic metal to this bioluminescent fungus (EC50(F)): Pb(II) > Ag(I) > Hg(I) > Cd(II) > Cu(II) > Co(II) Ni(II) > Mn(II) > Fe(II) approximate to Zn(II) > Mg(II) approximate to Ba(II) approximate to Cs(I) > Li(I) > K(I) approximate to Na(I) approximate to Sr(II)> Ca(II). Environ. Toxicol. Chem. 2010;29:2177-2181. (C) 2010 SETAC

EVALUATION OF METAL TOXICITY BY A MODIFIED METHOD BASED ON THE FUNGUS GERRONEMA VIRIDILUCENS BIOLUMINESCENCE IN AGAR MEDIUM

Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+), Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables LIS to propose a metal toxicity series to Gerronenia viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximate to Ni(2+) approximate to Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximate to Na(+) > Ca(2+), and to shed some li-ht on the mechanism of toxic action of metal cations to basidiomycete fungi. Environ. Toxicol. Chem. 2010;29:320-326. (C) 2009 SETAC

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

Xylarenones C-E from an Endophytic Fungus Isolated from Alibertia macrophylla

Xylarenones C-E (2-4), three new eremophilane sesquiterpenes, have been isolated from solid substrate cultures of a Camarops-like endophytic fungus isolated from Alibertia macrophylla. The structures were elucidated by analysis of spectroscopic data. Compounds were evaluated in subtilisin and pepsin protease assays, and compound 2 showed potent inhibitory activity against both proteases.

Isolation of Brazilian marine fungi capable of growing on DDD pesticide

The fungi Aspergillus sydowii Ce15, Aspergillus sydowii Ce19, Aspergillus sydowii Gc12, Bionectria sp. Ce5, Penicillium miczynskii Gc5, Penicillium raistrickii Ce16 and Trichoderma sp. Gc1, isolated from marine sponges Geodia corticostylifera and Chelonaplysylla erecta, were evaluated for their ability to grow in the presence of DDD pesticide. Increasing concentrations of DDD pesticide, i.e., 5.0 mg (1.56 x 10(-12) mmol), 10.0 mg (3.12 x 10(-2) mmol) and 15.0 mg (4.68 x 10(-2) mmol) in solid and liquid culture media were tested. The fungi Trichoderma sp. Gc1 and Penicillium miczynskii Gc5 were able to grow in the presence of up to 15.0 mg of DDD, suggesting their potential for biodegradation. A 100% degradation of DDD was attained in liquid culture medium when Trichoderma sp. Gc1 was previously cultivated for 5 days and supplemented with 5.0 mg of DDD in the presence of hydrogen peroxide. However, the quantitative analysis showed that DDD was accumulated on mycelium and biodegradation level reached a maximum value of 58% after 14 days.

Biotransformation of alpha-Bromoacetophenones by the Marine Fungus Aspergillus sydowii

The biotransformation reactions of alpha-bromoacetophenone (1), p-bromo-alpha-bromoacetophenone (2), and p-nitro-alpha-bromoacetophenone (3) by whole cells of the marine fungus Aspergillus sydowii Ce19 have been investigated. Fungal cells that had been grown in artificial sea water medium containing a high concentration of chloride ions (1.20 M) catalysed the biotransformation of 1 to 2-bromo-1-phenylethanol 4 (56%), together with the alpha-chlorohydrin 7 (9%), 1-phenylethan-1,2-diol 9 (26%), acetophenone 10 (4%) and phenylethanol 11 (5%) identified by GC-MS analysis. In addition, it was observed that the enzymatic reaction was accompanied by the spontaneous debromination of 1 to yield alpha-chloroacetophenone 5 (9%) and alpha-hydroxyacetophenone 6 (18%) identified by GC-FID analysis. When 2 and 3 were employed as substrates, various biotransformation products were detected but the formation of halohydrins was not observed. It is concluded that marine fungus A. sydowii Ce19 presents potential for the biotransformations of bromoacetophenone derivatives.

Sesquiterpenes from Xylaria sp., an endophytic fungus associated with Piper aduncum (Piperaceae)

Two new presilphiperfolane sesquiterpenes, 1 and 2, were isolated from the ethyl acetate extract of Xylaria sp., obtained from the leaves of Piper aduncum, along with two known eremophilane sesquiterpenes, phaseolinone (3) and phomenone (4). Chemical structures of 1 and 2 were established by analysis of spectroscopic data. The four compounds were tested in vitro for antifungal and cytotoxicity activities using CHO (Chinese hamster ovary). Compounds 1 and 2 did not show any antifungal and cytotoxic activity. Compounds 3 and 4 displayed moderate cytotoxic activities, as well as 4 antifungal activity. (C) 2010 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved.

Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases

Sunflower trypsin inhibitor-1 (SFI-1), a natural 14-residue cyclic peptide, and some of its synthetic acyclic variants are potent protease inhibitors displaying peculiar inhibitory profiles. Here we describe the synthesis and use of affinity sorbents prepared by coupling SFTI-1 analogues to agarose resin. Chymotrypsinand trypsin-like proteases could then be selectively isolated from pancreatin; similarly, other proteases were obtained from distinct biological sources. The binding capacity of [Lys5]-SFTI-1-agarose for trypsin was estimated at over 10 mg/mL of packed gel. SFTI-1-based resins could find application either to improve the performance of current purification protocols or as novel protease-discovery tools in different areas of biological investigation. (C) 2009 Elsevier B.V. All rights reserved.