Exposure of Aspergillus flavus NRRL 3357 to the Environmental Toxin, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin, Results in a Hyper Aflatoxicogenic Phenotype: A Possible Role for Caleosin/Peroxygenase (AfPXG).

Aflatoxins (AFs) as potent food contaminants are highly detrimental to human and animal health. The production of such biological toxins is influenced by environmental factors including pollutants, such as dioxins. Here, we report the biological feedback of an active AF-producer strain of A. flavus upon in vitro exposure to the most toxic congener of dioxins, the 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD). The phenotype of TCDD-exposed A. flavus was typified by a severe limitation in vegetative growth, activation of conidia formation and a significant boost in AF production. Furthermore, the level of reactive oxygen species (ROS) in fungal protoplast was increased (3.1- to 3.8-fold) in response to TCDD exposure at 10 and 50 ng mL-1, respectively. In parallel, superoxide dismutase (SOD) and catalase (CAT) activities were, respectively, increased by a factor of 2 and 3. In contrast to controls, transcript, protein and enzymatic activity of caleosin/peroxygenase (AfPXG) was also significantly induced in TCDD-exposed fungi. Subsequently, fungal cells accumulated fivefold more lipid droplets (LDs) than controls. Moreover, the TCDD-exposed fungi exhibited twofold higher levels of AFB1. Interestingly, TCDD-induced hyperaflatoxicogenicity was drastically abolished in the AfPXG-silencing strain of A. flavus, suggesting a role for AfPXG in fungal response to TCDD. Finally, TCDD-exposed fungi showed an increased in vitro virulence in terms of sporulation and AF production. The data highlight the possible effects of dioxin on aflatoxicogenicity of A. flavus and suggest therefore that attention should be paid in particular to the potential consequences of climate change on global food safety.

The cytochrome P450BM-1 of Bacillus megaterium A14K is induced by 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin: Biophysical, molecular and biochemical determinants.

The current study describes biological changes in Bacillus megaterium A14K cells growing in the presence of 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin (TCDD), the most potent congener of dioxins. The results indicate that the metabolizing of 2,3,7,8-TCDD by BmA14K was accompanied with a novel morphological and biophysical profile typified by the growth of single cells with high levels of biosurfactant production, surface hydrophobicity and cell membrane permeability. Moreover, the TCDD-grown bacteria exhibited a specific fatty acid profile characterized by low ratios of branched/straight chain fatty acids (BCFAs/SCFAs) and saturated/unsaturated fatty acids (SFAs/USFAs) with a specific "signature" due to the presence of branched chain unsaturated fatty acids (BCUFAs). This was synchronized with a significant induction of P450BM-1, an unsaturated fatty acid-metabolizing enzyme in B. megaterium. Subsequently, the profile of oxygenated fatty acids in the TCDD-grown bacteria was typified by the presence of 5,6-epoxy derived from unsaturated C15, C16 and C17 fatty acids, that were absent in control bacteria. A net increase was also detected in both hydroxylated and epoxidized fatty acids, especially those derived from C15:0 and C16:1, respectively, suggesting a specific TCDD-induced "signature" of oxygenated fatty acids in BmA14K. Overall, this study sheds light on the use of B. megaterium A14K as a promising bioindicator/biodegrader of dioxins.

Identification of a dioxin-responsive oxylipin signature in roots of date palm: involvement of a 9-hydroperoxide fatty acid reductase, caleosin/peroxygenase PdPXG2.

Dioxins are highly hazardous pollutants that have well characterized impacts on both animal and human health. However, the biological effects of dioxins on plants have yet to be described in detail. Here we describe a dioxin-inducible caleosin/peroxygenase isoform, PdPXG2, that is mainly expressed in the apical zone of date palm roots and specifically reduces 9-hydroperoxide fatty acids. A characteristic spectrum of 18 dioxin-responsive oxylipin (DROXYL) congeners was also detected in date palm roots after exposure to dioxin. Of particular interest, six oxylipins, mostly hydroxy fatty acids, were exclusively formed in response to TCDD. The DROXYL signature was evaluated in planta and validated in vitro using a specific inhibitor of PdPXG2 in a root-protoplast system. Comparative analysis of root suberin showed that levels of certain monomers, especially the mono-epoxides and tri-hydroxides of C16:3 and C18:3, were significantly increased after exposure to TCDD. Specific inhibition of PdPXG2 activity revealed a positive linear relationship between deposition of suberin in roots and their permeability to TCDD. The results highlight the involvement of this peroxygenase in the plant response to dioxin and suggest the use of dioxin-responsive oxylipin signatures as biomarkers for plant exposure to this important class of xenobiotic contaminants.

Evolutionary and genomic analysis of the caleosin/peroxygenase (CLO/PXG) gene/protein families in the Viridiplantae.

Bioinformatics analyses of caleosin/peroxygenases (CLO/PXG) demonstrated that these genes are present in the vast majority of Viridiplantae taxa for which sequence data are available. Functionally active CLO/PXG proteins with roles in abiotic stress tolerance and lipid droplet storage are present in some Trebouxiophycean and Chlorophycean green algae but are absent from the small number of sequenced Prasinophyceaen genomes. CLO/PXG-like genes are expressed during dehydration stress in Charophyte algae, a sister clade of the land plants (Embryophyta). CLO/PXG-like sequences are also present in all of the >300 sequenced Embryophyte genomes, where some species contain as many as 10-12 genes that have arisen via selective gene duplication. Angiosperm genomes harbour at least one copy each of two distinct CLO/PX isoforms, termed H (high) and L (low), where H-forms contain an additional C-terminal motif of about 30-50 residues that is absent from L-forms. In contrast, species in other Viridiplantae taxa, including green algae, non-vascular plants, ferns and gymnosperms, contain only one (or occasionally both) of these isoforms per genome. Transcriptome and biochemical data show that CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression. CLO/PXG proteins can associate with cytosolic lipid droplets and/or bilayer membranes. Many of the analysed isoforms also have peroxygenase activity and are involved in oxylipin metabolism. The distribution of CLO/PXG-like genes is consistent with an origin >1 billion years ago in at least two of the earliest diverging groups of the Viridiplantae, namely the Chlorophyta and the Streptophyta, after the Viridiplantae had already diverged from other Archaeplastidal groups such as the Rhodophyta and Glaucophyta. While algal CLO/PXGs have roles in lipid packaging and stress responses, the Embryophyte proteins have a much wider spectrum of roles and may have been instrumental in the colonisation of terrestrial habitats and the subsequent diversification as the major land flora.

Arabidopsis plants exposed to dioxin result in a WRINKLED seed phenotype due to 20S proteasomal degradation of WRI1.

Dioxins are highly toxic persistent organic pollutants bioaccumulated by both plants and animals that cause severe developmental abnormalities in humans. We investigated the effects of dioxins on seed development in Arabidopsis. Plants were exposed to various concentrations of the most toxic congener of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the effects on seed development were analysed in-depth at transcriptome, proteome and metabolome levels. Exposure to dioxin led to generalized effects on vegetative tissues plus a specific set of perturbations to seed development. Mature seeds from TCDD-treated plants had a characteristic 'wrinkled' phenotype, due to a two-thirds reduction in storage oil content. Transcriptional analysis of a panel of genes related to lipid and carbohydrate metabolism was consistent with the observed biochemical phenotypes. There were increases in WRI1 and LEC1 expression but decreases in ABI3 and FUS3 expression, which is puzzling in view of the low seed oil phenotype. This anomaly was explained by increased expression of 20S proteasome components that resulted in a substantial degradation of WRI1 protein, despite the up-regulation of the WRI1 gene. Our findings reveal novel effects of dioxins that lead to altered gene regulation patterns that profoundly affect seed development in Arabidopsis.

The Peroxygenase Activity of the Aspergillus flavus Caleosin, AfPXG, Modulates the Biosynthesis of Aflatoxins and Their Trafficking and Extracellular Secretion via Lipid Droplets.

Aflatoxins (AF) are highly detrimental to human and animal health. We recently demonstrated that the Aspergillus flavus caleosin, AfPXG, had peroxygenase activity and mediated fungal development and AF accumulation. We now report the characterization of an AfPXG-deficient line using reference strain NRRL3357. The resulting fungal phenotype included a severe decrease in mycelium growth, failure to sporulate, and reduced AF production. Increasing cellular oxidative status by administration of hydrogen peroxide and cumene hydroperoxide did not restore the AfPXG-deficient phenotype, which suggests that AfPXG-deficiency is not directly related to oxidative stress. To investigate possible alternative roles of AfPXG, a gain of function approach was used to overexpress AfPXG, with the reporter gene Gfp, in an AfPXG-deficient line, termed AfPXG+ . The resulting phenotype included elevated numbers of stable lipid droplets (LDs) plus enhanced AF production. Highly purified LDs from AfPXG+ cultures sequestered AF and this ability was positively correlated with overall LD number. Site-specific mutagenesis of AfPXG to delete Histidine 85 (AfPXGHis85), a residue essential for its catalytic activity, or deletion of the putative LD targeting domain (AfPXGD126-140), showed that AfPXG-peroxygenase activity was required for AF biosynthesis and that integration of AF into LDs was required for their export via a LD-dependent pathway. Ectopic expression in fungal cells of the plant LD-associated protein, oleosin, also resulted in both additional LD accumulation and enhanced AF secretion. These results suggest that both fungal LDs and their associated caleosin proteins are intimately involved in the biosynthesis, trafficking, and secretion of AF.

Biochemical, Molecular, and Transcriptional Highlights of the Biosynthesis of an Effective Biosurfactant Produced by Bacillus safensis PHA3, a Petroleum-Dwelling Bacteria.

Petroleum crude oil (PCO)-dwelling microorganisms have exceptional biological capabilities to tolerate the toxicity of petroleum contaminants and are therefore promising emulsifier and/or degraders of PCO. This study describes a set of PCO-inhabiting bacterial species, one of which, identified as Bacillus safensis PHA3, produces an efficient biosurfactant which was characterized as a glycolipid. Fourier transform infrared spectrometer, nuclear magnetic resonance, Thin layer chromatography, HPLC, and GC-MS analysis of the purified biosurfactant revealed that the extracted molecule under investigation is likely a mannolipid molecule with a hydrophilic part as mannose and a hydrophobic part as hexadecanoic acid (C16:0). The data reveal that: (i) PHA3 is a potential producer of biosurfactant (9.8 ± 0.5 mg mL-1); (ii) pre-adding 0.15% of the purified glycolipid enhanced the degradation of PCO by approximately 2.5-fold; (iii) the highest emulsifying activity of biosurfactant was found against the PCO and the lowest was against the naphthalene; (iv) the optimal PCO-emulsifying activity was found at 30-60°C, pH 8 and a high salinity. An orthologous gene encodes a putative ß-diglucosyldiacylglycerol synthase (ß-DGS) was identified in PHA3 and its transcripts were significantly up-regulated by exogenous PAHs, i.e., pyrene and benzo(e)pyrene but much less by mid-chain n-alkanes (ALKs) and fatty acids. Subsequently, the accumulation of ß-DGS transcripts coincided with an optimal growth of bacteria and a maximal accumulation of the biosurfactant. Of particular interest, we found that PHA3 actively catalyzed the degradation of PAHs notably the pyrene and benzo(e)pyrene but was much less effective in the mono-terminal oxidation of ALKs. Such characteristics make Bacillus safensis PHA3 a promising model for enhanced microbial oil recovery and environmental remediation.

Biochemical, Transcriptional, and Bioinformatic Analysis of Lipid Droplets from Seeds of Date Palm (Phoenix dactylifera L.) and Their Use as Potent Sequestration Agents against the Toxic Pollutant, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin.

Contamination of aquatic environments with dioxins, the most toxic group of persistent organic pollutants (POPs), is a major ecological issue. Dioxins are highly lipophilic and bioaccumulate in fatty tissues of marine organisms used for seafood where they constitute a potential risk for human health. Lipid droplets (LDs) purified from date palm, Phoenix dactylifera, seeds were characterized and their capacity to extract dioxins from aquatic systems was assessed. The bioaffinity of date palm LDs toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins was determined. Fractioned LDs were spheroidal with mean diameters of 2.5 µm, enclosing an oil-rich core of 392.5 mg mL(-1). Isolated LDs did not aggregate and/or coalesce unless placed in acidic media and were strongly associated with three major groups of polypeptides of relative mass 32-37, 20-24, and 16-18 kDa. These masses correspond to the LD-associated proteins, oleosins, caleosins, and steroleosins, respectively. Efficient partitioning of TCDD into LDs occurred with a coefficient of log K LB/w,TCDD = 7.528 ± 0.024; it was optimal at neutral pH and was dependent on the presence of the oil-rich core, but was independent of the presence of LD-associated proteins. Bioinformatic analysis of the date palm genome revealed nine oleosin-like, five caleosin-like, and five steroleosin-like sequences, with predicted structures having putative lipid-binding domains that match their LD stabilizing roles and use as bio-based encapsulation systems. Transcriptomic analysis of date palm seedlings exposed to TCDD showed strong up-regulation of several caleosin and steroleosin genes, consistent with increased LD formation. The results suggest that the plant LDs could be used in ecological remediation strategies to remove POPs from aquatic environments. Recent reports suggest that several fungal and algal species also use LDs to sequester both external and internally derived hydrophobic toxins, which indicates that our approach could be used as a broader biomimetic strategy for toxin removal.

Specific Caleosin/Peroxygenase and Lipoxygenase Activities Are Tissue-Differentially Expressed in Date Palm (Phoenix dactylifera L.) Seedlings and Are Further Induced Following Exposure to the Toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two caleosin/peroxygenase isoforms from date palm, Phoenix dactylifera L., PdCLO2 and PdCLO4, were characterized with respect to their tissue expression, subcellular localization, and oxylipin pathway substrate specificities in developing seedlings. Both PdCLO2 and PdCLO4 had peroxygenase activities that peaked at the mid-stage (radicle length of 2.5 cm) of seedling growth and were associated with the lipid droplet (LD) and microsomal fractions. Recombinant PdCLO2 and PdCLO4 proteins heterologously expressed in yeast cells were localized in both LD and microsomal fractions. Each of the purified recombinant proteins exhibited peroxygenase activity but they were catalytically distinct with respect to their specificity and product formation from fatty acid epoxide and hydroxide substrates. We recently showed that date palm CLO genes were upregulated following exposure to the potent toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hanano et al., 2016), and we show here that transcripts of 9- and 13-lipoxygenase (LOX) genes were also induced by TCDD exposure. At the enzyme level, 9-LOX and 13-LOX activities were present in a range of seedling tissues and responded differently to TCDD exposure, as did the 9- and 13-fatty acid hydroperoxide reductase activities. This demonstrates that at least two branches of the oxylipin pathway are involved in responses to the environmental organic toxin, TCDD in date palm.

Differential tissue accumulation of 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin in Arabidopsis thaliana affects plant chronology, lipid metabolism and seed yield.

BACKGROUND: Dioxins are one of the most toxic groups of persistent organic pollutants. Their biotransmission through the food chain constitutes a potential risk for human health. Plants as principal actors in the food chain can play a determinant role in removing dioxins from the environment. Due to the lack of data on dioxin/plant research, this study sets out to determine few responsive reactions adopted by Arabidopsis plant towards 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins. RESULTS: Using a high resolution gas chromatography/mass spectrometry, we demonstrated that Arabidopsis plant uptakes TCDD by the roots and accumulates it in the vegetative parts in a tissue-specific manner. TCDD mainly accumulated in rosette leaves and mature seeds and less in stem, flowers and immature siliques. Moreover, we observed that plants exposed to high doses of TCDD exhibited a delay in flowering and yielded fewer seeds of a reduced oil content with a low vitality. A particular focus on the plant fatty acid metabolism showed that TCDD caused a significant reduction in C18-unsaturated fatty acid level in plant tissues. Simultaneously, TCDD induced the expression of 9-LOX and 13-LOX genes and the formation of their corresponding hydroperoxides, 9- and 13-HPOD as well as 9- or 13-HPOT, derived from linoleic and linolenic acids, respectively. CONCLUSIONS: The current work highlights a side of toxicological effects resulting in the administration of 2,3,7,8-TCDD on the Arabidopsis plant. Similarly to animals, it seems that plants may accumulate TCDD in their lipids by involving few of the FA-metabolizing enzymes for sculpting a specific oxylipins "signature" typified to plant TCDD-tolerance. Together, our results uncover novel responses of Arabidopsis to dioxin, possibly emerging to overcome its toxicity.

A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection.

Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.

Saccharomyces cerevisiae SHSY detoxifies petroleum n-alkanes by an induced CYP52A58 and an enhanced order in cell surface hydrophobicity.

Environmental hydrocarbon contamination has a serious hazard to human health. Alkanes, the major component of hydrocarbons, can be consumed by various species of yeast. We previously identified a new strain SHSY of Saccharomyces cerevisiae with a remarkable ability to utilize the petroleum crude-oil (PCO) in aqueous solution. The current study demonstrated that the n-alkanes-assimilation activity of S. cerevisiae SHSY was related to an induced microsomal protein of 59 kDa approximately. The identified ORF encoded a protein of 517 amino acids and shared 93% sequence identity with an alkane-inducible hydroxylase CYP52A53 isolated from Scheffersomyces stipitis CBS. It was therefore referred as CYP52A58. The catalytic activity of the recombinant CYP52A58 was confirmed by the hydroxylation of n-alkanes, it showed an optimal mono-terminal hydroxylation activity toward n-hexadecane. Moreover, the ability of the yeast to use n-alkanes was accompanied with an increasing level in cell wall mannoproteins. Two differential protein bands were detected in the mannoproteins extracted from PCO-grown yeast. In parallel, a significant increase in the fatty acids content with a high degree of unsaturation was subsequently detected in the PCO-grown yeast. This study characterizes a safe and potential microorganism to remove n-alkanes from the aquatic environment.

Traceability of polychlorinated dibenzo-dioxins/furans pollutants in soil and their ecotoxicological effects on genetics, functions and composition of bacterial community.

Dioxins (PCDD/Fs) are persistent organic pollutants. Their accumulation in soil is a crucial step in their transmission through the ecosystem. Traceability of dioxin in soil was evaluated in four sites A, B, C and D considered as potential industrial PCDD/Fs sources in Syria. Our results showed that the highest pollution with dioxin (⩾50 ppt) was found in site C (vicinity of Homs refinery). In parallel, analysis of physicochemical proprieties and bacterial density of soil samples were carried out. Bacterial density differed significantly among samples between 68×10(4) and 64×10(6) CFU g(-1)DW. Analysis of 16S rRNA encoding sequences showed that the genus Bacillus was the most abundant (74.7%) in all samples, followed by the genera Arthrobacter and Klebsiella with 5.2% and 4.7%, respectively. The genera Microbacterium, Pantoea, Pseudomonas, Enterobacter and Exiguobacterium formed between 2.1% and 2.6%. Cellulomonas, Kocuria, Lysinibacillus, Staphylococcus and Streptomyces were in a minority (0.5-1%). The bacterial richness and biodiversity, estimated by DMg and H' index, were highest in the heavily polluted site. Molecular screening for angular dioxygenase (AD α-subunit) and the cytochrome P450 (CYPBM3) genes, led to identification of 41 strains as AD-positive and 31 strains as CYPBM3-positive. RT-real-time PCR analysis showed a significant abundance of AD α-subunit transcript in the heavily dioxin-polluted soils, while the expression of CYPBM3 was highest in the moderately polluted soils. Our results illustrate the microbial diversity and functionality in soil exposed to dioxin pollution. Identification of dioxin-degrading bacteria from polluted sites should allow bioremediation to be carried out.

Phytotoxicity effects and biological responses of Arabidopsis thaliana to 2,3,7,8-tetrachlorinated dibenzo-p-dioxin exposure.

Dioxins are persistent organic pollutants. Their bioaccumulation in the food chain makes dioxins a considerable risk for human health. The use of plants for removing toxic organic compounds, including dioxins, is a safe and efficient strategy. Herein we studied the toxicity effects and the biological responses in Arabidopsis thaliana to 2',3',7',8'-tetrachlorinated dibenzo-p-dioxin (TCDD) exposure. First, TCDD-induced toxicity was demonstrated using several parameters including, a decrease in seed germination, a loss in fresh weight with a striking decrease in chlorophyll content, but not in carotenoids, and an augmentation in the biomass of the lateral roots system, but not in the elongation of the primary root. Uptake of TCDD by Arabidopsis was confirmed. Responses to TCDD-exposure were marked by an enhanced level of hydrogen peroxide H2O2 production and a massive stimulation of anti-oxidative enzyme activities. Moreover, a significant variation in the transcript level of transcription factor genes, bHLH, MYB and AP2-EREBP was detected in Arabidopsis shoot and an up-regulation of WRKY, MYB and IAA was observed in the root. Our results illustrate the TCDD-induced toxicity effects and the biological responses of Arabidopsis to TCDD. Better understanding of the plants ability to detoxifydioxins would help to improve their use as a safe bioremediators.