Use of Selected Essential Oils to Control Aflatoxin Contaminated Stored Cashew and Detection of Aflatoxin Biosynthesis Gene

PubMed Central

Abd El-Aziz, Abeer R. M.; Mahmoud, Mohamed A.; Al-Othman, Monira R.; Al-Gahtani, Munirah F.

2015-01-01

Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method. PMID:25705718

Use of selected essential oils to control aflatoxin contaminated stored cashew and detection of aflatoxin biosynthesis gene.

PubMed

Abd El-Aziz, Abeer R M; Mahmoud, Mohamed A; Al-Othman, Monira R; Al-Gahtani, Munirah F

2015-01-01

Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method.

Citrate coated silver nanoparticles with modulatory effects on aflatoxin biosynthesis in Aspergillus parasiticus

NASA Astrophysics Data System (ADS)

Mitra, Chandrani

The manufacture and usage of silver nanoparticles has drastically increased in recent years (Fabrega et al. 2011a). Hence, the levels of nanoparticles released into the environment through various routes have measurably increased and therefore are concern to the environment and to public health (Panyala, Pena-Mendez and Havel 2008). Previous studies have shown that silver nanoparticles are toxic to various organisms such as bacteria (Kim et al. 2007), fungi (Kim et al. 2008), aquatic plants (He, Dorantes-Aranda and Waite 2012a), arthropods (Khan et al. 2015), and mammalian cells (Asharani, Hande and Valiyaveettil 2009) etc. Most of the toxicity studies are carried out using higher concentrations or lethal doses of silver nanoparticles. However, there is no information available on how the fungal community reacts to the silver nanoparticles at nontoxic concentrations. In this study, we have investigated the effect of citrate coated silver nanoparticles (AgNp-cit) at a size of 20nm on Aspergillus parasiticus, a popular plant pathogen and well-studied model for secondary metabolism (natural product synthesis). A. parasiticus produces 4 major types of aflatoxins. Among other aflatoxins, aflatoxin B1 is considered to be one of most potent naturally occurring liver carcinogen, and is associated with an estimated 155,000 liver cancer cases globally (Liu and Wu 2010); therefore, contaminated food and feed are a significant risk factor for liver cancer in humans and animals (CAST 2003; Liu and Wu 2010). In this study, we have demonstrated the uptake of AgNp-cit (20nm) by A. parasiticus cells from the growth medium using a time course ICP-OES experiment. It was observed that the uptake of AgNp-cit had no effect on fungal growth and significantly decreased intracellular oxidative stress. It also down-regulated aflatoxin biosynthesis at the level of gene expression of aflatoxin pathway genes and the global regulatory genes of secondary metabolism. We also observed that the

Inhibition of aflatoxin biosynthesis in Aspergillus flavus by phenolic compounds extracted of Piper betle L.

PubMed

Yazdani, Darab; Mior Ahmad, Zainal Abidin; Yee How, Tan; Jaganath, Indu Bala; Shahnazi, Sahar

2013-12-01

Food contamination by aflatoxins is an important food safety concern for agricultural products. In order to identify and develop novel antifungal agents, several plant extracts and isolated compounds have been evaluated for their bioactivities. Anti-infectious activity of Piper betle used in traditional medicine of Malaysia has been reported previously. Crude methanol extract from P. betel powdered leaves was partitioned between chloroform and water. The fractions were tested against A. flavus UPMC 89, a strong aflatoxin producing strain. Inhibition of mycelial growth and aflatoxin biosynthesis were tested by disk diffusion and macrodillution techniques, respectively. The presence of aflatoxin was determined by thin-layer chromatography (TLC) and fluorescence spectroscopy techniques using AFB1 standard. The chloroform soluble compounds were identified using HPLC-Tandem mass spectrometry technique. The results, evaluated by measuring the mycelial growth and quantification of aflatoxin B1(AFLB1) production in broth medium revealed that chloroform soluble compounds extract from P. betle dried leaves was able to block the aflatoxin biosynthesis pathway at concentration of 500μg/ml without a significant effect on mycelium growth. In analyzing of this effective fractions using HPLC-MS(2) with ESI ionization technique, 11 phenolic compounds were identified. The results showed that the certain phenolic compounds are able to decline the aflatoxin production in A. flavus with no significant effect on the fungus mycelia growth. The result also suggested P. betle could be used as potential antitoxin product.

Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

PubMed Central

Yabe, Kimiko; Nakamura, Miki; Hamasaki, Takashi

1999-01-01

We detected biosynthetic activity for aflatoxins G1 and G2 in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G1 and G2 were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B1, aflatoxin B2, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G2 from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G1 was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G1 and G2. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin. PMID:10473388

Genome-Wide Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Identifies Candidate Gene Signatures in Response to Aflatoxin Producing Fungus Aspergillus flavus.

PubMed

Bedre, Renesh; Rajasekaran, Kanniah; Mangu, Venkata Ramanarao; Sanchez Timm, Luis Eduardo; Bhatnagar, Deepak; Baisakh, Niranjan

2015-01-01

Aflatoxins are toxic and potent carcinogenic metabolites produced from the fungi Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. United States federal regulations restrict the use of aflatoxin contaminated cottonseed at >20 ppb for animal feed. Several strategies have been proposed for controlling aflatoxin contamination, and much success has been achieved by the application of an atoxigenic strain of A. flavus in cotton, peanut and maize fields. Development of cultivars resistant to aflatoxin through overexpression of resistance associated genes and/or knocking down aflatoxin biosynthesis of A. flavus will be an effective strategy for controlling aflatoxin contamination in cotton. In this study, genome-wide transcriptome profiling was performed to identify differentially expressed genes in response to infection with both toxigenic and atoxigenic strains of A. flavus on cotton (Gossypium hirsutum L.) pericarp and seed. The genes involved in antifungal response, oxidative burst, transcription factors, defense signaling pathways and stress response were highly differentially expressed in pericarp and seed tissues in response to A. flavus infection. The cell-wall modifying genes and genes involved in the production of antimicrobial substances were more active in pericarp as compared to seed. The genes involved in auxin and cytokinin signaling were also induced. Most of the genes involved in defense response in cotton were highly induced in pericarp than in seed. The global gene expression analysis in response to fungal invasion in cotton will serve as a source for identifying biomarkers for breeding, potential candidate genes for transgenic manipulation, and will help in understanding complex plant-fungal interaction for future downstream research.

Comparison of expression of secondary metabolite biosynthesis cluster genes in Aspergillus flavus, A. parasiticus, and A. oryzae.

PubMed

Ehrlich, Kenneth C; Mack, Brian M

2014-06-23

Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity.

Comparison of Expression of Secondary Metabolite Biosynthesis Cluster Genes in Aspergillus flavus, A. parasiticus, and A. oryzae

PubMed Central

Ehrlich, Kenneth C.; Mack, Brian M.

2014-01-01

Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity. PMID:24960201

Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production.

PubMed

Reverberi, M; Fabbri, A A; Zjalic, S; Ricelli, A; Punelli, F; Fanelli, C

2005-11-01

Biosynthesis of aflatoxins, toxic metabolites produced by Aspergillus parasiticus, is correlated to the fungal oxidative stress and cell ageing. In this paper, the mechanism underlying the aflatoxin-inhibiting effect of the Lentinula edodes culture filtrates was studied by analysing their anti-oxidant activity and beta-glucan content. Mushroom beta-glucans are pharmacologically active compounds stimulating anti-oxidant responses in animal cells. L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with beta-glucan content than with anti-oxidant activity of the filtrates. RT-PCR analyses on treated mycelia showed a delay in the activation of aflR, and norA, genes of aflatoxin cluster and a synchronous activation of hsf2-like, a homologue of a yeast transcription factor involved in oxidative stress responses. The first evidence of hsf2-like in A. parasiticus and its activation during aflatoxin biosynthesis is reported. L. edodes filtrates could play a role as external stimulus affecting the anti-oxidant status in the fungal cell that, in turn, leads to aflatoxin inhibition. In the fungal cell, beta-glucans present in the filtrates could stimulate the activation of transcription factors related to anti-oxidant response and anti-oxidant enzyme activity with a contemporaneous delay of aflatoxin genes transcription, which led to a marked reduction of aflatoxin production. This research suggests new perspectives to set suitable strategies against aflatoxins and L. edodes could be considered a promising tool.

Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B{sub 1}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Techapiesancharoenkij, Nirachara; Fiala, Jeannette L.A.; Navasumrit, Panida

Aflatoxin B{sub 1} (AFB{sub 1}) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB{sub 1}-DNA adducts in AFB{sub 1}-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB{sub 1} and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes inmore » gene regulation were observable 4 h after AFB{sub 1} administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB{sub 1}-induced hepatotoxicity. At 24 h after AFB{sub 1} administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB{sub 1}-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB{sub 1} hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer. - Highlights: • This study revealed sulforaphane (SF)-deregulated gene sets in aflatoxin B{sub 1} (AFB{sub 1})-treated rat livers. • SF redirects biochemical networks toward lipid biosynthesis in AFB{sub 1}-dosed rats. • SF enhanced gene sets that would be expected to favor cell repair and regeneration.« less

Effects of Zinc Chelators on Aflatoxin Production in Aspergillus parasiticus

PubMed Central

Wee, Josephine; Day, Devin M.; Linz, John E.

2016-01-01

Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented in the 1970s. In the present study, we utilized two zinc chelators, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) to explore the effect of zinc limitation on aflatoxin synthesis in Aspergillus parasiticus. TPEN but not DMPS decreased aflatoxin biosynthesis up to six-fold depending on whether A. parasiticus was grown on rich or minimal medium. Although we observed significant inhibition of aflatoxin production by TPEN, no detectable changes were observed in expression levels of the aflatoxin pathway gene ver-1 and the zinc binuclear cluster transcription factor, AflR. Treatment of growing A. parasiticus solid culture with a fluorescent zinc probe demonstrated an increase in intracellular zinc levels assessed by increases in fluorescent intensity of cultures treated with TPEN compared to controls. These data suggest that TPEN binds to cytoplasmic zinc therefore limiting fungal access to zinc. To investigate the efficacy of TPEN on food and feed crops, we found that TPEN effectively decreases aflatoxin accumulation on peanut medium but not in a sunflower seeds-derived medium. From an application perspective, these data provide the basis for biological differences that exist in the efficacy of different zinc chelators in various food and feed crops frequently contaminated by aflatoxin. PMID:27271668

Downregulation of transcription factor aflR in Aspergillus flavus confers reduction to aflatoxin accumulation in transgenic maize with alteration of host plant architecture.

PubMed

Masanga, Joel Okoyo; Matheka, Jonathan Mutie; Omer, Rasha Adam; Ommeh, Sheila Cecily; Monda, Ethel Oranga; Alakonya, Amos Emitati

2015-08-01

We report success of host-induced gene silencing in downregulation of aflatoxin biosynthesis in Aspergillus flavus infecting maize transformed with a hairpin construct targeting transcription factor aflR. Infestation of crops by aflatoxin-producing fungi results in economic losses as well as negative human and animal health effects. Currently, the control strategies against aflatoxin accumulation are not effective to the small holder farming systems in Africa and this has led to widespread aflatoxin exposure especially in rural populations of sub-Saharan Africa that rely on maize as a staple food crop. A recent strategy called host-induced gene silencing holds great potential for developing aflatoxin-resistant plant germplasm for the African context where farmers are unable to make further investments other than access to the germplasm. We transformed maize with a hairpin construct targeting the aflatoxin biosynthesis transcription factor aflR. The developed transgenic maize were challenged with an aflatoxigenic Aspergillus flavus strain from Eastern Kenya, a region endemic to aflatoxin outbreaks. Our results indicated that aflR was downregulated in A. flavus colonizing transgenic maize. Further, maize kernels from transgenic plants accumulated significantly lower levels of aflatoxins (14-fold) than those from wild type plants. Interestingly, we observed that our silencing cassette caused stunting and reduced kernel placement in the transgenic maize. This could have been due to "off-target" silencing of unintended genes in transformed plants by aflR siRNAs. Overall, this work indicates that host-induced gene silencing has potential in developing aflatoxin-resistant germplasm.

Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection.

PubMed

Chang, Perng-Kuang; Zhang, Qi; Scharfenstein, Leslie; Mack, Brian; Yoshimi, Akira; Miyazawa, Ken; Abe, Keietsu

2018-06-01

Many glycosylphosphatidylinositol-anchored proteins (GPI-APs) of fungi are membrane enzymes, organization components, and extracellular matrix adhesins. We analyzed eight Aspergillus flavus transcriptome sets for the GPI-AP gene family and identified AFLA_040110, AFLA_063860, and AFLA_113120 to be among the top 5 highly expressed genes of the 36 family genes analyzed. Disruption of the former two genes did not drastically affect A. flavus growth and development. In contrast, disruption of AFLA_113120, an orthologue of Saccharomyces cerevisiae ECM33, caused a significant decrease in vegetative growth and conidiation, promoted sclerotial production, and altered conidial pigmentation. The A. flavus ecm33 null mutant, compared with the wild type and the complemented strain, produced predominantly aflatoxin B 2 but accumulated comparable amounts of cyclopiazonic acid. It showed decreased sensitivity to Congo red at low concentrations (25-50 μg/mL) but had increased sensitivity to calcofluor white at high concentrations (250-500 μg/mL). Analyses of cell wall carbohydrates indicated that the α-glucan content was decreased significantly (p < 0.05), but the contents of chitin and ß-glucan were increased in the mutant strain. In a maize colonization study, the mutant was shown to be impaired in its infectivity and produced 3- to 4-fold lower amounts of conidia than the wild type and the complemented strain. A. flavus Ecm33 is required for proper cell wall composition and plays an important role in normal fungal growth and development, aflatoxin biosynthesis, and seed colonization.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

PubMed

Han, Guomin; Shao, Qian; Li, Cuiping; Zhao, Kai; Jiang, Li; Fan, Jun; Jiang, Haiyang; Tao, Fang

2018-05-01

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 10 6 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes.

PubMed

Hong, Seung-Beom; Lee, Mina; Kim, Dae-Ho; Chung, Soo-Hyun; Shin, Hyeon-Dong; Samson, Robert A

2013-12-01

Strains of the Aspergillus flavus/oryzae complex are frequently isolated from meju, a fermented soybean product, that is used as the starting material for ganjang (soy sauce) and doenjang (soybean paste) production. In this study, we examined the aflatoxin producing capacity of A. flavus/oryzae strains isolated from meju. 192 strains of A. flavus/oryzae were isolated from more than 100 meju samples collected from diverse regions of Korea from 2008 to 2011, and the norB-cypA, omtA, and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 178 strains (92.7%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and 14 strains (7.3%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). Only 7 strains (3.6%) in the aflatoxin-producible group produced aflatoxins on Czapek yeast-extract medium. The aflatoxin-producing capability of A. flavus/oryzae strains from other sources in Korea were also investigated, and 92.9% (52/56) strains from air, 93.9% (31/33) strains from rice straw, 91.7% (11/12) strains from soybean, 81.3% (13/16) strains from corn, 82% (41/50) strains from peanut, and 73.2% (41/56) strains from arable soil were included in the non-aflatoxigenic group. The proportion of non-aflatoxigenicity of meju strains was similar to that of strains from soybean, air and rice straw, all of which have an effect on the fermentation of meju. The data suggest that meju does not have a preference for non-aflatoxigenic or aflatoxin-producible strains of A. flavus/oryzae from the environment of meju. The non-aflatoxigenic meju strains are proposed to be named A. oryzae, while the meju strains that can produce aflatoxins should be referred to A. flavus in this study.

Aspergillus Volatiles Regulate Aflatoxin Synthesis and Asexual Sporulation in Aspergillus parasiticus▿

PubMed Central

Roze, Ludmila V.; Beaudry, Randolph M.; Arthur, Anna E.; Calvo, Ana M.; Linz, John E.

2007-01-01

Aspergillus parasiticus is one primary source of aflatoxin contamination in economically important crops. To prevent the potential health and economic impacts of aflatoxin contamination, our goal is to develop practical strategies to reduce aflatoxin synthesis on susceptible crops. One focus is to identify biological and environmental factors that regulate aflatoxin synthesis and to manipulate these factors to control aflatoxin biosynthesis in the field or during crop storage. In the current study, we analyzed the effects of aspergillus volatiles on growth, development, aflatoxin biosynthesis, and promoter activity in the filamentous fungus A. parasiticus. When colonies of Aspergillus nidulans and A. parasiticus were incubated in the same growth chamber, we observed a significant reduction in aflatoxin synthesis and asexual sporulation by A. parasiticus. Analysis of the headspace gases demonstrated that A. nidulans produced much larger quantities of 2-buten-1-ol (CA) and 2-ethyl-1-hexanol (EH) than A. parasiticus. In its pure form, EH inhibited growth and increased aflatoxin accumulation in A. parasiticus at all doses tested; EH also stimulated aflatoxin transcript accumulation. In contrast, CA exerted dose-dependent up-regulatory or down-regulatory effects on aflatoxin accumulation, conidiation, and aflatoxin transcript accumulation. Experiments with reporter strains carrying nor-1 promoter deletions and mutations suggested that the differential effects of CA were mediated through separate regulatory regions in the nor-1 promoter. The potential efficacy of CA as a tool for analysis of transcriptional regulation of aflatoxin biosynthesis is discussed. We also identify a novel, rapid, and reliable method to assess norsolorinic acid accumulation in solid culture using a Chroma Meter CR-300 apparatus. PMID:17890344

Lysine Succinylation Contributes to Aflatoxin Production and Pathogenicity in Aspergillus flavus*

PubMed Central

Ren, Silin; Yang, Mingkun; Yue, Yuewei; Ge, Feng; Li, Yu; Guo, Xiaodong; Zhang, Jia; Zhang, Feng; Nie, Xinyi; Wang, Shihua

2018-01-01

Aspergillus flavus (A. flavus) is a ubiquitous saprophytic and pathogenic fungus that produces the aflatoxin carcinogen, and A. flavus can have tremendous economic and health impacts worldwide. Increasing evidence demonstrates that lysine succinylation plays an important regulatory role in metabolic processes in both bacterial and human cells. However, little is known about the extent and function of lysine succinylation in A. flavus. Here, we performed a global succinylome analysis of A. flavus using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 985 succinylation sites on 349 succinylated proteins were identified in this pathogen. Bioinformatics analysis revealed that the succinylated proteins were involved in various biological processes and were particularly enriched in the aflatoxin biosynthesis process. Site-specific mutagenesis and biochemical studies showed that lysine succinylation on the norsolorinic acid reductase NorA (AflE), a key enzyme in aflatoxins biosynthesis, can affect the production of sclerotia and aflatoxins biosynthesis in A. flavus. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and aflatoxins biosynthesis in A. flavus. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this pathogen. PMID:29298838

Characterization of the maize lipoxygenase gene family in relation to aflatoxin accumulation resistance.

PubMed

Ogunola, Oluwaseun F; Hawkins, Leigh K; Mylroie, Erik; Kolomiets, Michael V; Borrego, Eli; Tang, Juliet D; Williams, W Paul; Warburton, Marilyn L

2017-01-01

Maize (Zea mays L.) is a globally important staple food crop prone to contamination by aflatoxin, a carcinogenic secondary metabolite produced by the fungus Aspergillus flavus. An efficient approach to reduce accumulation of aflatoxin is the development of germplasm resistant to colonization and toxin production by A. flavus. Lipoxygenases (LOXs) are a group of non-heme iron containing dioxygenase enzymes that catalyze oxygenation of polyunsaturated fatty acids (PUFAs). LOX derived oxylipins play critical roles in plant defense against pathogens including A. flavus. The objectives of this study were to summarize sequence diversity and expression patterns for all LOX genes in the maize genome, and map their effect on aflatoxin accumulation via linkage and association mapping. In total, 13 LOX genes were identified, characterized, and mapped. The sequence of one gene, ZmLOX10, is reported from 5 inbred lines. Genes ZmLOX1/2, 5, 8, 9, 10 and 12 (GRMZM2G156861, or V4 numbers ZM00001D042541 and Zm00001D042540, GRMZM2G102760, GRMZM2G104843, GRMZM2G017616, GRMZM2G015419, and GRMZM2G106748, respectively) fell under previously published QTL in one or more mapping populations and are linked to a measurable reduction of aflatoxin in maize grains. Association mapping results found 28 of the 726 SNPs tested were associated with reduced aflatoxin levels at p ≤ 9.71 x 10-4 according to association statistics. These fell within or near nine of the ZmLOX genes. This work confirms the importance of some lipoxygenases for resistance to aflatoxin accumulation and may be used to direct future genetic selection in maize.

Ethylene Inhibits Aflatoxin Biosynthesis in Aspergillus parasiticus Grown on Peanuts

PubMed Central

Gunterus, A.; Roze, L.V.; Beaudry, R.; Linz, J. E.

2007-01-01

The filamentous fungi Aspergillus parasiticus and A. flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had not discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage. PMID:17418318

Insight into Genes Regulating Postharvest Aflatoxin Contamination of Tetraploid Peanut from Transcriptional Profiling.

PubMed

Korani, Walid; Chu, Ye; Holbrook, C Corley; Ozias-Akins, Peggy

2018-05-01

Postharvest aflatoxin contamination is a challenging issue that affects peanut quality. Aflatoxin is produced by fungi belonging to the Aspergilli group, and is known as an acutely toxic, carcinogenic, and immune-suppressing class of mycotoxins. Evidence for several host genetic factors that may impact aflatoxin contamination has been reported, e.g. , genes for lipoxygenase (PnLOX1 and PnLOX2/PnLOX3 that showed either positive or negative regulation with Aspergillus infection), reactive oxygen species, and WRKY (highly associated with or differentially expressed upon infection of maize with Aspergillus flavus ); however, their roles remain unclear. Therefore, we conducted an RNA-sequencing experiment to differentiate gene response to the infection by A. flavus between resistant (ICG 1471) and susceptible (Florida-07) cultivated peanut genotypes. The gene expression profiling analysis was designed to reveal differentially expressed genes in response to the infection (infected vs. mock-treated seeds). In addition, the differential expression of the fungal genes was profiled. The study revealed the complexity of the interaction between the fungus and peanut seeds as the expression of a large number of genes was altered, including some in the process of plant defense to aflatoxin accumulation. Analysis of the experimental data with "keggseq," a novel designed tool for Kyoto Encyclopedia of Genes and Genomes enrichment analysis, showed the importance of α-linolenic acid metabolism, protein processing in the endoplasmic reticulum, spliceosome, and carbon fixation and metabolism pathways in conditioning resistance to aflatoxin accumulation. In addition, coexpression network analysis was carried out to reveal the correlation of gene expression among peanut and fungal genes. The results showed the importance of WRKY, toll/Interleukin1 receptor-nucleotide binding site leucine-rich repeat (TIR-NBS-LRR), ethylene, and heat shock proteins in the resistance mechanism. Copyright

Aflatoxins: mechanisms of inhibition by antagonistic plants and microorganisms

USDA-ARS?s Scientific Manuscript database

Aflatoxins are a family of toxic fungal secondary metabolites. The rapid expansion in our knowledge about inhibition of aflatoxin biosynthesis by compounds from plants and microorganisms has enabled us to utilize them as potential biocontrol agents. Substantial efforts have been devoted to identify ...

Two new aflatoxin producing species, and an overview of Aspergillus section Flavi

PubMed Central

Varga, J.; Frisvad, J.C.; Samson, R.A.

2011-01-01

Aspergillus subgenus Circumdati section Flavi includes species with usually biseriate conidial heads, in shades of yellow-green to brown, and dark sclerotia. Several species assigned to this section are either important mycotoxin producers including aflatoxins, cyclopiazonic acid, ochratoxins and kojic acid, or are used in oriental food fermentation processes and as hosts for heterologous gene expression. A polyphasic approach was applied using morphological characters, extrolite data and partial calmodulin, β-tubulin and ITS sequences to examine the evolutionary relationships within this section. The data indicate that Aspergillus section Flavi involves 22 species, which can be grouped into seven clades. Two new species, A. pseudocaelatus sp. nov. and A. pseudonomius sp. nov. have been discovered, and can be distinguished from other species in this section based on sequence data and extrolite profiles. Aspergillus pseudocaelatus is represented by a single isolate collected from Arachis burkartii leaf in Argentina, is closely related to the non-aflatoxin producing A. caelatus, and produces aflatoxins B & G, cyclopiazonic acid and kojic acid, while A. pseudonomius was isolated from insects and soil in the USA. This species is related to A. nomius, and produces aflatoxin B1 (but not G-type aflatoxins), chrysogine and kojic acid. In order to prove the aflatoxin producing abilities of the isolates, phylogenetic analysis of three genes taking part in aflatoxin biosynthesis, including the transcriptional regulator aflR, norsolonic acid reductase and O-methyltransferase were also carried out. A detailed overview of the species accepted in Aspergillus section Flavi is presented. PMID:21892243

The Aspergillus flavus Homeobox Gene, hbx1, is Required for Development and Aflatoxin Production.

PubMed

Cary, Jeffrey W; Harris-Coward, Pamela; Scharfenstein, Leslie; Mack, Brian M; Chang, Perng-Kuang; Wei, Qijian; Lebar, Matthew; Carter-Wientjes, Carol; Majumdar, Rajtilak; Mitra, Chandrani; Banerjee, Sourav; Chanda, Anindya

2017-10-12

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox ( hbx ) genes in the aflatoxin-producing ascomycete, Aspergillus flavus , and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1 , in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B₁ and B₂, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δ hbx1 mutants did not produce conidiophores. The inability of Δ hbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC , aflD , aflM and the cluster-specific regulatory gene, aflR . Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

2012-08-01

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut.

PubMed

Power, Imana L; Dang, Phat M; Sobolev, Victor S; Orner, Valerie A; Powell, Joseph L; Lamb, Marshall C; Arias, Renee S

2017-04-01

Aflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B 1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins. Copyright © 2017 Elsevier B.V. All rights reserved.

Fragmentation of an aflatoxin-like gene cluster in a forest pathogen

USDA-ARS?s Scientific Manuscript database

Secondary metabolic pathway genes are typically clustered in fungi. An exception to this paradigm is seen for genes required for the production of dothistromin, an aflatoxin-like virulence factor produced by the pine needle pathogen Dothistroma septosporum. In contrast to the tight clustering of gen...

Citrate-Coated Silver Nanoparticles Growth-Independently Inhibit Aflatoxin Synthesis in Aspergillus parasiticus.

PubMed

Mitra, Chandrani; Gummadidala, Phani M; Afshinnia, Kamelia; Merrifield, Ruth C; Baalousha, Mohammed; Lead, Jamie R; Chanda, Anindya

2017-07-18

Manufactured silver nanoparticles (Ag NPs) have long been used as antimicrobials. However, little is known about how these NPs affect fungal cell functions. While multiple previous studies reveal that Ag NPs inhibit secondary metabolite syntheses in several mycotoxin producing filamentous fungi, these effects are associated with growth repression and hence need sublethal to lethal NP doses, which besides stopping fungal growth, can potentially accumulate in the environment. Here we demonstrate that citrate-coated Ag NPs of size 20 nm, when applied at a selected nonlethal dose, can result in a >2 fold inhibition of biosynthesis of the carcinogenic mycotoxin and secondary metabolite, aflatoxin B 1 in the filamentous fungus and an important plant pathogen, Aspergillus parasiticus, without inhibiting fungal growth. We also show that the observed inhibition was not due to Ag ions, but was specifically associated with the mycelial uptake of Ag NPs. The NP exposure resulted in a significant decrease in transcript levels of five aflatoxin genes and at least two key global regulators of secondary metabolism, laeA and veA, with a concomitant reduction of total reactive oxygen species (ROS). Finally, the depletion of Ag NPs in the growth medium allowed the fungus to regain completely its ability of aflatoxin biosynthesis. Our results therefore demonstrate the feasibility of Ag NPs to inhibit fungal secondary metabolism at nonlethal concentrations, hence providing a novel starting point for discovery of custom designed engineered nanoparticles that can efficiently prevent mycotoxins with minimal risk to health and environment.

Isolation and identification of fungi from a meju contaminated with aflatoxins.

PubMed

Jung, Yu Jung; Chung, Soo Hyun; Lee, Hyo Ku; Chun, Hyang Sook; Hong, Seung Beom

2012-12-01

A home-made meju sample contaminated naturally with aflatoxins was used for isolation of fungal strains. Overall, 230 fungal isolates were obtained on dichloran rosebengal chloramphenicol (DRBC) and dichloran 18% glycerol (DG18) agar plates. Morphological characteristics and molecular analysis of a partial beta-tubulin gene and the internal transcribed spacer (ITS) of rDNA were used for the identification of the isolates. The fungal isolates were divided into 7 genera: Aspergillus, Eurotium, Penicillium, Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three strains from 56 isolates of the A. oryzae/flavus group were found to be aflatoxigenic A. flavus, by the presence of the aflatoxin biosynthesis genes and confirmatory aflatoxin production by high-performance liquid chromatography (HPLC). The predominant isolate from DRBC plates was A. oryzae (42 strains, 36.2%), whereas that from DG18 was A. candidus (61 strains, 53.5%). Out of the 230 isolates, the most common species was A. candidus (34.3%) followed by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. flavus and E. chevalieri presented occurrence levels of 2.2%, respectively. The remaining isolates of A. unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. inaequalis had lower occurrence levels of < 2.0%.

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

PubMed

Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

2015-09-01

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor

PubMed Central

Clevenger, Josh; Marasigan, Kathleen; Liakos, Vasileios; Sobolev, Victor; Vellidis, George; Holbrook, Corley; Ozias-Akins, Peggy

2016-01-01

Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC. PMID:27827875

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

PubMed

Gilbert, Matthew K; Majumdar, Rajtilak; Rajasekaran, Kanniah; Chen, Zhi-Yuan; Wei, Qijian; Sickler, Christine M; Lebar, Matthew D; Cary, Jeffrey W; Frame, Bronwyn R; Wang, Kan

2018-06-01

Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

The high-affinity phosphodiesterase PdeH regulates development and aflatoxin biosynthesis in Aspergillus flavus.

PubMed

Yang, Kunlong; Liu, Yinghang; Liang, Linlin; Li, Zhenguo; Qin, Qiuping; Nie, Xinyi; Wang, Shihua

2017-04-01

Cyclic AMP signaling controls a range of physiological processes in response to extracellular stimuli in organisms. Among the signaling cascades, cAMP, as a second messenger, is orchestrated by adenylate cyclase (biosynthesis) and cAMP phosphodiesterases (PDEs) (hydrolysis). In this study, we investigated the function of the high-affinity (PdeH) and low-affinity (PdeL) cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus Aspergillus flavus, and found that instead of PdeL, inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation. However, the ΔpdeL/ΔpdeH mutant exhibited an enhanced phenotype defects, a similar phenotype defects to wild-type strain treated with exogenous cAMP. The activation of PKA activity was inhibited in the ΔpdeH or ΔpdeL/ΔpdeH mutant, both of whom exhibited increasing AF production. Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain, and affected pdeL transcription. Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol, while PdeL-GFP localized mainly to the nucleus. Overall, our results indicated that PdeH plays a major role, but has overlapping function with PdeL, in vegetative growth, development and AF biosynthesis in A. flavus. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Roles of lignin biosynthesis and regulatory genes in plant development

PubMed Central

Yoon, Jinmi; Choi, Heebak

2015-01-01

Abstract Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non‐lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism. PMID:26297385

Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management

PubMed Central

Kumar, Pradeep; Mahato, Dipendra K.; Kamle, Madhu; Mohanta, Tapan K.; Kang, Sang G.

2017-01-01

The aflatoxin producing fungi, Aspergillus spp., are widely spread in nature and have severely contaminated food supplies of humans and animals, resulting in health hazards and even death. Therefore, there is great demand for aflatoxins research to develop suitable methods for their quantification, precise detection and control to ensure the safety of consumers’ health. Here, the chemistry and biosynthesis process of the mycotoxins is discussed in brief along with their occurrence, and the health hazards to humans and livestock. This review focuses on resources, production, detection and control measures of aflatoxins to ensure food and feed safety. The review is informative for health-conscious consumers and research experts in the fields. Furthermore, providing knowledge on aflatoxins toxicity will help in ensure food safety and meet the future demands of the increasing population by decreasing the incidence of outbreaks due to aflatoxins. PMID:28144235

Targeting mycotoxin biosynthesis pathway genes

USDA-ARS?s Scientific Manuscript database

Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities is mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requ...

Streptomyces-Aspergillus flavus interactions: impact on aflatoxin B accumulation.

PubMed

Verheecke, C; Liboz, T; Anson, P; Zhu, Y; Mathieu, F

2015-01-01

The aim of this work was to investigate the potential of Streptomyces sp. as biocontrol agents against aflatoxins in maize. As such, we assumed that Streptomyces sp. could provide a complementary approach to current biocontrol systems such as Afla-guard(®) and we focused on biocontrol that was able to have an antagonistic contact with A. flavus. A previous study showed that 27 (out of 38) Streptomyces sp. had mutual antagonism in contact with A. flavus. Among these, 16 Streptomyces sp. were able to reduce aflatoxin content to below 17% of the residual concentration. We selected six strains to understand the mechanisms involved in the prevention of aflatoxin accumulation. Thus, in interaction with A. flavus, we monitored by RT-qPCR the gene expression of aflD, aflM, aflP, aflR and aflS. All the Streptomyces sp. were able to reduce aflatoxin concentration (24.0-0.2% residual aflatoxin B1). They all impacted on gene expression, but only S35 and S38 were able to repress expression significantly. Indeed, S35 significantly repressed aflM expression and S38 significantly repressed aflR, aflM and aflP. S6 reduced aflatoxin concentrations (2.3% residual aflatoxin B1) and repressed aflS, aflM and enhanced aflR expression. In addition, the S6 strain (previously identified as the most reducing pure aflatoxin B1) was further tested to determine a potential adsorption mechanism. We did not observe any adsorption phenomenon. In conclusion, this study showed that Streptomyces sp. prevent the production of (aflatoxin gene expression) and decontamination of (aflatoxin B1 reduction) aflatoxins in vitro.

Expression Analysis of Stress-Related Genes in Kernels of Different Maize (Zea mays L.) Inbred Lines with Different Resistance to Aflatoxin Contamination

PubMed Central

Jiang, Tingbo; Zhou, Boru; Luo, Meng; Abbas, Hamed K.; Kemerait, Robert; Lee, Robert Dewey; Scully, Brian T.; Guo, Baozhu

2011-01-01

This research examined the expression patterns of 94 stress-related genes in seven maize inbred lines with differential expressions of resistance to aflatoxin contamination. The objective was to develop a set of genes/probes associated with resistance to A. flavus and/or aflatoxin contamination. Ninety four genes were selected from previous gene expression studies with abiotic stress to test the differential expression in maize lines, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6, using real-time RT-PCR. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups, which are highly expressed in the resistant Group 2 but down-regulated in susceptible Group 1. When further subjected to drought stress, Tex6 expressed more genes up-regulated and B73 has fewer genes up-regulated. The transcript patterns and interactions measured in these experiments indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as various host interactions with environmental factors, particularly, drought and high temperature. PMID:22069724

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

PubMed

Abdel-Hadi, Ahmed M; Caley, Daniel P; Carter, David R F; Magan, Naresh

2011-06-01

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize.

PubMed

Warburton, Marilyn L; Williams, William Paul; Hawkins, Leigh; Bridges, Susan; Gresham, Cathy; Harper, Jonathan; Ozkan, Seval; Mylroie, J Erik; Shan, Xueyan

2011-07-01

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.

Characterization of the Critical Amino Acids of an Aspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

PubMed Central

Yu, Jiujiang; Chang, Perng-Kuang; Ehrlich, Kenneth C.; Cary, Jeffrey W.; Montalbano, Beverly; Dyer, John M.; Bhatnagar, Deepak; Cleveland, Thomas E.

1998-01-01

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee. PMID:9835571

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

PubMed Central

Moore, Geromy G.; Elliott, Jacalyn L.; Singh, Rakhi; Horn, Bruce W.; Dorner, Joe W.; Stone, Eric A.; Chulze, Sofia N.; Barros, German G.; Naik, Manjunath K.; Wright, Graeme C.; Hell, Kerstin; Carbone, Ignazio

2013-01-01

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B1 being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B1-dominant and G1-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more

Peanuts that keep aflatoxin at bay: a threshold that matters.

PubMed

Sharma, Kiran K; Pothana, Arunima; Prasad, Kalyani; Shah, Dilip; Kaur, Jagdeep; Bhatnagar, Deepak; Chen, Zhi-Yuan; Raruang, Yenjit; Cary, Jeffrey W; Rajasekaran, Kanniah; Sudini, Hari Kishan; Bhatnagar-Mathur, Pooja

2018-05-01

Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub-Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut-Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host-induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE-Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species-scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near-immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi-arid regions. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Comparison of the aflR gene sequences of strains in Aspergillus section Flavi.

PubMed

Lee, Chao-Zong; Liou, Guey-Yuh; Yuan, Gwo-Fang

2006-01-01

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius and a few other species. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. Although Aspergillus oryzae and Aspergillus sojae, which are used in fermented foods and in ingredient manufacture, have no record of producing aflatoxin, they have been shown to possess an aflR gene. This study examined 34 strains of Aspergillus section Flavi. The aflR gene of 23 of these strains was successfully amplified and sequenced. No aflR PCR products were found in five A. sojae strains or six strains of A. oryzae. These PCR results suggested that the aflR gene is absent or significantly different in some A. sojae and A. oryzae strains. The sequenced aflR genes from the 23 positive strains had greater than 96.6 % similarity, which was particularly conserved in the zinc-finger DNA-binding domain. The aflR gene of A. sojae has two obvious characteristics: an extra CTCATG sequence fragment and a C to T transition that causes premature termination of AFLR protein synthesis. Differences between A. parasiticus/A. sojae and A. flavus/A. oryzae aflR genes were also identified. Some strains of A. flavus as well as A. flavus var. viridis, A. oryzae var. viridis and A. oryzae var. effuses have an A. oryzae-type aflR gene. For all strains with the A. oryzae-type aflR gene, there was no evidence of aflatoxin production. It is suggested that for safety reasons, the aflR gene could be examined to assess possible aflatoxin production by Aspergillus section Flavi strains.

Demonstration of Aflatoxin Inhibitory Activity in a Cotton Seed Coat Xylan

PubMed Central

Mellon, J. E.; Cotty, P. J.; Godshall, M. A.; Roberts, E.

1995-01-01

An inhibitor of aflatoxin biosynthesis localized in the seed coats of developing cotton was partially purified and characterized. Aqueous extracts from 25-day postanthesis seed coat tissue inhibited aflatoxin (B(inf1)) production in liquid cultures of Aspergillus flavus AF13. Inhibition was concentration dependent, with a 50% effective dose of 173 (mu)g of crude extract per ml of medium. The inhibitor was neutral in charge. Two active fractions were obtained from crude preparations by gel filtration chromatography (BioGel P-100). The purest fraction eluted in the void volume. Carbohydrate composition analysis of this void volume inhibitor indicated a composition of xylose (>90%) and mannose. Aflatoxin production in vitro was inversely related to inhibitor concentration in the fermentation medium (log of aflatoxin versus log of [inhibitor]; r(sup2) = 0.82; P < 0.002). The void volume inhibitor had a 50% effective dose of 6.2 (mu)g/ml, a 28-fold purification of the inhibitor material. These data support the hypothesis that seed coat inhibitory activity is associated with a cottonseed-specific xylan. PMID:16535194

Characterization of the Maize Chitinase Genes and Their Effect on Aspergillus flavus and Aflatoxin Accumulation Resistance

PubMed Central

Hawkins, Leigh K.; Mylroie, J. Erik; Oliveira, Dafne A.; Smith, J. Spencer; Ozkan, Seval; Windham, Gary L.; Williams, W. Paul; Warburton, Marilyn L.

2015-01-01

Maize (Zea mays L.) is a crop of global importance, but prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need for intervention by farmers. Chitinolytic enzymes respond to attack by potential pathogens and have been demonstrated to increase insect and fungal resistance in plants. Here, all chitinase genes in the maize genome were characterized via sequence diversity and expression patterns. Recent evolution within this gene family was noted. Markers from within each gene were developed and used to map the phenotypic effect on resistance of each gene in up to four QTL mapping populations and one association panel. Seven chitinase genes were identified that had alleles associated with increased resistance to aflatoxin accumulation and A. flavus infection in field grown maize. The chitinase in bin 1.05 identified a new and highly significant QTL, while chitinase genes in bins 2.04 and 5.03 fell directly beneath the peaks of previously published QTL. The expression patterns of these genes corroborate possible grain resistance mechanisms. Markers from within the gene sequences or very closely linked to them are presented to aid in the use of marker assisted selection to improve this trait. PMID:26090679

Characterization of the Maize Chitinase Genes and Their Effect on Aspergillus flavus and Aflatoxin Accumulation Resistance.

PubMed

Hawkins, Leigh K; Mylroie, J Erik; Oliveira, Dafne A; Smith, J Spencer; Ozkan, Seval; Windham, Gary L; Williams, W Paul; Warburton, Marilyn L

2015-01-01

Maize (Zea mays L.) is a crop of global importance, but prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need for intervention by farmers. Chitinolytic enzymes respond to attack by potential pathogens and have been demonstrated to increase insect and fungal resistance in plants. Here, all chitinase genes in the maize genome were characterized via sequence diversity and expression patterns. Recent evolution within this gene family was noted. Markers from within each gene were developed and used to map the phenotypic effect on resistance of each gene in up to four QTL mapping populations and one association panel. Seven chitinase genes were identified that had alleles associated with increased resistance to aflatoxin accumulation and A. flavus infection in field grown maize. The chitinase in bin 1.05 identified a new and highly significant QTL, while chitinase genes in bins 2.04 and 5.03 fell directly beneath the peaks of previously published QTL. The expression patterns of these genes corroborate possible grain resistance mechanisms. Markers from within the gene sequences or very closely linked to them are presented to aid in the use of marker assisted selection to improve this trait.

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces.

PubMed

Verheecke, C; Liboz, T; Anson, P; Diaz, R; Mathieu, F

2015-05-01

The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reverse-transcriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, βtub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression. © 2015 The Authors.

Interaction of Wild Strains of Aspergilla with Aspergillus parasiticus ATCC15517 and Aflatoxin Production †

PubMed Central

Martins, H. Marina; Almeida, Inês; Marques, Marta; Bernardo, Fernando

2008-01-01

Aflatoxins are secondary metabolites produced by some competent mould strains of Aspergillus flavus, A. parasiticus and A. nomius. These compounds have been extensively studied with regards to their toxicity for animals and humans; they are able to induce liver cancer and may cause a wide range of adverse effects in living organisms. Aflatoxins are found as natural contaminants of food and feed; the main line of the strategy to control them is based on the prevention of the mould growth in raw vegetable or during its storage and monitoring of each crop batch. Mould growth is conditioned by many ecological factors, including biotic ones. Hazard characterization models for aflatoxins in crops must take into consideration biotic interactions between moulds and their potential effects on growth development. The aim of this work is to study the effect of the biotic interaction of 14 different wild strains of Aspergilla (different species), with a competent strain (Aspergillus parasiticus ATCC 15517) using an in vitro production model. The laboratory model used was a natural matrix (humidified cracked corn), on which each wild strain challenged the aflatoxin production of a producer strain. Cultures were incubated at 28°C for 12 days and sampled at the 8th and 12th. Aflatoxin detection and quantification was performed by HPLC using a procedure with a MRPL = 1 μg/kg. Results of those interactive cultures revealed both synergic and antagonistic effects on aflatoxin biosynthesis. Productivity increases were particularly evident on the 8th day of incubation with wild strains of A. flavipes (+ 70.4 %), A. versicolor (+ 54.9 %) and A. flavus 3 (+ 62.6 %). Antagonistic effects were found with A. niger (− 69.5%), A. fumigatus (− 47.6 %) and A. terreus (− 47.6 %) on the 12th day. The increased effects were more evident on the 8th of incubation and the decreases were more patent on the 12th day. Results show that the development of Aspergilla strains concomitantly with

Cloning and bioinformatic analysis of lovastatin biosynthesis regulatory gene lovE.

PubMed

Huang, Xin; Li, Hao-ming

2009-08-05

Lovastatin is an effective drug for treatment of hyperlipidemia. This study aimed to clone lovastatin biosynthesis regulatory gene lovE and analyze the structure and function of its encoding protein. According to the lovastatin synthase gene sequence from genebank, primers were designed to amplify and clone the lovastatin biosynthesis regulatory gene lovE from Aspergillus terrus genomic DNA. Bioinformatic analysis of lovE and its encoding animo acid sequence was performed through internet resources and software like DNAMAN. Target fragment lovE, almost 1500 bp in length, was amplified from Aspergillus terrus genomic DNA and the secondary and three-dimensional structures of LovE protein were predicted. In the lovastatin biosynthesis process lovE is a regulatory gene and LovE protein is a GAL4-like transcriptional factor.

Survey of candidate genes for maize resistance to infection by Aspergillus flavus and/or aflatoxin contamination

Treesearch

Leigh Hawkins; Marilyn Warburton; Juliet Tang; John Tomashek; Dafne Alves Oliveira; Oluwaseun Ogunola; J. Smith; W. Williams

2018-01-01

Many projects have identified candidate genes for resistance to aflatoxin accumulation or Aspergillus flavus infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their relative contribution to...

A regulatory gene (ECO-orf4) required for ECO-0501 biosynthesis in Amycolatopsis orientalis.

PubMed

Shen, Yang; Huang, He; Zhu, Li; Luo, Minyu; Chen, Daijie

2014-02-01

ECO-0501 is a novel linear polyene antibiotic, which was discovered from Amycolatopsis orientalis. Recent study of ECO-0501 biosynthesis pathway revealed the presence of regulatory gene: ECO-orf4. The A. orientalis ECO-orf4 gene from the ECO-0501 biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. Database comparison revealed two hypothetical domains, a LuxR-type helix-turn-helix (HTH) DNA binding motif near the C-terminal and an N-terminal nucleotide triphosphate (NTP) binding motif included. Deletion of the corresponding gene (ECO-orf4) resulted in complete loss of ECO-0501 production. Complementation by one copy of intact ECO-orf4 restored the polyene biosynthesis demonstrating that ECO-orf4 is required for ECO-0501 biosynthesis. The results of overexpression ECO-orf4 on ECO-0501 production indicated that it is a positive regulatory gene. Gene expression analysis by reverse transcription PCR of the ECO-0501 gene cluster showed that the transcription of ECO-orf4 correlates with that of genes involved in polyketide biosynthesis. These results demonstrated that ECO-orf4 is a pathway-specific positive regulatory gene that is essential for ECO-0501 biosynthesis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An evaluation of aflatoxin and cyclopiazonic acid production in Aspergillus oryzae.

PubMed

Kim, Nam Yeun; Lee, Jin Hee; Lee, Inhyung; Ji, Geun Eog

2014-06-01

To date, edible fungi such as Aspergillus flavus var. oryzae (A. oryzae) has been considered as safe. However, some strains can produce mycotoxins. Thus, the biosynthetic ability to produce mycotoxins should be reevaluated to determine the safety of edible fungi. We analyzed the production of aflatoxins and cyclopiazonic acid (CPA) from edible fungi such as A. oryzae isolated from various Korean foods using multiplex PCR, enzyme-linked immunosorbent assay, and high-performance liquid chromatography (HPLC). In the multiplex PCR analysis of aflatoxin biosynthetic genes omtB, aflR, ver-1, and omtA, 5 of 19 Aspergillus strains produced all PCR products. Among them, aflatoxin B1 and aflatoxin B2 were detected from only A. flavus KACC 41403 by HPLC. Aflatoxins were not detected from the other four strains that produced all positive PCR bands. Aflatoxin also was not detected from 12 strains that had PCR patterns without aflR or ver-1 and from 2 strains that did not produce any of the expected PCR products. Only the seven A. oryzae strains that produced all of the positive PCR bands including the CPA biosynthetic genes maoA, dmaT, and pks-nrps produced CPA. CPA and aflatoxin production must be evaluated before A. oryzae strains are used for the development of fermented foods.

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

PubMed Central

Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing

2012-01-01

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity. PMID:22156425

Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements

PubMed Central

Lillehoj, E. B.; Garcia, W. J.; Lambrow, M.

1974-01-01

Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 μg of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 μg/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25 μg of cadmium per g or 500 μg of copper per g of germ. PMID:4216287

Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

USDA-ARS?s Scientific Manuscript database

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines w...

Comparative Analysis of Tocopherol Biosynthesis Genes and Its Transcriptional Regulation in Soybean Seeds.

PubMed

T, Vinutha; Bansal, Navita; Kumari, Khushboo; Prashat G, Rama; Sreevathsa, Rohini; Krishnan, Veda; Kumari, Sweta; Dahuja, Anil; Lal, S K; Sachdev, Archana; Praveen, Shelly

2017-12-20

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

Identification of Putative Genes Involved in Limonoids Biosynthesis in Citrus by Comparative Transcriptomic Analysis

PubMed Central

Wang, Fusheng; Wang, Mei; Liu, Xiaona; Xu, Yuanyuan; Zhu, Shiping; Shen, Wanxia; Zhao, Xiaochun

2017-01-01

Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L.) Osbeck) at different development stages with variances in limonoids contents were used for digital gene expression profiling analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s) and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC) for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing) demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids. PMID:28553308

Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions.

PubMed

Azuma, Akifumi; Yakushiji, Hiroshi; Koshita, Yoshiko; Kobayashi, Shozo

2012-10-01

Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15 °C) plus light treatment, whereas high temperature (35 °C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.

Characterization of the chitinase gene family and the effect on A. flavus and aflatoxin resistance in maize.

USDA-ARS?s Scientific Manuscript database

Maize (Zea mays L.) is a crop of global importance, but is prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need ...

Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

PubMed Central

Chang, Perng-Kuang; Ehrlich, Kenneth C.; Fujii, Isao

2009-01-01

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines what is currently known about the toxicity of CPA to animals and humans, both by itself or in combination with other mycotoxins. The review also discusses CPA biosynthesis and the genetic diversity of CPA production in A. flavus/oryzae populations. PMID:22069533

Deep sequencing of the Camellia chekiangoleosa transcriptome revealed candidate genes for anthocyanin biosynthesis.

PubMed

Wang, Zhong-Wei; Jiang, Cong; Wen, Qiang; Wang, Na; Tao, Yuan-Yuan; Xu, Li-An

2014-03-15

Camellia chekiangoleosa is an important species of genus Camellia. It provides high-quality edible oil and has great ornamental value. The flowers are big and red which bloom between February and March. Flower pigmentation is closely related to the accumulation of anthocyanin. Although anthocyanin biosynthesis has been studied extensively in herbaceous plants, little molecular information on the anthocyanin biosynthesis pathway of C. chekiangoleosa is yet known. In the present study, a cDNA library was constructed to obtain detailed and general data from the flowers of C. chekiangoleosa. To explore the transcriptome of C. chekiangoleosa and investigate genes involved in anthocyanin biosynthesis, a 454 GS FLX Titanium platform was used to generate an EST dataset. About 46,279 sequences were obtained, and 24,593 (53.1%) were annotated. Using Blast search against the AGRIS, 1740 unigenes were found homologous to 599 Arabidopsis transcription factor genes. Based on the transcriptome dataset, nine anthocyanin biosynthesis pathway genes (PAL, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. The spatio-temporal expression patterns of these genes were also analyzed using quantitative real-time polymerase chain reaction. The study results not only enrich the gene resource but also provide valuable information for further studies concerning anthocyanin biosynthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

PubMed Central

Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling

2017-01-01

The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This

Regulatory genes and their roles for improvement of antibiotic biosynthesis in Streptomyces.

PubMed

Lu, Fengjuan; Hou, Yanyan; Zhang, Heming; Chu, Yiwen; Xia, Haiyang; Tian, Yongqiang

2017-08-01

The numerous secondary metabolites in Streptomyces spp. are crucial for various applications. For example, cephamycin C is used as an antibiotic, and avermectin is used as an insecticide. Specifically, antibiotic yield is closely related to many factors, such as the external environment, nutrition (including nitrogen and carbon sources), biosynthetic efficiency and the regulatory mechanisms in producing strains. There are various types of regulatory genes that work in different ways, such as pleiotropic (or global) regulatory genes, cluster-situated regulators, which are also called pathway-specific regulatory genes, and many other regulators. The study of regulatory genes that influence antibiotic biosynthesis in Streptomyces spp. not only provides a theoretical basis for antibiotic biosynthesis in Streptomyces but also helps to increase the yield of antibiotics via molecular manipulation of these regulatory genes. Currently, more and more emphasis is being placed on the regulatory genes of antibiotic biosynthetic gene clusters in Streptomyces spp., and many studies on these genes have been performed to improve the yield of antibiotics in Streptomyces. This paper lists many antibiotic biosynthesis regulatory genes in Streptomyces spp. and focuses on frequently investigated regulatory genes that are involved in pathway-specific regulation and pleiotropic regulation and their applications in genetic engineering.

Aflatoxins

Cancer.gov

Learn about aflatoxins, a family of toxins associated with an increased risk of liver cancer. Aflatoxins are produced by certain fungi found on agricultural crops such as maize (corn), peanuts, cottonseed, and tree nuts.

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

PubMed Central

Buchanan, R L; Lewis, D F

1984-01-01

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes. PMID:6091545

[Expression of saponin biosynthesis related genes in different tissues of Panax quinquefolius].

PubMed

Wang, Kang-Yu; Liu, Wei-Can; Zhang, Mei-Ping; Zhao, Ming-Zhu; Wang, Yan-Fang; Li, Li; Sun, Chun-Yu; Hu, Ke-Xin; Cong, Yue-Yi; Wang, Yi

2018-01-01

The relationship between saponin content of Panax quinquefolius in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of P. quinquefolius were studied, six saponins in P. quinquefolius. Samples (ginsenoside Rg₁, Re, Rb₁, Rc, Rb₂ and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes ( SQS, OSC, DS, β-AS, SQE, P450 and FPS ) in different tissues of P. quinquefolius were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the P. quinquefolius involved in ginsenoside synthesis, the expression of β-AS and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, FPS, SQS, OSC, DS and SQE had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS ( P <0.05 or P <0.01). The biosynthesis of partial saponins, grouping saponins and total saponins in P. quinquefolius was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of P. quinquefolius was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that FPS, SQS, OSC, DS and SQE was the key enzyme to control the synthesis of saponins in P. quinquefolius by correlation analysis, the biosynthesis of ginsenosides in P. quinquefolius was regulated by these five kind of enzymes in cluster co-expression of interaction mode. Copyright© by the Chinese Pharmaceutical Association.

Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

PubMed Central

Ternes, Chad M.; Schönknecht, Gerald

2014-01-01

NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

Enzymatic formation of the bisfuran structure in aflatoxin biosynthesis.

PubMed Central

Wan, N C; Hsieh, D P

1980-01-01

A relatively stable enzyme system that converts versiconal hemiacetal acetate to versicolorin A was isolated from the soluble fraction of the homogenized cells of Aspergillus parasiticus ATCC 15517. The cell-free preparation did not require oxygen or oxidized nicotinamide adenine dinucleotide phosphate for activity, nor did it require dithiothreitol, polyclar (polyvinyl pyrrolidone), or glycerol for stabilization of activity. It was susceptible to inhibition by dichlorvos and cysteine. Isotope tracer studies revealed involvement of several intermediates in the conversion of versiconal hemiacetal acetate to versicolorin A. These findings confirm the biogenetic relationship of versiconal hemiacetal acetate and versicolorin A, and they confirm that the bisfuran ring structure in aflatoxins and related fungal metabolites is derived from the hemiacetal structure of versiconal hemiacetal acetate. Images PMID:7356313

Identification of the Anti-Aflatoxinogenic Activity of Micromeria graeca and Elucidation of Its Molecular Mechanism in Aspergillus flavus

PubMed Central

El Khoury, Rhoda; Caceres, Isaura; Puel, Olivier; Bailly, Sylviane; Atoui, Ali; Oswald, Isabelle P.; El Khoury, André; Bailly, Jean-Denis

2017-01-01

Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B1 (AFB1), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca—known as hyssop—completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB1 synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB1 contamination. PMID:28257049

A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize

USDA-ARS?s Scientific Manuscript database

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of any maize gene sequence with resistance under field conditions. Reso...

Characterization of the maize lipoxygenase gene family in relation to aflatoxin accumulation resistance

Treesearch

Oluwaseun F. Ogunola; Leigh K. Hawkins; Erik Mylroie; Michael V. Kolomiets; Eli Borrego; Juliet D. Tang; Paul W. Williams; Marilyn L. Warburton

2017-01-01

Maize (Zea mays L.) is a globally important staple food crop prone to contamination by aflatoxin, a carcinogenic secondary metabolite produced by the fungus Aspergillus flavus. An efficient approach to reduce accumulation of aflatoxin is the development of germplasm resistant to colonization and toxin...

Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale

USDA-ARS?s Scientific Manuscript database

The worldwide costs associated with aflatoxin monitoring and crop losses are in the hundreds of millions of dollars. Aflatoxins also account for considerable health risks, even in countries where food contamination is regulated. Aspergillus flavus and A. parasiticus are the most common agents of af...

Genome-Wide Survey of Flavonoid Biosynthesis Genes and Gene Expression Analysis between Black- and Yellow-Seeded Brassica napus

PubMed Central

Qu, Cunmin; Zhao, Huiyan; Fu, Fuyou; Wang, Zhen; Zhang, Kai; Zhou, Yan; Wang, Xin; Wang, Rui; Xu, Xinfu; Tang, Zhanglin; Lu, Kun; Li, Jia-Na

2016-01-01

Flavonoids, the compounds that impart color to fruits, flowers, and seeds, are the most widespread secondary metabolites in plants. However, a systematic analysis of these loci has not been performed in Brassicaceae. In this study, we isolated 649 nucleotide sequences related to flavonoid biosynthesis, i.e., the Transparent Testa (TT) genes, and their associated amino acid sequences in 17 Brassicaceae species, grouped into Arabidopsis or Brassicaceae subgroups. Moreover, 36 copies of 21 genes of the flavonoid biosynthesis pathway were identified in Arabidopsis thaliana, 53 were identified in Brassica rapa, 50 in Brassica oleracea, and 95 in B. napus, followed the genomic distribution, collinearity analysis and genes triplication of them among Brassicaceae species. The results showed that the extensive gene loss, whole genome triplication, and diploidization that occurred after divergence from the common ancestor. Using qRT-PCR methods, we analyzed the expression of 18 flavonoid biosynthesis genes in 6 yellow- and black-seeded B. napus inbred lines with different genetic background, found that 12 of which were preferentially expressed during seed development, whereas the remaining genes were expressed in all B. napus tissues examined. Moreover, 14 of these genes showed significant differences in expression level during seed development, and all but four of these (i.e., BnTT5, BnTT7, BnTT10, and BnTTG1) had similar expression patterns among the yellow- and black-seeded B. napus. Results showed that the structural genes (BnTT3, BnTT18, and BnBAN), regulatory genes (BnTTG2 and BnTT16) and three encoding transfer proteins (BnTT12, BnTT19, and BnAHA10) might play an crucial roles in the formation of different seed coat colors in B. napus. These data will be helpful for illustrating the molecular mechanisms of flavonoid biosynthesis in Brassicaceae species. PMID:27999578

Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review.

PubMed

Leong, Yin-Hui; Latiff, Aishah A; Ahmad, Nurul Izzah; Rosma, Ahmad

2012-05-01

Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed.

A reference gene set for sex pheromone biosynthesis and degradation genes from the diamondback moth, Plutella xylostella, based on genome and transcriptome digital gene expression analyses.

PubMed

He, Peng; Zhang, Yun-Fei; Hong, Duan-Yang; Wang, Jun; Wang, Xing-Liang; Zuo, Ling-Hua; Tang, Xian-Fu; Xu, Wei-Ming; He, Ming

2017-03-01

Female moths synthesize species-specific sex pheromone components and release them to attract male moths, which depend on precise sex pheromone chemosensory system to locate females. Two types of genes involved in the sex pheromone biosynthesis and degradation pathways play essential roles in this important moth behavior. To understand the function of genes in the sex pheromone pathway, this study investigated the genome-wide and digital gene expression of sex pheromone biosynthesis and degradation genes in various adult tissues in the diamondback moth (DBM), Plutella xylostella, which is a notorious vegetable pest worldwide. A massive transcriptome data (at least 39.04 Gb) was generated by sequencing 6 adult tissues including male antennae, female antennae, heads, legs, abdomen and female pheromone glands from DBM by using Illumina 4000 next-generation sequencing and mapping to a published DBM genome. Bioinformatics analysis yielded a total of 89,332 unigenes among which 87 transcripts were putatively related to seven gene families in the sex pheromone biosynthesis pathway. Among these, seven [two desaturases (DES), three fatty acyl-CoA reductases (FAR) one acetyltransferase (ACT) and one alcohol dehydrogenase (AD)] were mainly expressed in the pheromone glands with likely function in the three essential sex pheromone biosynthesis steps: desaturation, reduction, and esterification. We also identified 210 odorant-degradation related genes (including sex pheromone-degradation related genes) from seven major enzyme groups. Among these genes, 100 genes are new identified and two aldehyde oxidases (AOXs), one aldehyde dehydrogenase (ALDH), five carboxyl/cholinesterases (CCEs), five UDP-glycosyltransferases (UGTs), eight cytochrome P450 (CYP) and three glutathione S-transferases (GSTs) displayed more robust expression in the antennae, and thus are proposed to participate in the degradation of sex pheromone components and plant volatiles. To date, this is the most

Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures

PubMed Central

Chen, Ruibing; Li, Qing; Tan, Hexin; Chen, Junfeng; Xiao, Ying; Ma, Ruifang; Gao, Shouhong; Zerbe, Philipp; Chen, Wansheng; Zhang, Lei

2015-01-01

Root and leaf tissue of Isatis indigotica shows notable anti-viral efficacy, and are widely used as “Banlangen” and “Daqingye” in traditional Chinese medicine. The plants' pharmacological activity is attributed to phenylpropanoids, especially a group of lignan metabolites. However, the biosynthesis of lignans in I. indigotica remains opaque. This study describes the discovery and analysis of biosynthetic genes and AP2/ERF-type transcription factors involved in lignan biosynthesis in I. indigotica. MeJA treatment revealed differential expression of three genes involved in phenylpropanoid backbone biosynthesis (IiPAL, IiC4H, Ii4CL), five genes involved in lignan biosynthesis (IiCAD, IiC3H, IiCCR, IiDIR, and IiPLR), and 112 putative AP2/ERF transcription factors. In addition, four intermediates of lariciresinol biosynthesis were found to be induced. Based on these results, a canonical correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite networks and identify putative key genes and rate-limiting reactions in lignan biosynthesis. Over-expression of IiC3H, identified as a key pathway gene, was used for metabolic engineering of I. indigotica hairy roots, and resulted in an increase in lariciresinol production. These findings illustrate the utility of canonical correlation analysis for the discovery and metabolic engineering of key metabolic genes in plants. PMID:26579184

Effects of Heracleum persicum ethyl acetate extract on the growth, hyphal ultrastructure and aflatoxin biosynthesis in Aspergillus parasiticus.

PubMed

Razzaghi-Abyaneh, Mehdi; Saberi, Reza; Sharifan, Anosheh; Rezaee, Mohammad-Bagher; Seifili, Roghayeh; Hosseini, Seyed-Ibrahim; Shams-Ghahfarokhi, Masoomeh; Nikkhah, Mehdi; Saberi, Ismail; Amani, Akram

2013-11-01

The ethyl acetate extract of leaves, seeds and flowers of Heracleum persicum, a medicinal plant of Iran (family Apiaceae) inhibited growth and aflatoxin (AF) production of Aspergillus parasiticus. On the basis of total dry weight growth inhibition by the leaf extract ranged from 17.1 to 36.9 %, by the flower extract from 32.2 to 75.6 %, and by the seed extract from 27.5 to 74.9 %. Production of AFB1 and AFG1 was inhibited in a dose-dependent manner, with a reduction of 88.5-100 % at the highest concentration of 8,000 μg/ml tested. The flower extract decreased ergosterol content of hyphae most significantly. Electron microscopy further revealed structural defects in the treated A. parasiticus including disruption of cytoplasmic membranous compartments, detachment of plasma membrane from the cell wall, and disorganization of hyphal compartments. Collapsed hyphae without conidiation, shorter branches and undifferentiated hyphal tips were also evident. The results indicate that H. persicum extract exerts antifungal and anti-AF activities by disrupting plasma membrane integrity and permeability mainly through interference with ergosterol biosynthesis. These results show that H. persicum can serve as a potent and safe alternative for inhibiting toxigenic aspergilli growth and thus preventing AF contamination of foods and feeds.

Aflatoxin variability in pistachios.

PubMed Central

Mahoney, N E; Rodriguez, S B

1996-01-01

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus. PMID:8919781

Aflatoxin decomposition in various soils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angle, J.S.

The persistence of aflatoxin in the soil environment could potentially result in a number of adverse environmental consequences. To determine the persistence of aflatoxin in soil, /sup 14/C-labeled aflatoxin B1, was added to silt loam, sandy loam, and silty clay loam soils and the subsequent release of /sup 14/CO/sub 2/ was determined. After 120 days of incubation, 8.1% of the original aflatoxin added to the silt loam soil was released as CO/sub 2/. Aflatoxin decomposition in the sandy loam soil proceeded more quickly than the other two soils for the first 20 days of incubation. After this time, the decompositionmore » rate declined and by the end of the study, 4.9% of the aflatoxin was released as CO/sub 2/. Aflatoxin decomposition proceeded most slowly in the silty clay loam soil. Only 1.4% of aflatoxin added to the soil was released as CO/sub 2/ after 120 days incubation. To determine whether aflatoxin was bound to the silty clay loam soil, aflatoxin B1 was added to this soil and incubated for 20 days. The soil was periodically extracted and the aflatoxin species present were determined using thin layer chromatographic (TLC) procedures. After one day of incubation, the degradation products, aflatoxins B2 and G2, were observed. It was also found that much of the aflatoxin extracted from the soil was not mobile with the TLC solvent system used. This indicated that a conjugate may have formed and thus may be responsible for the lack of aflatoxin decomposition.« less

A review on aflatoxin contamination and its implications in the developing world: a sub-Saharan African perspective.

PubMed

Gnonlonfin, G J B; Hell, K; Adjovi, Y; Fandohan, P; Koudande, D O; Mensah, G A; Sanni, A; Brimer, L

2013-01-01

Mycotoxins contamination in some agricultural food commodities seriously impact human and animal health and reduce the commercial value of crops. Mycotoxins are toxic secondary metabolites produced by fungi that contaminate agricultural commodities pre- or postharvest. Africa is one of the continents where environmental, agricultural and storage conditions of food commodities are conducive of Aspergillus fungi infection and aflatoxin biosynthesis. This paper reviews the commodity-wise aetiology and contamination process of aflatoxins and evaluates the potential risk of exposure from common African foods. Possible ways of reducing risk for fungal infection and aflatoxin development that are relevant to the African context. The presented database would be useful as benchmark information for development and prioritization of future research. There is need for more investigations on food quality and safety by making available advanced advanced equipments and analytical methods as well as surveillance and awareness creation in the region.

Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

PubMed Central

Guleria, Praveen; Yadav, Sudesh Kumar

2013-01-01

Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

Aflatoxin B1 inhibition in Aspergillus flavus by Aspergillus niger through down-regulating expression of major biosynthetic genes and AFB1 degradation by atoxigenic A. flavus.

PubMed

Xing, Fuguo; Wang, Limin; Liu, Xiao; Selvaraj, Jonathan Nimal; Wang, Yan; Zhao, Yueju; Liu, Yang

2017-09-01

Twenty Aspergillus niger strains were isolated from peanuts and 14 strains were able to completely inhibit AFB 1 production with co-cultivation. By using a Spin-X centrifuge system, it was confirmed that there are some soluble signal molecules or antibiotics involved in the inhibition by A. niger, although they are absent during the initial 24h of A. flavus growth when it is sensitive to inhibition. In A. flavus, 19 of 20 aflatoxin biosynthetic genes were down-regulated by A. niger. Importantly, the expression of aflS was significantly down-regulated, resulting in a reduction of AflS/AflR ratio. The results suggest that A. niger could directly inhibit AFB 1 biosynthesis through reducing the abundance of aflS to aflR mRNAs. Interestingly, atoxigenic A. flavus JZ2 and GZ15 effectively degrade AFB 1 . Two new metabolites were identified and the key toxic lactone and furofuran rings both were destroyed and hydrogenated, meaning that lactonase and reductase might be involved in the degradation process. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibition of aflatoxin metabolism and growth of Aspergillus flavus in liquid culture by a DNA methylation inhibitor.

PubMed

Yang, Kunlong; Zhuang, Zhenhong; Zhang, Feng; Song, Fengqin; Zhong, Hong; Ran, Fanlei; Yu, Song; Xu, Gaopo; Lan, Faxiu; Wang, Shihua

2015-01-01

Aflatoxins (AFs) are a group of highly oxygenated polyketidese-derived toxins mainly produced by Aspergillus flavus and A. parasiticus, whose biosynthesis mechanisms are extremely sophisticated. Methylation is known as the major form of epigenetic regulation, which is correlated with gene expression. As the DNA methylation inhibitor 5-azacytidine (5-AC) blocks AF production, we studied AFB1 metabolism and morphological changes of A. flavus by treatment with 5-AC in liquid culture. The results show that 5-AC caused a decrease in AF production and concurrent changes in morphology. In addition, we isolated a non-aflatoxigenic mutant of A. flavus, showing a significant reduction in pigment production, after 5-AC treatment. This mutant showed significant reduction in the expression of genes in the AF biosynthesis pathway, and conidia formation. Furthermore, as AF biosynthesis and oxidative stress are intimately related events, we assessed the viability of A. flavus to oxidative stress after treatment with 5-AC, which showed that the mutant was more sensitive to the strong oxidant hydrogen peroxide. We found that the non-aflatoxigenic mutant showed a decrease in reactive oxygen species (ROS) and metabolites indicative of oxidative stress, which may be caused by the disruption of the defence system against excessive ROS formation after 5-AC treatment. These data indicate that 5-AC, as an inactivator of DNA methyltransferase, plays a very important role in AFB1 metabolism and the development of A. flavus, which might provide an effective strategy to pre- or post-harvest control of AFs.

Accumulation of Charantin and Expression of Triterpenoid Biosynthesis Genes in Bitter Melon (Momordica charantia).

PubMed

Cuong, Do Manh; Jeon, Jin; Morgan, Abubaker M A; Kim, Changsoo; Kim, Jae Kwang; Lee, Sook Young; Park, Sang Un

2017-08-23

Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of McSE and McCAS1, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength.

Study of the genetic diversity of the aflatoxin biosynthesis cluster in Aspergillus section Flavi using insertion/deletion markers in peanut seeds from Georgia, USA

USDA-ARS?s Scientific Manuscript database

Aflatoxins are among the most powerful carcinogens in nature. The major aflatoxin-producing fungi are Aspergillus flavus and A. parasiticus. Numerous crops, including peanut, are susceptible to aflatoxin contamination by these fungi. There has been an increased use of RNA interference (RNAi) technol...

Effect of Various Compounds Blocking the Colony Pigmentation on the Aflatoxin B1 Production by Aspergillus flavus.

PubMed

Dzhavakhiya, Vitaly G; Voinova, Tatiana M; Popletaeva, Sofya B; Statsyuk, Natalia V; Limantseva, Lyudmila A; Shcherbakova, Larisa A

2016-10-28

Aflatoxins and melanins are the products of a polyketide biosynthesis. In this study, the search of potential inhibitors of the aflatoxin B1 (AFB1) biosynthesis was performed among compounds blocking the pigmentation in fungi. Four compounds-three natural (thymol, 3-hydroxybenzaldehyde, compactin) and one synthetic (fluconazole)-were examined for their ability to block the pigmentation and AFB1 production in Aspergillus flavus . All compounds inhibited the mycelium pigmentation of a fungus growing on solid medium. At the same time, thymol, fluconazole, and 3-hydroxybenzaldehyde stimulated AFB1 accumulation in culture broth of A. flavus under submerged fermentation, whereas the addition of 2.5 μg/mL of compactin resulted in a 50× reduction in AFB1 production. Moreover, compactin also suppressed the sporulation of A. flavus on solid medium. In vivo treatment of corn and wheat grain with compactin (50 μg/g of grain) reduced the level of AFB1 accumulation 14 and 15 times, respectively. Further prospects of the compactin study as potential AFB1 inhibitor are discussed.

Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination in crops.

PubMed

Bhatnagar-Mathur, Pooja; Sunkara, Sowmini; Bhatnagar-Panwar, Madhurima; Waliyar, Farid; Sharma, Kiran Kumar

2015-05-01

Aflatoxins are toxic, carcinogenic, mutagenic, teratogenic and immunosuppressive byproducts of Aspergillus spp. that contaminate a wide range of crops such as maize, peanut, and cotton. Aflatoxin not only affects crop production but renders the produce unfit for consumption and harmful to human and livestock health, with stringent threshold limits of acceptability. In many crops, breeding for resistance is not a reliable option because of the limited availability of genotypes with durable resistance to Aspergillus. Understanding the fungal/crop/environment interactions involved in aflatoxin contamination is therefore essential in designing measures for its prevention and control. For a sustainable solution to aflatoxin contamination, research must be focused on identifying and improving knowledge of host-plant resistance factors to aflatoxin accumulation. Current advances in genetic transformation, proteomics, RNAi technology, and marker-assisted selection offer great potential in minimizing pre-harvest aflatoxin contamination in cultivated crop species. Moreover, developing effective phenotyping strategies for transgenic as well as precision breeding of resistance genes into commercial varieties is critical. While appropriate storage practices can generally minimize post-harvest aflatoxin contamination in crops, the use of biotechnology to interrupt the probability of pre-harvest infection and contamination has the potential to provide sustainable solution. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Influences of climate on aflatoxin producing fungi and aflatoxin contamination.

PubMed

Cotty, Peter J; Jaime-Garcia, Ramon

2007-10-20

Aflatoxins are potent mycotoxins that cause developmental and immune system suppression, cancer, and death. As a result of regulations intended to reduce human exposure, crop contamination with aflatoxins causes significant economic loss for producers, marketers, and processors of diverse susceptible crops. Aflatoxin contamination occurs when specific fungi in the genus Aspergillus infect crops. Many industries frequently affected by aflatoxin contamination know from experience and anecdote that fluctuations in climate impact the extent of contamination. Climate influences contamination, in part, by direct effects on the causative fungi. As climate shifts, so do the complex communities of aflatoxin-producing fungi. This includes changes in the quantity of aflatoxin-producers in the environment and alterations to fungal community structure. Fluctuations in climate also influence predisposition of hosts to contamination by altering crop development and by affecting insects that create wounds on which aflatoxin-producers proliferate. Aflatoxin contamination is prevalent both in warm humid climates and in irrigated hot deserts. In temperate regions, contamination may be severe during drought. The contamination process is frequently broken down into two phases with the first phase occurring on the developing crop and the second phase affecting the crop after maturation. Rain and temperature influence the phases differently with dry, hot conditions favoring the first and warm, wet conditions favoring the second. Contamination varies with climate both temporally and spatially. Geostatistics and multiple regression analyses have shed light on influences of weather on contamination. Geostatistical analyses have been used to identify recurrent contamination patterns and to match these with environmental variables. In the process environmental conditions with the greatest impact on contamination are identified. Likewise, multiple regression analyses allow ranking of

Genetic characterization of the maize lipoxygenase gene family in relation to aflatoxin accumuation resistance.

USDA-ARS?s Scientific Manuscript database

Maize (Zea mays L.) is a globally important staple food crop. It is prone to contamination by aflatoxin, a secondary carcinogenic metabolite produced by the fungus Aspergillus flavus. An efficient approach to combat the accumulation of aflatoxin is the development of germplasm resistant to infection...

Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015

PubMed Central

Zhang, Chunxiao; Sheng, Chaolan; Wang, Wei; Hu, Hongbo; Peng, Huasong; Zhang, Xuehong

2015-01-01

Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces. PMID:26305803

Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli.

PubMed

Choi, J I; Lee, S Y; Han, K

1998-12-01

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, beta-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

Aflatoxin-free transgenic maize using host-induced gene silencing

USDA-ARS?s Scientific Manuscript database

Aflatoxins are potent carcinogenic anti-nutritionals that suppress immune systems and contaminate staple foods in warm regions across the globe as the result of crop infection by certain Aspergillus species. Despite decades of control efforts, millions of tons of crops are produced annually that exc...

Identification of candidate genes in Populus cell wall biosynthesis using text-mining, co-expression network and comparative genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiaohan; Ye, Chuyu; Bisaria, Anjali

2011-01-01

Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of ethanol from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidences supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database and additionalmore » genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional genomics in relation to cell wall biosynthesis.« less

Production of Aflatoxin on Soybeans

PubMed Central

Gupta, S. K.; Venkitasubramanian, T. A.

1975-01-01

Probable factors influencing resistance to aflatoxin synthesis in soybeans have been investigated by using cultures of Aspergillus parasiticus NRRL 3240. Soybeans contain a small amount of zinc (0.01 μg/g) bound to phytic acid. Autoclaving soybeans at 15 pounds (6803.88 g) for 15 min increases the aflatoxin production, probably by making zinc available. Addition of zinc to both autoclaved and nonautoclaved soybeans promotes aflatoxin production. However, addition of varying levels of phytic acid at a constant concentration of zinc depresses aflatoxin synthesis with an increase in the added phytic acid. In a synthetic medium known to give good yields of aflatoxin, the addition of phytic acid (10 mM) decreases aflatoxin synthesis. PMID:1171654

Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

PubMed

Agarwal, Aditya Vikram; Singh, Deeksha; Dhar, Yogeshwar Vikram; Michael, Rahul; Gupta, Parul; Chandra, Deepak; Trivedi, Prabodh Kumar

2018-02-01

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

Transcriptome Sequencing of Codonopsis pilosula and Identification of Candidate Genes Involved in Polysaccharide Biosynthesis

PubMed Central

Gao, Jian Ping; Wang, Dong; Cao, Ling Ya; Sun, Hai Feng

2015-01-01

Background Codonopsis pilosula (Franch.) Nannf. is one of the most widely used medicinal plants. Although chemical and pharmacological studies have shown that codonopsis polysaccharides (CPPs) are bioactive compounds and that their composition is variable, their biosynthetic pathways remain largely unknown. Next-generation sequencing is an efficient and high-throughput technique that allows the identification of candidate genes involved in secondary metabolism. Principal Findings To identify the components involved in CPP biosynthesis, a transcriptome library, prepared using root and other tissues, was assembled with the help of Illumina sequencing. A total of 9.2 Gb of clean nucleotides was obtained comprising 91,175,044 clean reads, 102,125 contigs, and 45,511 unigenes. After aligning the sequences to the public protein databases, 76.1% of the unigenes were annotated. Among these annotated unigenes, 26,189 were assigned to Gene Ontology categories, 11,415 to Clusters of Orthologous Groups, and 18,848 to Kyoto Encyclopedia of Genes and Genomes pathways. Analysis of abundance of transcripts in the library showed that genes, including those encoding metallothionein, aquaporin, and cysteine protease that are related to stress responses, were in the top list. Among genes involved in the biosynthesis of CPP, those responsible for the synthesis of UDP-L-arabinose and UDP-xylose were highly expressed. Significance To our knowledge, this is the first study to provide a public transcriptome dataset prepared from C. pilosula and an outline of the biosynthetic pathway of polysaccharides in a medicinal plant. Identified candidate genes involved in CPP biosynthesis provide understanding of the biosynthesis and regulation of CPP at the molecular level. PMID:25719364

The rubber tree genome shows expansion of gene family associated with rubber biosynthesis

PubMed Central

Lau, Nyok-Sean; Makita, Yuko; Kawashima, Mika; Taylor, Todd D.; Kondo, Shinji; Othman, Ahmad Sofiman; Shu-Chien, Alexander Chong; Matsui, Minami

2016-01-01

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis’s capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree. PMID:27339202

The rubber tree genome shows expansion of gene family associated with rubber biosynthesis.

PubMed

Lau, Nyok-Sean; Makita, Yuko; Kawashima, Mika; Taylor, Todd D; Kondo, Shinji; Othman, Ahmad Sofiman; Shu-Chien, Alexander Chong; Matsui, Minami

2016-06-24

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis's capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.

Loss of msnA, a putative stress regulatory gene, in Aspergillus parasiticus and Aspergillus flavus increased production of conidia, aflatoxins and kojic acid

USDA-ARS?s Scientific Manuscript database

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene, the ortholog of Saccharomyces cerevisiae MSN2 associated with multi-stress response, of the two species was disrupted....

Regulation of aflatoxin biosynthesis and branched-chain amino acids metabolism in Aspergillus flavus by 2-phenylethanol reveal biocontrol mechanism of Pichia anomala

USDA-ARS?s Scientific Manuscript database

Pichia anomala WRL-076 is a biocontrol yeast which has been shown to inhibit growth and aflatoxin production of A. flavus. Using the SPME-GC/MS analysis we identified that the volatile, 2-phenylethanol (2-PE) produced by this yeast and demonstrated that the compound inhibited aflatoxin production. W...

Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi

PubMed Central

Cook, Daniel; Donzelli, Bruno G. G.; Creamer, Rebecca; Baucom, Deana L.; Gardner, Dale R.; Pan, Juan; Moore, Neil; Krasnoff, Stuart B.; Jaromczyk, Jerzy W.; Schardl, Christopher L.

2017-01-01

Swainsonine—a cytotoxic fungal alkaloid and a potential cancer therapy drug—is produced by the insect pathogen and plant symbiont Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated “SWN,” which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a β-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete’s foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals. PMID:28381497

Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi.

PubMed

Cook, Daniel; Donzelli, Bruno G G; Creamer, Rebecca; Baucom, Deana L; Gardner, Dale R; Pan, Juan; Moore, Neil; Krasnoff, Stuart B; Jaromczyk, Jerzy W; Schardl, Christopher L

2017-06-07

Swainsonine-a cytotoxic fungal alkaloid and a potential cancer therapy drug-is produced by the insect pathogen and plant symbiont Metarhizium robertsii , the clover pathogen Slafractonia leguminicola , locoweed symbionts belonging to Alternaria sect. Undifilum , and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated " SWN ," which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a β-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆ swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete's foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals. Copyright © 2017 Cook et al.

Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the Developing Embryo of Pecan (Carya illinoinensis).

PubMed

Huang, Ruimin; Huang, Youjun; Sun, Zhichao; Huang, Jianqin; Wang, Zhengjia

2017-05-24

Pecan (Carya illinoinensis) is an important woody tree species because of the high content of healthy oil in its nut. Thus far, the pathways and key genes related to oil biosynthesis in developing pecan seeds remain largely unclear. Our analyses revealed that mature pecan embryo accumulated more than 80% oil, in which 90% was unsaturated fatty acids with abundant oleic acid. RNA sequencing generated 84,643 unigenes in three cDNA libraries prepared from pecan embryos collected at 105, 120, and 165 days after flowering (DAF). We identified 153 unigenes associated with lipid biosynthesis, including 107 unigenes for fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, 7 for oil bodies, and 5 for transcription factors involved in oil synthesis. The genes associated with fatty acid synthesis were the most abundantly expressed genes at 120 DAF. Additionally, the biosynthesis of oil began to increase while crude fat contents increased from 16.61 to 74.45% (165 DAF). We identified four SAD, two FAD2, one FAD6, two FAD7, and two FAD8 unigenes responsible for unsaturated fatty acid biosynthesis. However, FAD3 homologues were not detected. Consequently, we inferred that the linolenic acid in developing pecan embryos is generated by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic reticula. During pecan embryo development, different unigenes are expressed for plastidial and cytosolic glycolysis. Plastidial glycolysis is more relevant to lipid synthesis than cytosolic glycolysis. The 18 most important genes associated with lipid biosynthesis were evaluated in five stages of developing embryos using quantitative PCR (qPCR). The qPCR data were well consistent with their expression in transcriptomic analyses. Our data would be important for the metabolic engineering of pecans to increase oil contents and modify fatty acid composition.

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

PubMed Central

Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

2015-01-01

ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many

Computational search for aflatoxin binding proteins

NASA Astrophysics Data System (ADS)

Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

2017-10-01

Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction.

PubMed

Tetteh, Antonia Y; Sun, Katherine H; Hung, Chiu-Yueh; Kittur, Farooqahmed S; Ibeanu, Gordon C; Williams, Daniel; Xie, Jiahua

2014-01-01

Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se(0)), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼ 50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼ 30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

2010-01-07

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3more » (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

PubMed

Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

2010-02-01

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

Biological Controls for Aflatoxin Reduction

USDA-ARS?s Scientific Manuscript database

Aflatoxin exposure is frequent and widespread in most African countries where the key staples, maize and groundnut, are particularly vulnerable to aflatoxin contamination. Aflatoxin-producing fungi are ubiquitous in Africa where they occupy soil and colonize diverse organic matter while producing sp...

Identification and Characterization of Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in Coptis Species

PubMed Central

He, Si-Mei; Liang, Yan-Li; Cong, Kun; Chen, Geng; Zhao, Xiu; Zhao, Qi-Ming; Zhang, Jia-Jin; Wang, Xiao; Dong, Yang; Yang, Jian-Li; Zhang, Guang-Hui; Qian, Zhi-Long; Fan, Wei; Yang, Sheng-Chao

2018-01-01

The dried rhizomes of Coptis chinensis have been extensively used in heat clearing, dampness drying, fire draining, and detoxification by virtue of their major bioactive components, benzylisoquinoline alkaloids (BIAs). However, C. teeta and C. chinensis are occasionally interchanged, and current understanding of the molecular basis of BIA biosynthesis in these two species is limited. Here, berberine, coptisine, jatrorrhizine, and palmatine were detected in two species, and showed the highest contents in the roots, while epiberberine were found only in C. chinensis. Comprehensive transcriptome analysis of the roots and leaves of C. teeta and C. chinensis, respectively, identified 53 and 52 unigenes encoding enzymes potentially involved in BIA biosynthesis. By integrating probable biosynthetic pathways for BIAs, the jatrorrhizine biosynthesis ill-informed previously was further characterized. Two genes encoding norcoclaurine/norlaudanosoline 6-O-methyltransferases (Cc6OMT1 and Cc6OMT2) and one gene encoding norcoclaurine-7OMT (Ct7OMT) catalyzed enzymatically O-methylate (S)-norcoclaurine at C6 that yield (S)-coclaurine, along with a smaller amount of O-methylation occurred at C7, thereby forming its isomer (isococlaurine). In addition, scoulerine 9-OMT (CtSOMT) was determined to show strict substrate specificity, targeting (S)-scoulerine to yield (S)-tetrahydrocolumbamine. Taken together, the integration of the transcriptome and enzyme activity assays further provides new insight into molecular mechanisms underlying BIA biosynthesis in plants and identifies candidate genes for the study of synthetic biology in microorganisms. PMID:29915609

Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes.

PubMed

Kamiya, Regianne Umeko; Höfling, José Francisco; Gonçalves, Reginaldo Bruno

2008-05-01

The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I-IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=-0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.

Salt tolerance and methionine biosynthesis in Saccharomyces cerevisiae involve a putative phosphatase gene.

PubMed Central

Gläser, H U; Thomas, D; Gaxiola, R; Montrichard, F; Surdin-Kerjan, Y; Serrano, R

1993-01-01

The progressive salinization of irrigated land poses a threat to the future of agriculture in arid regions. The identification of crucial metabolic steps in salt tolerance is important for the understanding of stress physiology and may provide the tools for its genetic engineering. In the yeast Saccharomyces cerevisiae we have isolated a gene, HAL2, which upon increase in gene dosage improves growth under NaCl and LiCl stresses. The HAL2 protein is homologous to inositol phosphatases, enzymes known to be inhibited by lithium salts. Complementation analysis demonstrated that HAL2 is identical to MET22, a gene involved in methionine biosynthesis. Accordingly, methionine supplementation improves the tolerance of yeast to NaCl and LiCl. These results demonstrate an unsuspected interplay between methionine biosynthesis and salt tolerance. Images PMID:8393782

Manual sorting to eliminate aflatoxin from peanuts.

PubMed

Galvez, F C F; Francisco, M L D L; Villarino, B J; Lustre, A O; Resurreccion, A V A

2003-10-01

A manual sorting procedure was developed to eliminate aflatoxin contamination from peanuts. The efficiency of the sorting process in eliminating aflatoxin-contaminated kernels from lots of raw peanuts was verified. The blanching of 20 kg of peanuts at 140 degrees C for 25 min in preheated roasters facilitated the manual sorting of aflatoxin-contaminated kernels after deskinning. The manual sorting of raw materials with initially high aflatoxin contents (300 ppb) resulted in aflatoxin-free peanuts (i.e., peanuts in which no aflatoxin was detected). Verification procedures showed that the sorted sound peanuts contained no aflatoxin or contained low levels (<15 ppb) of aflatoxin. The results obtained confirmed that the sorting process was effective in separating contaminated peanuts whether or nor contamination was extensive. At the commercial level, when roasters were not preheated, the dry blanching of 50 kg of peanuts for 45 to 55 min facilitated the proper deskinning and subsequent manual sorting of aflatoxin-contaminated peanut kernels from sound kernels.

Kinetics of aflatoxin degradation during peanut roasting.

PubMed

Martins, Ligia M; Sant'Ana, Anderson S; Iamanaka, Beatriz T; Berto, Maria Isabel; Pitt, John I; Taniwaki, Marta H

2017-07-01

This study investigated aflatoxin degradation during peanut roasting. First, peanuts contaminated with three initial aflatoxin concentrations (35, 332 and 695μg/kg) were roasted at 180°C for up to 20min. The percentage of aflatoxin degradation after 20min were 55, 64 and 81% for peanuts contaminated with aflatoxin at 35, 332 and 695μg/kg, respectively. This difference was statistically significant (p<0.05), showing that initial concentration influences aflatoxin reduction. Thereafter, peanut samples contaminated with an initial aflatoxin concentration of 85μg/kg were roasted at 160, 180 and 200°C for 5, 10, 15, 20 and 25min, then residual concentrations of aflatoxin were determined. Roasting at 160, 180 and 200°C resulted in an aflatoxin reduction of 61.6, 83.6 and 89.7%, respectively. This study has provided quantitative data reinforcing the fact that roasting alone is not enough to control aflatoxins in peanuts. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Josse, Rozenn; Dumont, Julie; Fautrel, Alain

Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cellmore » cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered

Host-Induced Gene Silencing (HIGS) of aflatoxin synthesis genes in peanut and maize: use of RNA interference and genetic diversity of Aspergillus

USDA-ARS?s Scientific Manuscript database

Approximately 4.5 billion people are chronically exposed to aflatoxins, these are powerful carcinogens produced by Aspergillus flavus and A. parasiticus. High levels of aflatoxins in crops result in approximately 100 million metric tons of cereals, ¬nuts, root crops and other agricultural products ...

Occurrence of Aflatoxins and Aflatoxin-Producing Strains of Aspergillus spp. in Soybeans 1

PubMed Central

Bean, George A.; Schillinger, John A.; Klarman, William L.

1972-01-01

Above average rainfall in Maryland during August, September, and October 1971 resulted in heavy mold growth in soybeans while still in the field. Of 28 samples of soybean seed, aflatoxins were found in 14, 2 of which had been used in poultry feed. Aflatoxins were identified by thin-layer chromatography, spectrophotometry, and chicken embryo bioassay. Aspergillus spp. were isolated from 11 samples, and 5 of these isolates produced aflatoxins when grown in liquid culture. PMID:4673021

RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes.

PubMed

Wei, Yunxie; Chang, Yanli; Zeng, Hongqiu; Liu, Guoyin; He, Chaozu; Shi, Haitao

2018-01-01

With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus-induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation-PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2-melatonin biosynthesis genes-melatonin level in plant disease resistance against cassava bacterial blight. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Present and future directions of translational research on aflatoxin and hepatocellular carcinoma. A review.

PubMed

Wogan, Gerald N; Kensler, Thomas W; Groopman, John D

2012-01-01

The aflatoxins were discovered in toxic peanut meal causing "turkey X" disease, which killed large numbers of turkey poults, ducklings and chicks in the UK in the early 1960s. Extracts of toxic feed induced the symptoms in experimental animals, and purified metabolites with properties identical to aflatoxins B(1) and G(1) (AFB(1) and AFG(1)) were isolated from Aspergillus flavus cultures. Structure elucidation of aflatoxin B(1) was accomplished and confirmed by total synthesis in 1963. AFB(1) is a potent liver carcinogen in rodents, non-human primates, fish and birds, operating through a genotoxic mechanism involving metabolic activation to an epoxide, formation of DNA adducts and, in humans, modification of the p53 gene. Aflatoxins are unique among environmental carcinogens, in that elucidation of their mechanisms of action combined with molecular epidemiology provides a foundation for quantitative risk assessment; extensive evidence confirms that contamination of the food supply by AFB(1) puts an exposed population at increased risk of developing hepatocellular carcinoma (HCC). Molecular biomarkers to quantify aflatoxin exposure in individuals were essential to link aflatoxin exposure with liver cancer risk. Biomarkers were validated in populations with high HCC incidence in China and The Gambia, West Africa; urinary AFB(1)-N (7)-Guanine excretion was linearly related to aflatoxin intake, and levels of aflatoxin-serum albumin adducts also reflected aflatoxin intake. Two major cohort studies employing aflatoxin biomarkers identified their causative role in HCC etiology. Results of a study in Shanghai men strongly support a causal relationship between HCC risk and the presence of biomarkers for aflatoxin and HBV infection, and also show that the two risk factors act synergistically. Subsequent cohort studies in Taiwan confirm these results. IARC classified aflatoxin as a Group 1 human carcinogen in 1993, based on sufficient evidence in humans and experimental

7 CFR 983.150 - Aflatoxin regulations.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE PISTACHIOS GROWN IN CALIFORNIA... handler shall ship for domestic human consumption, pistachios that exceed an aflatoxin level of 15 ppb... covered by an aflatoxin inspection certificate. Pistachios that fail to meet the aflatoxin requirements...

Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit

USDA-ARS?s Scientific Manuscript database

Little is known about how plants regulate their folate content, including whether the expression of folate biosynthesis genes is orchestrated during development or modulated by folate levels. Nor is much known about how folate levels impact the expression of other genes. These points were addressed ...

Phospholipid biosynthesis genes and susceptibility to obesity: analysis of expression and polymorphisms.

PubMed

Sharma, Neeraj K; Langberg, Kurt A; Mondal, Ashis K; Das, Swapan K

2013-01-01

Recent studies have identified links between phospholipid composition and altered cellular functions in animal models of obesity, but the involvement of phospholipid biosynthesis genes in human obesity are not well understood. We analyzed the transcript of four phospholipid biosynthesis genes in adipose and muscle from 170 subjects. We examined publicly available genome-wide association data from the GIANT and MAGIC cohorts to investigate the association of SNPs in these genes with obesity and glucose homeostasis traits, respectively. Trait-associated SNPs were genotyped to evaluate their roles in regulating expression in adipose. In adipose tissue, expression of PEMT, PCYT1A, and PTDSS2 were positively correlated and PCYT2 was negatively correlated with percent fat mass and body mass index (BMI). Among the polymorphisms in these genes, SNP rs4646404 in PEMT showed the strongest association (p = 3.07E-06) with waist-to-hip ratio (WHR) adjusted for BMI. The WHR-associated intronic SNP rs4646343 in the PEMT gene showed the strongest association with its expression in adipose. Allele "C" of this SNP was associated with higher WHR (p = 2.47E-05) and with higher expression (p = 4.10E-04). Our study shows that the expression of PEMT gene is high in obese insulin-resistant subjects. Intronic cis-regulatory polymorphisms may increase the genetic risk of obesity by modulating PEMT expression.

Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5.

PubMed

Mihali, Troco K; Kellmann, Ralf; Neilan, Brett A

2009-03-30

Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of

Two divergent Symbiodinium genomes reveal conservation of a gene cluster for sunscreen biosynthesis and recently lost genes.

PubMed

Shoguchi, Eiichi; Beedessee, Girish; Tada, Ipputa; Hisata, Kanako; Kawashima, Takeshi; Takeuchi, Takeshi; Arakaki, Nana; Fujie, Manabu; Koyanagi, Ryo; Roy, Michael C; Kawachi, Masanobu; Hidaka, Michio; Satoh, Noriyuki; Shinzato, Chuya

2018-06-14

The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

PubMed

de Jonge, Ronnie; Ebert, Malaika K; Huitt-Roehl, Callie R; Pal, Paramita; Suttle, Jeffrey C; Spanner, Rebecca E; Neubauer, Jonathan D; Jurick, Wayne M; Stott, Karina A; Secor, Gary A; Thomma, Bart P H J; Van de Peer, Yves; Townsend, Craig A; Bolton, Melvin D

2018-06-12

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis ( CTB ) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae , the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide. Copyright © 2018 the Author(s). Published by PNAS.

Lipopolysaccharide and cAMP modify placental calcitriol biosynthesis reducing antimicrobial peptides gene expression.

PubMed

Olmos-Ortiz, Andrea; García-Quiroz, Janice; Avila, Euclides; Caldiño-Soto, Felipe; Halhali, Ali; Larrea, Fernando; Díaz, Lorenza

2018-06-01

Calcitriol, the hormonal form of vitamin D 3 (VD), stimulates placental antimicrobial peptides expression; nonetheless, the regulation of calcitriol biosynthesis in the presence of bacterial products and its consequence on placental innate immunity have scarcely been addressed. We investigated how some bacterial products modify placental VD metabolism and its ability to induce antimicrobial peptides gene expression. Cultured human trophoblasts biosynthesized calcitriol only in the presence of its precursor calcidiol, a process that was inhibited by cyclic-AMP but stimulated by lipopolysaccharide (LPS). Intracrine calcitriol upregulated cathelicidin, S100A9, and β-defensins (HBDs) gene expression, while LPS further stimulated HBD2 and S100A9. Unexpectedly, LPS significantly repressed cathelicidin basal mRNA levels and drastically diminished calcidiol ability to induce it. Meanwhile, cyclic-AMP, which is used by many microbes to avoid host defenses, suppressed calcitriol biosynthesis, resulting in significant inhibition of most VD-dependent microbicidal peptides gene expression. While LPS stimulated calcitriol biosynthesis, cyclic-AMP inhibited it. LPS downregulated cathelicidin mRNA expression, whereas cyclic-AMP antagonized VD-dependent-upregulation of most antimicrobial peptides. These findings reveal LPS and cyclic-AMP involvement in dampening placental innate immunity, highlighting the importance of cyclic-AMP in the context of placental infection and suggesting its participation to facilitate bacterial survival. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Putative Gene Cluster from a Lyngbya wollei Bloom that Encodes Paralytic Shellfish Toxin Biosynthesis

PubMed Central

Mihali, Troco K.; Carmichael, Wayne W.; Neilan, Brett A.

2011-01-01

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds. PMID:21347365

Present and future directions of translational research on aflatoxin and hepatocellular carcinoma. A review

PubMed Central

Wogan, Gerald N.; Kensler, Thomas W.; Groopman, John D.

2015-01-01

The aflatoxins were discovered in toxic peanut meal causing “turkey X” disease, which killed large numbers of turkey poults, ducklings and chicks in the UK in the early 1960s. Extracts of toxic feed induced the symptoms in experimental animals, and purified metabolites with properties identical to aflatoxins B1 and G1 (AFB1 and AFG1) were isolated from Aspergillus flavus cultures. Structure elucidation of aflatoxin B1 was accomplished and confirmed by total synthesis in 1963. AFB1 is a potent liver carcinogen in rodents, non-human primates, fish and birds, operating through a genotoxic mechanism involving metabolic activation to an epoxide, formation of DNA adducts and, in humans, modification of the p53 gene. Aflatoxins are unique among environmental carcinogens, in that elucidation of their mechanisms of action combined with molecular epidemiology provides a foundation for quantitative risk assessment; extensive evidence confirms that contamination of the food supply by AFB1 puts an exposed population at increased risk of developing hepatocellular carcinoma (HCC). Molecular biomarkers to quantify aflatoxin exposure in individuals were essential to link aflatoxin exposure with liver cancer risk. Biomarkers were validated in populations with high HCC incidence in China and The Gambia, West Africa; urinary AFB1–N7-Guanine excretion was linearly related to aflatoxin intake, and levels of aflatoxin– serum albumin adducts also reflected aflatoxin intake. Two major cohort studies employing aflatoxin biomarkers identified their causative role in HCC etiology. Results of a study in Shanghai men strongly support a causal relationship between HCC risk and the presence of biomarkers for aflatoxin and HBV infection, and also show that the two risk factors act synergistically. Subsequent cohort studies in Taiwan confirm these results. IARC classified aflatoxin as a Group 1 human carcinogen in 1993, based on sufficient evidence in humans and experimental animals

Aflatoxins, hepatocellular carcinoma and public health.

PubMed

Magnussen, Arvin; Parsi, Mansour A

2013-03-14

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide, primarily affecting populations in the developing countries. Aflatoxin, a food contaminant produced by the fungi Aspergillus flavus and Aspergillus parasiticus, is a known human carcinogen that has been shown to be a causative agent in the pathogenesis of HCC. Aflatoxin can affect a wide range of food commodities including corns, oilseeds, spices, and tree nuts as well as milk, meat, and dried fruit. Many factors affect the growth of Aspergillus fungi and the level of aflatoxin contamination in food. Drought stress is one of the factors that increase susceptibility of plants to Aspergillus and thus aflatoxin contamination. A recent drought is thought to be responsible for finding of trace amounts of aflatoxin in some of the corn harvested in the United States. Although it's too soon to know whether aflatoxin will be a significant problem, since United States is the world's largest corn producer and exporter, this has raised alarm bells. Strict regulations and testing of finished foods and feeds in the United States should prevent a major health scare, and prevent human exposure to deleterious levels of aflatoxin. Unfortunately, such regulations and testing are not in place in many countries. The purpose of this editorial is to summarize the current knowledge on association of aflatoxin and HCC, encourage future research and draw attention to this global public health issue.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

A systems approach to model the relationship between aflatoxin gene cluster expression, environmental factors, growth and toxin production by Aspergillus flavus

PubMed Central

Abdel-Hadi, Ahmed; Schmidt-Heydt, Markus; Parra, Roberto; Geisen, Rolf; Magan, Naresh

2012-01-01

A microarray analysis was used to examine the effect of combinations of water activity (aw, 0.995–0.90) and temperature (20–42°C) on the activation of aflatoxin biosynthetic genes (30 genes) in Aspergillus flavus grown on a conducive YES (20 g yeast extract, 150 g sucrose, 1 g MgSO4·7H2O) medium. The relative expression of 10 key genes (aflF, aflD, aflE, aflM, aflO, aflP, aflQ, aflX, aflR and aflS) in the biosynthetic pathway was examined in relation to different environmental factors and phenotypic aflatoxin B1 (AFB1) production. These data, plus data on relative growth rates and AFB1 production under different aw × temperature conditions were used to develop a mixed-growth-associated product formation model. The gene expression data were normalized and then used as a linear combination of the data for all 10 genes and combined with the physical model. This was used to relate gene expression to aw and temperature conditions to predict AFB1 production. The relationship between the observed AFB1 production provided a good linear regression fit to the predicted production based in the model. The model was then validated by examining datasets outside the model fitting conditions used (37°C, 40°C and different aw levels). The relationship between structural genes (aflD, aflM) in the biosynthetic pathway and the regulatory genes (aflS, aflJ) was examined in relation to aw and temperature by developing ternary diagrams of relative expression. These findings are important in developing a more integrated systems approach by combining gene expression, ecophysiological influences and growth data to predict mycotoxin production. This could help in developing a more targeted approach to develop prevention strategies to control such carcinogenic natural metabolites that are prevalent in many staple food products. The model could also be used to predict the impact of climate change on toxin production. PMID:21880616

Chlorophyll Biosynthesis Gene Evolution Indicates Photosystem Gene Duplication, Not Photosystem Merger, at the Origin of Oxygenic Photosynthesis

PubMed Central

Sousa, Filipa L.; Shavit-Grievink, Liat; Allen, John F.; Martin, William F.

2013-01-01

An open question regarding the evolution of photosynthesis is how cyanobacteria came to possess the two reaction center (RC) types, Type I reaction center (RCI) and Type II reaction center (RCII). The two main competing theories in the foreground of current thinking on this issue are that either 1) RCI and RCII are related via lineage divergence among anoxygenic photosynthetic bacteria and became merged in cyanobacteria via an event of large-scale lateral gene transfer (also called "fusion" theories) or 2) the two RC types are related via gene duplication in an ancestral, anoxygenic but protocyanobacterial phototroph that possessed both RC types before making the transition to using water as an electron donor. To distinguish between these possibilities, we studied the evolution of the core (bacterio)chlorophyll biosynthetic pathway from protoporphyrin IX (Proto IX) up to (bacterio)chlorophyllide a. The results show no dichotomy of chlorophyll biosynthesis genes into RCI- and RCII-specific chlorophyll biosynthetic clades, thereby excluding models of fusion at the origin of cyanobacteria and supporting the selective-loss hypothesis. By considering the cofactor demands of the pathway and the source genes from which several steps in chlorophyll biosynthesis are derived, we infer that the cell that first synthesized chlorophyll was a cobalamin-dependent, heme-synthesizing, diazotrophic anaerobe. PMID:23258841

Chlorophyll biosynthesis gene evolution indicates photosystem gene duplication, not photosystem merger, at the origin of oxygenic photosynthesis.

PubMed

Sousa, Filipa L; Shavit-Grievink, Liat; Allen, John F; Martin, William F

2013-01-01

An open question regarding the evolution of photosynthesis is how cyanobacteria came to possess the two reaction center (RC) types, Type I reaction center (RCI) and Type II reaction center (RCII). The two main competing theories in the foreground of current thinking on this issue are that either 1) RCI and RCII are related via lineage divergence among anoxygenic photosynthetic bacteria and became merged in cyanobacteria via an event of large-scale lateral gene transfer (also called "fusion" theories) or 2) the two RC types are related via gene duplication in an ancestral, anoxygenic but protocyanobacterial phototroph that possessed both RC types before making the transition to using water as an electron donor. To distinguish between these possibilities, we studied the evolution of the core (bacterio)chlorophyll biosynthetic pathway from protoporphyrin IX (Proto IX) up to (bacterio)chlorophyllide a. The results show no dichotomy of chlorophyll biosynthesis genes into RCI- and RCII-specific chlorophyll biosynthetic clades, thereby excluding models of fusion at the origin of cyanobacteria and supporting the selective-loss hypothesis. By considering the cofactor demands of the pathway and the source genes from which several steps in chlorophyll biosynthesis are derived, we infer that the cell that first synthesized chlorophyll was a cobalamin-dependent, heme-synthesizing, diazotrophic anaerobe.

Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis.

PubMed

Hwang, Hwan-Su; Lee, Hyoshin; Choi, Yong Eui

2015-03-14

Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant. In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway. The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.

RNAi control of aflatoxins in peanut plants, a multifactorial system

USDA-ARS?s Scientific Manuscript database

RNA-interference (RNAi)-mediated control of aflatoxin contamination in peanut plants is a multifactorial and hyper variable system. The use of RNAi biotechnology to silence single genes in plants has inherently high-variability among transgenic events. Also the level of expression of small interfe...

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

PubMed Central

Zhang, Gaiyun; Zhang, Haibo; Li, Sumei; Xiao, Ji; Zhang, Guangtao; Zhu, Yiguang; Niu, Siwen; Ju, Jianhua

2012-01-01

Amicetin, an antibacterial and antiviral agent, belongs to a group of disaccharide nucleoside antibiotics featuring an α-(1→4)-glycoside bond in the disaccharide moiety. In this study, the amicetin biosynthesis gene cluster was cloned from Streptomyces vinaceusdrappus NRRL 2363 and localized on a 37-kb contiguous DNA region. Heterologous expression of the amicetin biosynthesis gene cluster in Streptomyces lividans TK64 resulted in the production of amicetin and its analogues, thereby confirming the identity of the ami gene cluster. In silico sequence analysis revealed that 21 genes were putatively involved in amicetin biosynthesis, including 3 for regulation and transportation, 10 for disaccharide biosynthesis, and 8 for the formation of the amicetin skeleton by the linkage of cytosine, p-aminobenzoic acid (PABA), and the terminal (+)-α-methylserine moieties. The inactivation of the benzoate coenzyme A (benzoate-CoA) ligase gene amiL and the N-acetyltransferase gene amiF led to two mutants that accumulated the same two compounds, cytosamine and 4-acetamido-3-hydroxybenzoic acid. These data indicated that AmiF functioned as an amide synthethase to link cytosine and PABA. The inactivation of amiR, encoding an acyl-CoA-acyl carrier protein transacylase, resulted in the production of plicacetin and norplicacetin, indicating AmiR to be responsible for attachment of the terminal methylserine moiety to form another amide bond. These findings implicated two alternative strategies for amide bond formation in amicetin biosynthesis. PMID:22267658

The Distant Siblings-A Phylogenomic Roadmap Illuminates the Origins of Extant Diversity in Fungal Aromatic Polyketide Biosynthesis.

PubMed

Koczyk, Grzegorz; Dawidziuk, Adam; Popiel, Delfina

2015-11-03

In recent years, the influx of newly sequenced fungal genomes has enabled sampling of secondary metabolite biosynthesis on an unprecedented scale. However, explanations of extant diversity which take into account both large-scale phylogeny reconstructions and knowledge gained from multiple genome projects are still lacking. We analyzed the evolutionary sources of genetic diversity in aromatic polyketide biosynthesis in over 100 model fungal genomes. By reconciling the history of over 400 nonreducing polyketide synthases (NR-PKSs) with corresponding species history, we demonstrate that extant fungal NR-PKSs are clades of distant siblings, originating from a burst of duplications in early Pezizomycotina and thinned by extensive losses. The capability of higher fungi to biosynthesize the simplest precursor molecule (orsellinic acid) is highlighted as an ancestral trait underlying biosynthesis of aromatic compounds. This base activity was modified during early evolution of filamentous fungi, toward divergent reaction schemes associated with biosynthesis of, for example, aflatoxins and fusarubins (C4-C9 cyclization) or various anthraquinone derivatives (C6-C11 cyclization). The functional plasticity is further shown to have been supplemented by modularization of domain architecture into discrete pieces (conserved splice junctions within product template domain), as well as tight linkage of key accessory enzyme families and divergence in employed transcriptional factors. Although the majority of discord between species and gene history is explained by ancient duplications, this landscape has been altered by more recent duplications, as well as multiple horizontal gene transfers. The 25 detected transfers include previously undescribed events leading to emergence of, for example, fusarubin biosynthesis in Fusarium genus. Both the underlying data and the results of present analysis (including alternative scenarios revealed by sampling multiple reconciliation optima) are

De Novo Transcriptome Assembly and Characterization of Lithospermum officinale to Discover Putative Genes Involved in Specialized Metabolites Biosynthesis.

PubMed

Rai, Amit; Nakaya, Taiki; Shimizu, Yohei; Rai, Megha; Nakamura, Michimi; Suzuki, Hideyuki; Saito, Kazuki; Yamazaki, Mami

2018-05-29

Lithospermum officinale is a valuable source of bioactive metabolites with medicinal and industrial values. However, little is known about genes involved in the biosynthesis of these metabolites, primarily due to the lack of genome or transcriptome resources. This study presents the first effort to establish and characterize de novo transcriptome assembly resource for L. officinale and expression analysis for three of its tissues, namely leaf, stem, and root. Using over 4Gbps of RNA-sequencing datasets, we obtained de novo transcriptome assembly of L. officinale , consisting of 77,047 unigenes with assembly N50 value as 1524 bps. Based on transcriptome annotation and functional classification, 52,766 unigenes were assigned with putative genes functions, gene ontology terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene ontology enrichment analysis using highly expressed unigenes across three tissues and targeted metabolome analysis showed active secondary metabolic processes enriched specifically in the root of L. officinale . Using co-expression analysis, we also identified 20 and 48 unigenes representing different enzymes of lithospermic/chlorogenic acid and shikonin biosynthesis pathways, respectively. We further identified 15 candidate unigenes annotated as cytochrome P450 with the highest expression in the root of L. officinale as novel genes with a role in key biochemical reactions toward shikonin biosynthesis. Thus, through this study, we not only generated a high-quality genomic resource for L. officinale but also propose candidate genes to be involved in shikonin biosynthesis pathways for further functional characterization. Georg Thieme Verlag KG Stuttgart · New York.

Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates.

PubMed

Rosic, Nedeljka N

2012-04-01

Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310-362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B₁-lysine albumin biomarkers.

PubMed

Groopman, John D; Egner, Patricia A; Schulze, Kerry J; Wu, Lee S-F; Merrill, Rebecca; Mehra, Sucheta; Shamim, Abu A; Ali, Hasmot; Shaikh, Saijuddin; Gernand, Alison; Khatry, Subarna K; LeClerq, Steven C; West, Keith P; Christian, Parul

2014-12-01

Aflatoxin B1 is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B1-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B1-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B1-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B1-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B1-lysine/mg albumin at age two years. Aflatoxin B1-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illustrates exposure over the first 1000 days of life. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5

PubMed Central

Mihali, Troco K; Kellmann, Ralf; Neilan, Brett A

2009-01-01

Background Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. Results We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. Conclusion The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed Central

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B1-lysine albumin biomarkers

PubMed Central

Groopman, John D.; Egner, Patricia A.; Schulze, Kerry J.; Wu, Lee S-F; Merrill, Rebecca; Mehra, Sucheta; Shamim, Abu A.; Ali, Hasmot; Shaikh, Saijuddin; Gernand, Alison; Khatry, Subarna K.; LeClerq, Steven C.; West, Keith P.; Christian, Parul

2015-01-01

Aflatoxin B1 is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B1-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B1-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B1-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B1-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B1-lysine/mg albumin at age two years. Aflatoxin B1-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illus trates exposure over the first 1000 days of life. PMID:25308602

Identification and Characterization of a Novel Biotin Biosynthesis Gene in Saccharomyces cerevisiae

PubMed Central

Wu, Hong; Ito, Kiyoshi; Shimoi, Hitoshi

2005-01-01

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae. PMID:16269718

The Product of the fimI Gene Is Necessary for Escherichia coli Type 1 Pilus Biosynthesis

PubMed Central

Valenski, Mary L.; Harris, Sandra L.; Spears, Patricia A.; Horton, John R.; Orndorff, Paul E.

2003-01-01

Site-directed mutagenesis was employed to create lesions in fimI, a gene of uncertain function located in the chromosomal gene cluster (fim) involved in Escherichia coli type 1 pilus biosynthesis. Chromosomal fimI mutations produced a piliation-negative phenotype. Complementation analysis indicated that a fimI′-kan insertion mutation and a fimI frameshift mutation produced polarity-like effects not seen with an in-frame fimI deletion mutation. Minicell analysis associated fimI with a 16.4-kDa noncytoplasmic protein product (FimI). We conclude that FimI has a required role in normal pilus biosynthesis. PMID:12897022

Aflatoxins in Iran: Nature, Hazards and Carcinogenicity

PubMed Central

Khoshpey, B; Farhud, DD; Zaini, F

2011-01-01

Many studies have shown that mycotoxin contamination of agricultural products is a challenge for individual’s health especially in developing countries. Improper production and storage of foods, prepare conditions for aflatoxin production in crops, especially rice, wheat, pistachio, walnut, almond, etc which are the main sources of foods for people. Feeding livestock by contaminated bread is another way of human exposure to mycotoxins, especially aflatoxin and because of expensive methods for detecting and analyzing aflatoxin in laboratory; it is not measured in foods. This manuscript is a review of some Iranian and nonIranian reports about aflatoxin, its exposure ways, its adverse effect on human health and nutrition, as well as methods for reducing its exposure. Based on studies on foods, aflatoxin exposure is high in Iran. Since livestock feeding by contaminated bread is one of the potential ways for milk contamination, we should control and reduce aflatoxin contamination by improving production process, storage condition and livestock feeding as soon as possible. Pistachio is one of the most important exporting products of Iran and to maintain Iran’s position in exporting of this product, specific regulations on lowering its contamination with aflatoxin should be considered seriously. Finally, effective controlling of all food and feedstuffs which are vulnerable to aflatoxin contamination is necessary to prevent its effects. PMID:23113099

Population structure and aflatoxin production by Aspergillus Sect. Flavi from maize in Nigeria and Ghana.

PubMed

Perrone, Giancarlo; Haidukowski, Miriam; Stea, Gaetano; Epifani, Filomena; Bandyopadhyay, Ranajit; Leslie, John F; Logrieco, Antonio

2014-08-01

Aflatoxins are highly toxic carcinogens that contaminate crops worldwide. Previous studies conducted in Nigeria and Ghana found high concentrations of aflatoxins in pre- and post-harvest maize. However, little information is available on the population structure of Aspergillus Sect. Flavi in West Africa. We determined the incidence of Aspergillus Sect. Flavi and the level of aflatoxin contamination in 91 maize samples from farms and markets in Nigeria and Ghana. Aspergillus spp. were recovered from 61/91 maize samples and aflatoxins B1 and/or B2 occurred in 36/91 samples. Three samples from the farms also contained aflatoxin G1 and/or G2. Farm samples were more highly contaminated than were samples from the market, in terms of both the percentage of the samples contaminated and the level of mycotoxin contamination. One-hundred-and-thirty-five strains representative of the 1163 strains collected were identified by using a multilocus sequence analysis of portions of the genes encoding calmodulin, β-tubulin and actin, and evaluated for aflatoxin production. Of the 135 strains, there were 110 - Aspergillus flavus, 20 - Aspergillus tamarii, 2 - Aspergillus wentii, 2 - Aspergillus flavofurcatus, and 1 - Aspergillus parvisclerotigenus. Twenty-five of the A. flavus strains and the A. parvisclerotigenus strain were the only strains that produced aflatoxins. The higher contamination of the farm than the market samples suggests that the aflatoxin exposure of rural farmers is even higher than previously estimated based on reported contamination of market samples. The relative infrequency of the A. flavus SBG strains, producing small sclerotia and high levels of both aflatoxins (B and G), suggests that long-term chronic exposure to this mycotoxin are a much higher health risk in West Africa than is the acute toxicity due to very highly contaminated maize in east Africa. Copyright © 2014 Elsevier Ltd. All rights reserved.

Aflatoxin

MedlinePlus

... found in the following foods: Peanuts and peanut butter Tree nuts such as pecans Corn Wheat Oil ... foods that may contain aflatoxin. Peanuts and peanut butter are some of the most rigorously tested products ...

7 CFR 996.11 - Negative aflatoxin content.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Negative aflatoxin content. 996.11 Section 996.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING... aflatoxin content. Negative aflatoxin content means 15 parts per billion (ppb) or less for peanuts that have...

7 CFR 996.11 - Negative aflatoxin content.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Negative aflatoxin content. 996.11 Section 996.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING... aflatoxin content. Negative aflatoxin content means 15 parts per billion (ppb) or less for peanuts that have...

7 CFR 996.11 - Negative aflatoxin content.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Negative aflatoxin content. 996.11 Section 996.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing... aflatoxin content. Negative aflatoxin content means 15 parts per billion (ppb) or less for peanuts that have...

Aflatoxigenic Fungi and Aflatoxins in Portuguese Almonds

PubMed Central

Rodrigues, P.; Venâncio, A.; Lima, N.

2012-01-01

Aflatoxin contamination of nuts is an increasing concern to the consumer's health. Portugal is a big producer of almonds, but there is no scientific knowledge on the safety of those nuts, in terms of mycotoxins. The aim of this paper was to study the incidence of aflatoxigenic fungi and aflatoxin contamination of 21 samples of Portuguese almonds, and its evolution throughout the various stages of production. All fungi belonging to Aspergillus section Flavi were identified and tested for their aflatoxigenic ability. Almond samples were tested for aflatoxin contamination by HPLC-fluorescence. In total, 352 fungi belonging to Aspergillus section Flavi were isolated from Portuguese almonds: 127 were identified as A. flavus (of which 28% produced aflatoxins B), 196 as typical or atypical A. parasiticus (all producing aflatoxins B and G), and 29 as A. tamarii (all nonaflatoxigenic). Aflatoxins were detected in only one sample at 4.97 μg/kg. PMID:22666128

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness

PubMed Central

Lee, Wing-Sham; Malitsky, Sergey; Almekias-Siegl, Efrat; Levy, Matan; Ben-Zvi, Gil; Alkan, Noam; Uauy, Cristobal; Jetter, Reinhard

2016-01-01

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response. PMID:27225753

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness.

PubMed

Hen-Avivi, Shelly; Savin, Orna; Racovita, Radu C; Lee, Wing-Sham; Adamski, Nikolai M; Malitsky, Sergey; Almekias-Siegl, Efrat; Levy, Matan; Vautrin, Sonia; Bergès, Hélène; Friedlander, Gilgi; Kartvelishvily, Elena; Ben-Zvi, Gil; Alkan, Noam; Uauy, Cristobal; Kanyuka, Kostya; Jetter, Reinhard; Distelfeld, Assaf; Aharoni, Asaph

2016-06-01

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response. © 2016 American Society of Plant Biologists. All rights reserved.

Identification and reduction of urinary aflatoxin metabolites in dogs.

PubMed

Bingham, A K; Huebner, H J; Phillips, T D; Bauer, J E

2004-11-01

Hydrated sodium calcium aluminosilicate (HSCAS) is a phyllosilicate clay commonly used as an anticaking agent in animal feeds. HSCAS tightly and selectively adsorbs aflatoxin. In 1998, 55 dogs died in Texas after eating dog food containing aflatoxin (150-300 ppb). The corn in the diets was contaminated with aflatoxin. Six dogs were given a low-level, sub-clinical dose of aflatoxin B(1). On average, 71.5% of aflatoxin M(1) cleared within 6 h after dosing, increasing to 90.4% after 12 h. Aflatoxin M(1) was no longer detectable in urine after 48 h. Aflatoxin P(1) was not found in urine compared to large amounts of M(1) and trace amounts of Q(1). In a crossover study, six dogs randomly fed a commercial dog food (no-clay control) or coated with HSCAS (0.5% by weight) were subsequently administered a sub-clinical dose of aflatoxin B(1). Diets were switched and the process repeated. The HSCAS-coated diet significantly reduced urinary aflatoxin M(1) by 48.4%+/-16.6 SD versus the control diet. In conclusion, HSCAS protects dogs fed diets with even minimal aflatoxin contamination. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may provide the pet food industry further assurance of canine diet safety.

Inhibitory Effects of Respiration Inhibitors on Aflatoxin Production

PubMed Central

Sakuda, Shohei; Prabowo, Diyan Febri; Takagi, Keiko; Shiomi, Kazuro; Mori, Mihoko; Ōmura, Satoshi; Nagasawa, Hiromichi

2014-01-01

Aflatoxin production inhibitors, which do not inhibit the growth of aflatoxigenic fungi, may be used to control aflatoxin without incurring a rapid spread of resistant strains. A respiration inhibitor that inhibits aflatoxin production was identified during a screening process for natural, aflatoxin-production inhibitors. This prompted us to evaluate respiration inhibitors as potential aflatoxin control agents. The inhibitory activities of four natural inhibitors, seven synthetic miticides, and nine synthetic fungicides were evaluated on aflatoxin production in Aspergillus parasiticus. All of the natural inhibitors (rotenone, siccanin, aptenin A5, and antimycin A) inhibited fungal aflatoxin production with IC50 values around 10 µM. Among the synthetic miticides, pyridaben, fluacrypyrim, and tolfenpyrad exhibited strong inhibitory activities with IC50 values less than 0.2 µM, whereas cyflumetofen did not show significant inhibitory activity. Of the synthetic fungicides, boscalid, pyribencarb, azoxystrobin, pyraclostrobin, and kresoxim-methyl demonstrated strong inhibitory activities, with IC50 values less than 0.5 µM. Fungal growth was not significantly affected by any of the inhibitors tested at concentrations used. There was no correlation observed between the targets of respiration inhibitors (complexes I, II, and III) and their IC50 values for aflatoxin-production inhibitory activity. This study suggests that respiration inhibitors, including commonly used pesticides, are useful for aflatoxin control. PMID:24674936

Differential expression of jasmonate biosynthesis genes in cacao genotypes contrasting for resistance against Moniliophthora perniciosa.

PubMed

Litholdo, Celso G; Leal, Gildemberg A; Albuquerque, Paulo S B; Figueira, Antonio

2015-10-01

The resistance mechanism of cacao against M. perniciosa is likely to be mediated by JA/ET-signaling pathways due to the preferential TcAOS and TcSAM induction in a resistant genotype. The basidiomycete Moniliophthora perniciosa causes a serious disease in cacao (Theobroma cacao L.), and the use of resistant varieties is the only sustainable long-term solution. Cacao resistance against M. perniciosa is characterized by pathogen growth inhibition with reduced colonization and an attenuation of disease symptoms, suggesting a regulation by jasmonate (JA)/ethylene (ET) signaling pathways. The hypothesis that genes involved in JA biosynthesis would be active in the interaction of T. cacao and M. perniciosa was tested here. The cacao JA-related genes were evaluated for their relative quantitative expression in susceptible and resistant genotypes upon the exogenous application of ET, methyl-jasmonate (MJ), and salicylic acid (SA), or after M. perniciosa inoculation. MJ treatment triggered changes in the expression of genes involved in JA biosynthesis, indicating that the mechanism of positive regulation by exogenous MJ application occurs in cacao. However, a higher induction of these genes was observed in the susceptible genotype. Further, a contrast in JA-related transcriptional expression was detected between susceptible and resistant plants under M. perniciosa infection, with the induction of the allene oxide synthase gene (TcAOS), which encodes a key enzyme in the JA biosynthesis pathway in the resistant genotype. Altogether, this work provides additional evidences that the JA-dependent signaling pathway is modulating the defense response against M. perniciosa in a cacao-resistant genotype.

Evaluation of the expression genes associated with resistance to Aspergillus flavus colonization and aflatoxin production in different maize lines.

USDA-ARS?s Scientific Manuscript database

Aflatoxins are carcinogenic toxic compounds produced by Aspergillus flavus during infection of crops including maize (Zea mays L.). Contamination of maize with aflatoxin is exacerbated by late season drought stress. Previous studies have implicated numerous resistance-associated proteins (RAPs) that...

Testing an aflatoxin B1 gene signature in rat archival tissues.

PubMed

Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

2012-05-21

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced

Genome-wide comparison of genes involved in the biosynthesis, metabolism, and signaling of juvenile hormone between silkworm and other insects

PubMed Central

Cheng, Daojun; Meng, Meng; Peng, Jian; Qian, Wenliang; Kang, Lixia; Xia, Qingyou

2014-01-01

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects. PMID:25071411

Aflatoxin Control in Maize by Trametes versicolor

PubMed Central

Scarpari, Marzia; Bello, Cristiano; Pietricola, Chiara; Zaccaria, Marco; Bertocchi, Luigi; Angelucci, Alessandra; Ricciardi, Maria Rosaria; Scala, Valeria; Parroni, Alessia; Fabbri, Anna A.; Reverberi, Massimo; Zjalic, Slaven; Fanelli, Corrado

2014-01-01

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1. PMID:25525683

Aflatoxin control in maize by Trametes versicolor.

PubMed

Scarpari, Marzia; Bello, Cristiano; Pietricola, Chiara; Zaccaria, Marco; Bertocchi, Luigi; Angelucci, Alessandra; Ricciardi, Maria Rosaria; Scala, Valeria; Parroni, Alessia; Fabbri, Anna A; Reverberi, Massimo; Zjalic, Slaven; Fanelli, Corrado

2014-12-17

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.

Effects of Pistacia atlantica subsp. kurdica on Growth and Aflatoxin Production by Aspergillus parasiticus

PubMed Central

Khodavaisy, Sadegh; Rezaie, Sassan; Noorbakhsh, Fatemeh; Baghdadi, Elham; Sharifynia, Somayeh; Aala, Farzad

2016-01-01

Background Aflatoxins are highly toxic secondary metabolites mainly produced by Aspergillus parasiticus. This species can contaminate a wide range of agricultural commodities, including cereals, peanuts, and crops in the field. In recent years, research on medicinal herbs, such as Pistacia atlantica subsp. kurdica, have led to reduced microbial growth, and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds. Objectives In this study, we to examine P. atlantica subsp. kurdica as a natural compound used to inhibit the growth of A. parasiticus and to act as an anti-mycotoxin. Materials and Methods In vitro antifungal susceptibility testing of P. atlantica subsp. kurdica for A. parasiticus was performed according to CLSI document M38-A2. The rate of aflatoxin production was determined using the HPLC technique after exposure to different concentrations (62.5 - 125 mg/mL) of the gum. The changes in expression levels of the aflR gene were analyzed with a quantitative real-time PCR assay. Results The results showed that P. atlantica subsp. kurdica can inhibit A. parasiticus growth at a concentration of 125 mg/mL. HPLC results revealed a significant decrease in aflatoxin production with 125 mg/mL of P. atlantica subsp. kurdica, and AFL-B1 production was entirely inhibited. Based on quantitative real-time PCR results, the rate of aflR gene expression was significantly decreased after treatment with P. atlantica subsp. kurdica. Conclusions Pistacia atlantica subsp. kurdica has anti-toxic properties in addition to an inhibitory effect on A. parasiticus growth, and is able to decrease aflatoxin production effectively in a dose-dependent manner. Therefore, this herbal extract maybe considered a potential anti-mycotoxin agent in medicine or industrial agriculture. PMID:27800127

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

PubMed

Ziemons, Sandra; Koutsantas, Katerina; Becker, Kordula; Dahlmann, Tim; Kück, Ulrich

2017-02-16

Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

Effects of a Calcium Bentonite Clay in Diets Containing Aflatoxin when Measuring Liver Residues of Aflatoxin B₁ in Starter Broiler Chicks.

PubMed

Fowler, Justin; Li, Wei; Bailey, Christopher

2015-08-26

Research has shown success using clay-based binders to adsorb aflatoxin in animal feeds; however, no adsorbent has been approved for the prevention or treatment of aflatoxicosis. In this study, growth and relative organ weights were evaluated along with a residue analysis for aflatoxin B₁ in liver tissue collected from broiler chickens consuming dietary aflatoxin (0, 600, 1200, and 1800 µg/kg) both with and without 0.2% of a calcium bentonite clay additive (TX4). After one week, only the combined measure of a broiler productivity index was significantly affected by 1800 µg/kg aflatoxin. However, once birds had consumed treatment diets for two weeks, body weights and relative kidney weights were affected by the lowest concentration. Then, during the third week, body weights, feed conversion, and the productivity index were affected by the 600 µg/kg level. Results also showed that 0.2% TX4 was effective at reducing the accumulation of aflatoxin B₁ residues in the liver and improving livability in birds fed aflatoxin. The time required to clear all residues from the liver was less than one week. With evidence that the liver's ability to process aflatoxin becomes relatively efficient within three weeks, this would imply that an alternative strategy for handling aflatoxin contamination in feed could be to allow a short, punctuated exposure to a higher level, so long as that exposure is followed by at least a week of a withdrawal period on a clean diet free of aflatoxin.

Identification and Expression Profiles of Sex Pheromone Biosynthesis and Transport Related Genes in Spodoptera litura

PubMed Central

Zhang, Ya-Nan; Zhu, Xiu-Yun; Fang, Li-Ping; He, Peng; Wang, Zhi-Qiang; Chen, Geng; Sun, Liang; Ye, Zhan-Feng; Deng, Dao-Gui; Li, Jin-Bu

2015-01-01

Although the general pathway of sex pheromone synthesis in moth species has been established, the molecular mechanisms remain poorly understood. The common cutworm Spodoptera litura is an important agricultural pest worldwide and causes huge economic losses annually. The female sex pheromone of S. litura comprises Z9,E11-14:OAc, Z9,E12-14:OAc, Z9-14:OAc, and E11-14:OAc. By sequencing and analyzing the transcriptomic data of the sex pheromone glands, we identified 94 candidate genes related to pheromone biosynthesis (55 genes) or chemoreception (39 genes). Gene expression patterns and phylogenetic analysis revealed that two desaturase genes (SlitDes5 and SlitDes11) and one fatty acyl reductase gene (SlitFAR3) showed pheromone gland (PG) biased or specific expression, and clustered with genes known to be involved in pheromone synthesis in other moth species. Furthermore, 4 chemoreception related genes (SlitOBP6, SlitOBP11, SlitCSP3, and SlitCSP14) also showed higher expression in the PG, and could be additional candidate genes involved in sex pheromone transport. This study provides the first solid background information that should facilitate further elucidation of sex pheromone biosynthesis and transport, and indicates potential targets to disrupt sexual communication in S. litura for a novel pest management strategy. PMID:26445454

Altered Expression of Genes Implicated in Xylan Biosynthesis Affects Penetration Resistance against Powdery Mildew.

PubMed

Chowdhury, Jamil; Lück, Stefanie; Rajaraman, Jeyaraman; Douchkov, Dimitar; Shirley, Neil J; Schwerdt, Julian G; Schweizer, Patrick; Fincher, Geoffrey B; Burton, Rachel A; Little, Alan

2017-01-01

Heteroxylan has recently been identified as an important component of papillae, which are formed during powdery mildew infection of barley leaves. Deposition of heteroxylan near the sites of attempted fungal penetration in the epidermal cell wall is believed to enhance the physical resistance to the fungal penetration peg and hence to improve pre-invasion resistance. Several glycosyltransferase (GT) families are implicated in the assembly of heteroxylan in the plant cell wall, and are likely to work together in a multi-enzyme complex. Members of key GT families reported to be involved in heteroxylan biosynthesis are up-regulated in the epidermal layer of barley leaves during powdery mildew infection. Modulation of their expression leads to altered susceptibility levels, suggesting that these genes are important for penetration resistance. The highest level of resistance was achieved when a GT43 gene was co-expressed with a GT47 candidate gene, both of which have been predicted to be involved in xylan backbone biosynthesis. Altering the expression level of several candidate heteroxylan synthesis genes can significantly alter disease susceptibility. This is predicted to occur through changes in the amount and structure of heteroxylan in barley papillae.

A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

PubMed Central

Schumacher, Julia; Gautier, Angélique; Morgant, Guillaume; Studt, Lena; Ducrot, Paul-Henri; Le Pêcheur, Pascal; Azeddine, Saad; Fillinger, Sabine; Leroux, Pierre; Tudzynski, Bettina; Viaud, Muriel

2013-01-01

The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus. PMID:23308280

Present status of the aflatoxin situation in the Philippines.

PubMed

Arim, R H

1995-01-01

Aflatoxin research in the Philippines started at the FNRI in 1967 with a survey on the aflatoxin content of various foods. Local researchers from government institutions and academe also conducted studies on the aflatoxin contamination of agricultural crops and their products/by-products. The data indicated that corn and peanuts are the two commodities that contain toxic levels of aflatoxin. Past and current research in the country is documented. Problems and research needs for the surveillance and/or control of aflatoxin contamination are discussed.

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli.

PubMed

Iguchi, Atsushi; Shirai, Hiroki; Seto, Kazuko; Ooka, Tadasuke; Ogura, Yoshitoshi; Hayashi, Tetsuya; Osawa, Kayo; Osawa, Ro

2011-01-01

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.

Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa.

PubMed

Luo, Yushuang; Kou, Xiaoxiao; Ding, Xuezhi; Hu, Shengbiao; Tang, Ying; Li, Wenping; Huang, Fan; Yang, Qi; Chen, Hanna; Xia, Liqiu

2012-02-01

To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.

Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production.

PubMed

Boulnois, G J; Roberts, I S; Hodge, R; Hardy, K R; Jann, K B; Timmis, K N

1987-06-01

Transposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1+ isolate from which the genes were cloned.

Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis1[C][W

PubMed Central

Pouvreau, Benjamin; Baud, Sébastien; Vernoud, Vanessa; Morin, Valérie; Py, Cyrille; Gendrot, Ghislaine; Pichon, Jean-Philippe; Rouster, Jacques; Paul, Wyatt; Rogowsky, Peter M.

2011-01-01

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs. PMID:21474435

Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

PubMed

Yang, Fan; Korban, Schuyler S; Pusey, P Lawrence; Elofsson, Michael; Sundin, George W; Zhao, Youfu

2014-01-01

The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Transformation of adsorbed aflatoxin B1 on smectite at elevated temperatures

USDA-ARS?s Scientific Manuscript database

Aflatoxins cause liver damage and suppress immunity. Smectites can be used to reduce the bioavailability of aflatoxins through adsorption. To further reduce the toxicity of aflatoxins and to eliminate the treatments of aflatoxin-loaded smectites, degrading the adsorbed aflatoxin to nontoxic or less ...

Dip-strip method for monitoring environmental contamination of aflatoxin in food and feed: use of a portable aflatoxin detection kit.

PubMed

Sashidhar, R B

1993-10-01

Aflatoxin contamination of food and feed have gained global significance due to its deleterious effect on human and animal health and its importance in the international trade. The potential of aflatoxin as a carcinogen, mutagen, teratogen, and immunosuppressive agent is well documented. The problem of aflatoxin contamination of food and feed has led to the enactment of various legislation. However, meaningful strategies for implementation of this legislation is limited by nonavailability of simple, cost-effective method for screening and detection of aflatoxin under field conditions. Keeping in mind the analytical constraints in developing countries, a simple-to-operate, rapid, reliable, and cost-effective portable aflatoxin detection kit has been developed. The important components of the kit include a hand-held UV lamp (365 nm, 4 W output), a solvent blender (12,000 rpm) for toxin extraction, and adsorbent-coated dip-strips (polyester film) for detecting and quantifying aflatoxin. Analysis of variance indicates that there were no significant differences between various batches of dip-strips (p > 0.05). The minimum detection limit for aflatoxin B1 was 10 ppb per spot. The kit may find wide application as a research tool in public health laboratories, environmental monitoring agencies, and in the poultry industry.

Dip-strip method for monitoring environmental contamination of aflatoxin in food and feed: use of a portable aflatoxin detection kit.

PubMed Central

Sashidhar, R B

1993-01-01

Aflatoxin contamination of food and feed have gained global significance due to its deleterious effect on human and animal health and its importance in the international trade. The potential of aflatoxin as a carcinogen, mutagen, teratogen, and immunosuppressive agent is well documented. The problem of aflatoxin contamination of food and feed has led to the enactment of various legislation. However, meaningful strategies for implementation of this legislation is limited by nonavailability of simple, cost-effective method for screening and detection of aflatoxin under field conditions. Keeping in mind the analytical constraints in developing countries, a simple-to-operate, rapid, reliable, and cost-effective portable aflatoxin detection kit has been developed. The important components of the kit include a hand-held UV lamp (365 nm, 4 W output), a solvent blender (12,000 rpm) for toxin extraction, and adsorbent-coated dip-strips (polyester film) for detecting and quantifying aflatoxin. Analysis of variance indicates that there were no significant differences between various batches of dip-strips (p > 0.05). The minimum detection limit for aflatoxin B1 was 10 ppb per spot. The kit may find wide application as a research tool in public health laboratories, environmental monitoring agencies, and in the poultry industry. Images FIGURE 1. PMID:8143644

Aflatoxins, discolouration and insect damage in dried cowpea and pigeon pea in Malawi and the effectiveness of flotation/washing operation in eliminating the aflatoxins.

PubMed

Matumba, Limbikani; Singano, Lazarus; Pungulani, Lawrent; Mvula, Naomi; Matumba, Annie; Singano, Charles; Matita, Grey

2017-05-01

Aflatoxin contamination and biodeterioration were examined in 302 samples of dry cowpeas and pigeon peas that were randomly purchased from 9 districts of the Southern Region of Malawi during July and November 2015. Further, the impact of flotation/washing on aflatoxin levels on the pulses was elucidated. Aflatoxin analyses involved immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD) while legume biodeterioration assessments were done by visual inspection. Aflatoxins were frequently detected in cowpea (24%, max., 66 μg/kg) and pigeon pea (22%, max., 80 μg/kg) samples that were collected in the month of July. Lower aflatoxin incidence of 15% in cowpeas (max., 470 μg/kg) and 14% in pigeon peas (max., 377 μg/kg) was recorded in the November collection. Overall, aflatoxin levels were significantly higher in the pulses that were collected in November. However, there were no significant differences in the total aflatoxin (aflatoxin B 1 (AFB 1 ) + AFB 2 + AFG 1 + AFG 2 ) levels between the two types of pulses. Remarkably, in 76.2% of the aflatoxin positive cowpea and in 41.7% of the aflatoxin positive pigeon pea samples, aflatoxin G 1 concentration exceeded aflatoxin B 1. Insect damage percentage averaged at 18.1 ± 18.2% (mean ± SD) in the cowpeas and 16.1 ± 19.4% in pigeon peas. Mean discolouration percentage (number of pulses) of the cowpeas and pigeon peas was found to be at 6.7 ± 4.9 and 8.7 ± 6.2%, respectively. Washing and discarding the buoyant fraction was highly efficient in reducing aflatoxin levels; only 5.2 ± 11.1% of the initial aflatoxin level was found in the cleaned samples. In conclusion, cowpeas and pigeon peas sold on the local market in Malawi may constitute a hazard especially if floatation/washing step is skipped.

Mining of the Uncharacterized Cytochrome P450 Genes Involved in Alkaloid Biosynthesis in California Poppy Using a Draft Genome Sequence

PubMed Central

Hori, Kentaro; Yamada, Yasuyuki; Purwanto, Ratmoyo; Minakuchi, Yohei; Toyoda, Atsushi; Hirakawa, Hideki

2018-01-01

Abstract Land plants produce specialized low molecular weight metabolites to adapt to various environmental stressors, such as UV radiation, pathogen infection, wounding and animal feeding damage. Due to the large variety of stresses, plants produce various chemicals, particularly plant species-specific alkaloids, through specialized biosynthetic pathways. In this study, using a draft genome sequence and querying known biosynthetic cytochrome P450 (P450) enzyme-encoding genes, we characterized the P450 genes involved in benzylisoquinoline alkaloid (BIA) biosynthesis in California poppy (Eschscholzia californica), as P450s are key enzymes involved in the diversification of specialized metabolism. Our in silico studies showed that all identified enzyme-encoding genes involved in BIA biosynthesis were found in the draft genome sequence of approximately 489 Mb, which covered approximately 97% of the whole genome (502 Mb). Further analyses showed that some P450 families involved in BIA biosynthesis, i.e. the CYP80, CYP82 and CYP719 families, were more enriched in the genome of E. californica than in the genome of Arabidopsis thaliana, a plant that does not produce BIAs. CYP82 family genes were highly abundant, so we measured the expression of CYP82 genes with respect to alkaloid accumulation in different plant tissues and two cell lines whose BIA production differs to estimate the functions of the genes. Further characterization revealed two highly homologous P450s (CYP82P2 and CYP82P3) that exhibited 10-hydroxylase activities with different substrate specificities. Here, we discuss the evolution of the P450 genes and the potential for further genome mining of the genes encoding the enzymes involved in BIA biosynthesis. PMID:29301019

Evaluation of the expression of genes associated with resistance to Aspergillus flavus colonization and aflatoxin production in different maize lines

USDA-ARS?s Scientific Manuscript database

Aflatoxins are carcinogenic toxic compounds produced by Aspergillus flavus during infection of crops including maize (Zea mays L.). Contamination of maize with aflatoxin is exacerbated by late season drought stress. Previous studies have implicated numerous resistance-associated proteins (RAPs) that...

Detoxification of Aflatoxin B1 by Antifungal Compounds from Lactobacillus brevis and Lactobacillus paracasei, Isolated from Dairy Products.

PubMed

Gomaa, Eman Zakaria; Abdelall, Manal Farouk; El-Mahdy, Omima Mohammed

2018-06-01

Aflatoxins are a large group of highly toxic, mutagenic, and carcinogenic mycotoxins produced by specific species of fungi. Potential contamination of food commodities by these compounds causes extensive damage that lead to great economic losses. This study explored the potential use of antifungal compounds, produced by Lactobacillus brevis and Lactobacillus paracasei, for growth inhibition and subsequent aflatoxin B1 production from select strains of Aspergillus flavus and Aspergillus parasiticus. Lactobacilli strains were isolated from traditional Egyptian dairy products, whereas fungal strains were isolated from infected cereal seeds. There were noticeable decreases in mycelium biomass and aflatoxin production as well. L. brevis exhibited the highest reduction of aflatoxin B1 production by A. flavus and A. parasiticus, 96.31 and 90.43%, respectively. The concentrations of amino acids of the antifungal compound produced by L. brevis were significantly higher than that produced by L. paracasei. Asparagine, glutamine, glycine, alanine, and leucine were the most concentrated amino acids for both strains. The antifungal compounds produced by L. brevis and L. paracasei were active in a wide range of pH, heat stable and inactivated by proteolytic enzymes (protease K and trypsin A). The expression of Omt-A gene that involved in the later step of aflatoxin production was evaluated by real-time PCR. There was a vigorous reduction at transcriptional level of Omt-A gene observed in A. flavus that is treated by L. brevis and L. paracasei (80 and 70%, respectively). However, the reduction of Omt-A gene observed in A. parasiticus that is treated by L. brevis and L. paracasei was 64.5 and 52%, respectively. Treating maize seeds with antifungal compounds exhibited great efficiency in controlling fungal infection and increasing seed germination. The results confirmed that lactic acid bacteria are a promising strategy to control food contamination of fermented food and dairy

Occurrence of aflatoxins in oilseeds providing cocoa-butter substitutes.

PubMed Central

Kershaw, S J

1982-01-01

Four oilseeds providing cocoa-butter substitutes--shea, pentadecima, illipe, and salseed--when tested as substrates for aflatoxin production by two strains of Aspergillus parasiticus, gave varying levels of aflatoxin. Aflatoxins were found at low levels occurring naturally in moldy shea-nuts, but none of 21 commercial shea-nut samples contained greater than 20 micrograms of aflatoxin B1 per kg. PMID:6808919

Occurrence of aflatoxins in oilseeds providing cocoa-butter substitutes.

PubMed

Kershaw, S J

1982-05-01

Four oilseeds providing cocoa-butter substitutes--shea, pentadecima, illipe, and salseed--when tested as substrates for aflatoxin production by two strains of Aspergillus parasiticus, gave varying levels of aflatoxin. Aflatoxins were found at low levels occurring naturally in moldy shea-nuts, but none of 21 commercial shea-nut samples contained greater than 20 micrograms of aflatoxin B1 per kg.

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

PubMed

Han, Rongchun; Takahashi, Hiroki; Nakamura, Michimi; Bunsupa, Somnuk; Yoshimoto, Naoko; Yamamoto, Hirobumi; Suzuki, Hideyuki; Shibata, Daisuke; Yamazaki, Mami; Saito, Kazuki

2015-01-01

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

A Biotin Biosynthesis Gene Restricted to Helicobacter

PubMed Central

Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E.

2016-01-01

In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections. PMID:26868423

Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK228 in Chromobacterium violaceum No. 968.

PubMed

Cheng, Yi-Qiang; Yang, Min; Matter, Andrea M

2007-06-01

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

PubMed Central

2011-01-01

Abstract Background Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. Results One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues. Conclusions A collection of high

Assembly and expression analysis of oat vitamin E biosynthesis gene homeologs during seed development

USDA-ARS?s Scientific Manuscript database

Among the cereal grains, hexaploid oats (Avena sativa L.) are particularly rich in vitamin E, an essential liposoluble vitamin that maintains membrane stability and possesses antioxidant and anti-inflammatory properties. To date, no gene sequences involved in vitamin E biosynthesis have been reporte...

Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis

PubMed Central

Chiniquy, Dawn; Varanasi, Patanjali; Oh, Taeyun; Harholt, Jesper; Katnelson, Jacob; Singh, Seema; Auer, Manfred; Simmons, Blake; Adams, Paul D.; Scheller, Henrik V.; Ronald, Pamela C.

2013-01-01

Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength. PMID:23596448

Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors

PubMed Central

2014-01-01

Background Microalgae can accumulate considerable amounts of lipids under different nutrient-deficient conditions, making them as one of the most promising sustainable sources for biofuel production. These inducible processes provide a powerful experimental basis for fully understanding the mechanisms of physiological acclimation, lipid hyperaccumulation and gene expression in algae. In this study, three nutrient-deficiency strategies, viz nitrogen-, phosphorus- and iron-deficiency were applied to trigger the lipid hyperaccumulation in an oleaginous Chlorella pyrenoidosa. Regular patterns of growth characteristics, lipid accumulation, physiological parameters, as well as the expression patterns of lipid biosynthesis-related genes were fully analyzed and compared. Results Our results showed that all the nutrient stress conditions could enhance the lipid content considerably compared with the control. The total lipid and neutral lipid contents exhibit the most marked increment under nitrogen deficiency, achieving 50.32% and 34.29% of dry cell weight at the end of cultivation, respectively. Both photosynthesis indicators and reactive oxygen species parameters reveal that physiological stress turned up when exposed to nutrient depletions. Time-course transcript patterns of lipid biosynthesis-related genes showed that diverse expression dynamics probably contributes to the different lipidic phenotypes under stress conditions. By analyzing the correlation between lipid content and gene expression level, we pinpoint several genes viz. rbsL, me g6562, accA, accD, dgat g2354, dgat g3280 and dgat g7063, which encode corresponding enzymes or subunits of malic enzyme, ACCase and diacylglycerol acyltransferase in the de novo TAG biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification. Conclusion This study provided us not only a comprehensive picture of adaptive mechanisms from physiological perspective, but

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

NASA Astrophysics Data System (ADS)

Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

Genomic and Coexpression Analyses Predict Multiple Genes Involved in Triterpene Saponin Biosynthesis in Medicago truncatula[C][W

PubMed Central

Naoumkina, Marina A.; Modolo, Luzia V.; Huhman, David V.; Urbanczyk-Wochniak, Ewa; Tang, Yuhong; Sumner, Lloyd W.; Dixon, Richard A.

2010-01-01

Saponins, an important group of bioactive plant natural products, are glycosides of triterpenoid or steroidal aglycones (sapogenins). Saponins possess many biological activities, including conferring potential health benefits for humans. However, most of the steps specific for the biosynthesis of triterpene saponins remain uncharacterized at the molecular level. Here, we use comprehensive gene expression clustering analysis to identify candidate genes involved in the elaboration, hydroxylation, and glycosylation of the triterpene skeleton in the model legume Medicago truncatula. Four candidate uridine diphosphate glycosyltransferases were expressed in Escherichia coli, one of which (UGT73F3) showed specificity for multiple sapogenins and was confirmed to glucosylate hederagenin at the C28 position. Genetic loss-of-function studies in M. truncatula confirmed the in vivo function of UGT73F3 in saponin biosynthesis. This report provides a basis for future studies to define genetically the roles of multiple cytochromes P450 and glycosyltransferases in triterpene saponin biosynthesis in Medicago. PMID:20348429

Transcriptome, antioxidant enzyme activity and histopathology analysis of hepatopancreas from the white shrimp Litopenaeus vannamei fed with aflatoxin B1(AFB1).

PubMed

Zhao, Wei; Wang, Lei; Liu, Mei; Jiang, Keyong; Wang, Mengqiang; Yang, Guang; Qi, Cancan; Wang, Baojie

2017-09-01

Aflatoxin produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing and storage. Aflatoxins cause severe health problems reducing the yield and profitability of shrimp cultures. We sought to understand the interaction between shrimp immunity and aflatoxin B1 (AFB1), analyzing transcriptome expression, antioxidant enzyme activity, and histological features of the hepatopancreas of shrimp fed with AFB1. From over 4 million high-quality reads, de novo unigene assembly produced 103,644 fully annotated genes. A total of 1024 genes were differentially expressed in shrimp fed with AFB1, being involved in functions, such as peroxidase metabolism, signal transduction, transcriptional control, apoptosis, proteolysis, endocytosis, and cell adhesion and cell junction. Upon AFB1 challenge, there were severe histological alterations in shrimp hepatopancreas. AFB1 challenge increased the activity of several antioxidant enzymes. Our data contribute to improve the current understanding of host-AFB1 interaction, providing an abundant source for identification of novel genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Profiling and Quantifying Differential Gene Transcription Provide Insights into Ganoderic Acid Biosynthesis in Ganoderma lucidum in Response to Methyl Jasmonate

PubMed Central

Shi, Liang; Mu, Da-Shuai; Jiang, Ai-Liang; Han, Qin; Zhao, Ming-Wen

2013-01-01

Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum. PMID:23762280

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis.

PubMed

Wang, Shiqiang; Wang, Bin; Hua, Wenping; Niu, Junfeng; Dang, Kaikai; Qiang, Yi; Wang, Zhezhi

2017-09-12

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase ( sacA ), fructokinase ( scrK ), UDP-glucose 4-epimerase ( GALE ), Mannose-1-phosphate guanylyltransferase ( GMPP ), and UDP-glucose 6-dehydrogenase ( UGDH ), but negatively correlated with the expression of Hexokinase ( HK ). Through qRT-PCR validation and comprehensive analysis, we determined that sacA , HK , and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions.

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis

PubMed Central

Wang, Shiqiang; Wang, Bin; Hua, Wenping; Niu, Junfeng; Dang, Kaikai; Qiang, Yi; Wang, Zhezhi

2017-01-01

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase (sacA), fructokinase (scrK), UDP-glucose 4-epimerase (GALE), Mannose-1-phosphate guanylyltransferase (GMPP), and UDP-glucose 6-dehydrogenase (UGDH), but negatively correlated with the expression of Hexokinase (HK). Through qRT-PCR validation and comprehensive analysis, we determined that sacA, HK, and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions. PMID:28895881

Global transcriptome analysis of Huperzia serrata and identification of critical genes involved in the biosynthesis of huperzine A.

PubMed

Yang, Mengquan; You, Wenjing; Wu, Shiwen; Fan, Zhen; Xu, Baofu; Zhu, Mulan; Li, Xuan; Xiao, Youli

2017-03-22

Huperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed. In this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms.

PubMed

Nara, Ayako; Hashimoto, Takuya; Komatsu, Mamoru; Nishiyama, Makoto; Kuzuyama, Tomohisa; Ikeda, Haruo

2017-05-01

Bafilomycins A 1 , C 1 and B 1 (setamycin) produced by Kitasatospora setae KM-6054 belong to the plecomacrolide family, which exhibit antibacterial, antifungal, antineoplastic and immunosuppressive activities. An analysis of gene clusters from K. setae KM-6054 governing the biosynthesis of bafilomycins revealed that it contains five large open reading frames (ORFs) encoding the multifunctional polypeptides of bafilomycin polyketide synthases (PKSs). These clustered PKS genes, which are responsible for bafilomycin biosynthesis, together encode 11 homologous sets of enzyme activities, each catalyzing a specific round of polyketide chain elongation. The region contains an additional 13 ORFs spanning a distance of 73 287 bp, some of which encode polypeptides governing other key steps in bafilomycin biosynthesis. Five ORFs, BfmB, BfmC, BfmD, BfmE and BfmF, were involved in the formation of methoxymalonyl-acyl carrier protein (ACP). Two possible regulatory genes, bfmR and bfmH, were found downstream of the above genes. A gene-knockout analysis revealed that BfmR was only a transcriptional regulator for the transcription of bafilomycin biosynthetic genes. Two genes, bfmI and bfmJ, were found downstream of bfmH. An analysis of these gene-disruption mutants in addition to an enzymatic analysis of BfmI and BfmJ revealed that BfmJ activated fumarate and BfmI functioned as a catalyst to form a fumaryl ester at the C21 hydroxyl residue of bafilomycin A 1 . A comparative analysis of bafilomycin gene clusters in K. setae KM-6054, Streptomyces lohii JCM 14114 and Streptomyces griseus DSM 2608 revealed that each ORF of both gene clusters in two Streptomyces strains were quite similar to each other. However, each ORF of gene cluster in K. setae KM-6054 was of lower similarity to that of corresponding ORF in the two Streptomyces species.

Acute effects of aflatoxin on northern bobwhites (Colinus virginianus).

PubMed

Moore, Deana L; Henke, Scott E; Fedynich, Alan M; Laurenz, Jamie C; Morgan, Robert

2013-07-01

Aflatoxin is a widely occurring and harmful mycotoxin produced by strains of Aspergillus spp. growing on vegetable matter. We investigated the concentration of aflatoxin needed to impair normal physiologic responses and induce acute morbidity and mortality in Northern Bobwhites (Colinus virginianus). Ten wild-caught adult bobwhites (five males and five females) from southern Texas were randomly assigned to each treatment group (0, 100, 500, 1,000, and 2,000 parts per billion (ppb) aflatoxin; n=50). We orally administered 100 μL of aflatoxin, derived from Aspergillus flavus, once per week for 4 wk and monitored bird mass, daily feed consumption, liver histology, and blood chemistries. An in vitro white blood cell proliferation test was conducted using spleen tissue to determine the effect of aflatoxin on the immune system. There was no mortality in the control groups, whereas mortalities occurred in all treatment groups except in the 100 ppb aflatoxin treatment. Immunosuppression, reduction in gamma-globulin, glucose, and gamma-glutamyltransferase blood levels, and abnormal liver histology were observed in aflatoxin-exposed quail. Blood chemistry indicated cellular damage to the liver and kidneys. We concluded that short-term, acute doses of aflatoxin as low as 100 ppb can be detrimental to the health of Northern Bobwhites.

Aflatoxin Exposure During Pregnancy, Maternal Anemia, and Adverse Birth Outcomes

PubMed Central

Smith, Laura E.; Prendergast, Andrew J.; Turner, Paul C.; Humphrey, Jean H.; Stoltzfus, Rebecca J.

2017-01-01

Pregnant women and their developing fetuses are vulnerable to multiple environmental insults, including exposure to aflatoxin, a mycotoxin that may contaminate as much as 25% of the world food supply. We reviewed and integrated findings from studies of aflatoxin exposure during pregnancy and evaluated potential links to adverse pregnancy outcomes. We identified 27 studies (10 human cross-sectional studies and 17 animal studies) assessing the relationship between aflatoxin exposure and adverse birth outcomes or anemia. Findings suggest that aflatoxin exposure during pregnancy may impair fetal growth. Only one human study investigated aflatoxin exposure and prematurity, and no studies investigated its relationship with pregnancy loss, but animal studies suggest aflatoxin exposure may increase risk for prematurity and pregnancy loss. The fetus could be affected by maternal aflatoxin exposure through direct toxicity as well as indirect toxicity, via maternal systemic inflammation, impaired placental growth, or elevation of placental cytokines. The cytotoxic and systemic effects of aflatoxin could plausibly mediate maternal anemia, intrauterine growth restriction, fetal loss, and preterm birth. Given the widespread exposure to this toxin in developing countries, longitudinal studies in pregnant women are needed to provide stronger evidence for the role of aflatoxin in adverse pregnancy outcomes, and to explore biological mechanisms. Potential pathways for intervention to reduce aflatoxin exposure are urgently needed, and this might reduce the global burden of stillbirth, preterm birth, and low birthweight. PMID:28500823

Aflatoxin Exposure During Pregnancy, Maternal Anemia, and Adverse Birth Outcomes.

PubMed

Smith, Laura E; Prendergast, Andrew J; Turner, Paul C; Humphrey, Jean H; Stoltzfus, Rebecca J

2017-04-01

AbstractPregnant women and their developing fetuses are vulnerable to multiple environmental insults, including exposure to aflatoxin, a mycotoxin that may contaminate as much as 25% of the world food supply. We reviewed and integrated findings from studies of aflatoxin exposure during pregnancy and evaluated potential links to adverse pregnancy outcomes. We identified 27 studies (10 human cross-sectional studies and 17 animal studies) assessing the relationship between aflatoxin exposure and adverse birth outcomes or anemia. Findings suggest that aflatoxin exposure during pregnancy may impair fetal growth. Only one human study investigated aflatoxin exposure and prematurity, and no studies investigated its relationship with pregnancy loss, but animal studies suggest aflatoxin exposure may increase risk for prematurity and pregnancy loss. The fetus could be affected by maternal aflatoxin exposure through direct toxicity as well as indirect toxicity, via maternal systemic inflammation, impaired placental growth, or elevation of placental cytokines. The cytotoxic and systemic effects of aflatoxin could plausibly mediate maternal anemia, intrauterine growth restriction, fetal loss, and preterm birth. Given the widespread exposure to this toxin in developing countries, longitudinal studies in pregnant women are needed to provide stronger evidence for the role of aflatoxin in adverse pregnancy outcomes, and to explore biological mechanisms. Potential pathways for intervention to reduce aflatoxin exposure are urgently needed, and this might reduce the global burden of stillbirth, preterm birth, and low birthweight.

Cytotoxicity of aflatoxin on red blood corpuscles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verma, R.J.; Raval, P.J.

The exact mechanism of aflatoxin action is not clearly understood. In the present investigation the authors report morphological aberrations and increased rate of hemolysis caused by aflatoxins in vitro.

Identification of a Gene Cluster for Biosynthesis of Mannosylerythritol Lipids in the Basidiomycetous Fungus Ustilago maydis

PubMed Central

Hewald, Sandra; Linne, Uwe; Scherer, Mario; Marahiel, Mohamed A.; Kämper, Jörg; Bölker, Michael

2006-01-01

Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis. PMID:16885300

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery.

PubMed

Chang, Perng-Kuang; Scharfenstein, Leslie L; Mack, Brian; Yu, Jiujiang; Ehrlich, Kenneth C

2014-07-01

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early

[Correlation of gene expression related to amount of ginseng saponin in 15 tissues and 6 kinds of ginseng saponin biosynthesis].

PubMed

Wang, Kang-yu; Zhang, Mei-ping; Li, Chuang; Jiang, Shi-cui; Yin, Rui; Sun, Chun-yu; Wang, Yi

2015-08-01

Fifteen tissues of 4-year-old fruit repining stage Jilin ginseng were chosen as materials, six kinds of monomer saponins (ginsenosides Rg1, Re, Rb1, Rc, Rb2 and Rd) content in 15 tissues was measured by HPLC and vanillin-sulfuric acid method. The relative expression of FPS, SQS, SQE, OSC, β-AS and P450 genes in 15 tissues was analyzed by real-time PCR. The correlations between ginseng saponin content in 15 tissues of Jilin ginseng and biosynthetic pathway -related genes were obtained. The results showed that was a synergistic increase and decrease trend of positive linear correlation among six kinds of monomer saponin content, and there was a significantly (P < 0.01) positive correlation between monomer saponin content and total saponins content. Monomer saponin content and 6 kinds of enzyme gene correlation were different. Biosynthesis of ginseng total saponins and monomer saponin were regulated by six kinds of participation ginsenoside biosynthesis enzyme genes, the expression of these six kinds of genes in different tissues of ginseng showed collaborative increase and decrease trend, and regulated biosynthesis of ginseng ginsenoside by group coordinative manner.

Determination of aflatoxin risk components for in-shell Brazil nuts.

PubMed

Vargas, E A; dos Santos, E A; Whitaker, T B; Slate, A B

2011-09-01

A study was conducted on the risk from aflatoxins associated with the kernels and shells of Brazil nuts. Samples were collected from processing plants in Amazonia, Brazil. A total of 54 test samples (40 kg) were taken from 13 in-shell Brazil nut lots ready for market. Each in-shell sample was shelled and the kernels and shells were sorted in five fractions: good kernels, rotten kernels, good shells with kernel residue, good shells without kernel residue, and rotten shells, and analysed for aflatoxins. The kernel:shell ratio mass (w/w) was 50.2/49.8%. The Brazil nut shell was found to be contaminated with aflatoxin. Rotten nuts were found to be a high-risk fraction for aflatoxin in in-shell Brazil nut lots. Rotten nuts contributed only 4.2% of the sample mass (kg), but contributed 76.6% of the total aflatoxin mass (µg) in the in-shell test sample. The highest correlations were found between the aflatoxin concentration in in-shell Brazil nuts samples and the aflatoxin concentration in all defective fractions (R(2)=0.97). The aflatoxin mass of all defective fractions (R(2)=0.90) as well as that of the rotten nut (R(2)=0.88) were also strongly correlated with the aflatoxin concentration of the in-shell test samples. Process factors of 0.17, 0.16 and 0.24 were respectively calculated to estimate the aflatoxin concentration in the good kernels (edible) and good nuts by measuring the aflatoxin concentration in the in-shell test sample and in all kernels, respectively. © 2011 Taylor & Francis

Human aflatoxin exposure in Kenya, 2007: a cross-sectional study

PubMed Central

Yard, Ellen E.; Daniel, Johnni H.; Lewis, Lauren S.; Rybak, Michael E.; Paliakov, Ekaterina M.; Kim, Andrea A.; Montgomery, Joel M.; Bunnell, Rebecca; Abudo, Mamo Umuro; Akhwale, Willis; Breiman, Robert F.; Sharif, Shahnaaz K.

2013-01-01

Aflatoxins contaminate approximately 25% of agricultural products worldwide. They can cause liver failure and liver cancer. Kenya has experienced multiple aflatoxicosis outbreaks in recent years, often resulting in fatalities. However, the full extent of aflatoxin exposure in Kenya has been unknown. Our objective was to quantify aflatoxin exposure across Kenya. We analysed aflatoxin levels in serum specimens from the 2007 Kenya AIDS Indicator Survey – a nationally representative, cross-sectional serosurvey. KAIS collected 15,853 blood specimens. Of the 3180 human immunodeficiency virus-negative specimens with ≥1 mL sera, we randomly selected 600 specimens stratified by province and sex. We analysed serum specimens for aflatoxin albumin adducts by using isotope dilution MS/MS to quantify aflatoxin B1-lysine, and normalised with serum albumin. Aflatoxin concentrations were then compared by demographic, socioeconomic and geographic characteristics. We detected serum aflatoxin B1-lysine in 78% of serum specimens (range = Aflatoxin exposure did not vary by sex, age group, marital status, religion or socioeconomic characteristics. Aflatoxin exposure varied by province (p < 0.05); it was highest in Eastern (median = 7.87 pg/mg albumin) and Coast (median = 3.70 pg/mg albumin) provinces and lowest in Nyanza (median = aflatoxin exposure is a public health problem throughout Kenya, and it could be substantially impacting human health. Wide-scale, evidence-based interventions are urgently needed to decrease exposure and subsequent health effects. PMID:23767939

Food Safety Legislation Regarding Of Aflatoxins Contamination

NASA Astrophysics Data System (ADS)

Ketney, Otto

2015-09-01

The main objective of the European Union (EU) is to reduce certain contaminants in foodstuffs to acceptable levels. The occurrence of aflatoxin B1 in food was considered to be one of the most important issues of global food security to protect the health of humans and animals, over 100 nations have established maximum tolerable levels for aflatoxin in food. Although EU legislation covers many aspects of food safety was not legally establish an integrated framework that could effectively combat and cover all sectors of the food chain. Monitoring and reporting levels of aflatoxins after controls are essential actions that assist to identify potential risks to human health. The review process for aflatoxin regulations is a complex activity involving many factors and stakeholders.

Identification of the first diphenyl ether gene cluster for pestheic acid biosynthesis in plant endophyte Pestalotiopsis fici.

PubMed

Xu, Xinxin; Liu, Ling; Zhang, Fan; Wang, Wenzhao; Li, Jinyang; Guo, Liangdong; Che, Yongsheng; Liu, Gang

2014-01-24

The diphenyl ether pestheic acid was isolated from the endophytic fungus Pestalotiopsis fici, which is proposed to be the biosynthetic precursor of the unique chloropupukeananes. The pestheic acid biosynthetic gene (pta) cluster was identified in the fungus through genome scanning. Sequence analysis revealed that this gene cluster encodes a nonreducing polyketide synthase, a number of modification enzymes, and three regulators. Gene disruption and intermediate analysis demonstrated that the biosynthesis proceeded through formation of the polyketide backbone, cyclization of a polyketo acid to a benzophenone, chlorination, and formation of the diphenyl ether skeleton through oxidation and hydrolyzation. A dihydrogeodin oxidase gene, ptaE, was essential for diphenyl ether formation, and ptaM encoded a flavin-dependent halogenase catalyzing chlorination in the biosynthesis. Identification of the pta cluster laid the foundation to decipher the genetic and biochemical mechanisms involved in the pathway. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. Methods: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. Results: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. Conclusion: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells. PMID:24312879

Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

USDA-ARS?s Scientific Manuscript database

Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins.

PubMed

Siciliano, Ilenia; Spadaro, Davide; Prelle, Ambra; Vallauri, Dario; Cavallero, Maria Chiara; Garibaldi, Angelo; Gullino, Maria Lodovica

2016-04-26

Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N₂, 0.1% O₂ and 1% O₂, 21% O₂), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB₁ of over 70% was obtained. Aflatoxins B₁ and G₁ were more sensitive to plasma treatments compared to aflatoxins B₂ and G₂, respectively. Under plasma treatment, aflatoxin B₁ was more sensitive compared to aflatoxin G₁. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics.

Occurrence of aflatoxin B1 in natural products

PubMed Central

Prado, Guilherme; Altoé, Aline F.; Gomes, Tatiana C. B.; Leal, Alexandre S.; Morais, Vanessa A. D.; Oliveira, Marize S.; Ferreira, Marli B.; Gomes, Mateus B.; Paschoal, Fabiano N.; von S. Souza, Rafael; Silva, Daniela A.; Cruz Madeira, Jovita E. G.

2012-01-01

The media claims for the consumption of natural resource-based food have gradually increased in both developing and developed countries. The interest in the safety of these products is partially due to the possible presence of toxigenic fungi acting as mycotoxin producers, such as aflatoxins produced during the secondary metabolism of Aspergillus flavus, A. parasiticus and A. nomius. Aflatoxins, mainly aflatoxin B1, are directly associated with liver cancer in human beings. This paper is aimed at evaluating the presence of aflatoxin B1 in a few vegetable drugs, dried plant extracts and industrialized products traded in 2010 in the city of Belo Horizonte, State of Minas Gerais, Brazil. The method used for the quantification of aflatoxin B1 was based on extraction through acetone:water (85:15), immunoaffinity column purification followed by separation and detection in high efficiency liquid chromatography. Under the conditions of analysis, the Limits of Detection and Quantification were 0.6 µg kg-1 and 1.0 µg kg-1 respectively. The complete sets of analyses were carried out in duplicate. Aflatoxin B1 was noticed in a single sample (< 1.0 µg kg-1). The results revealed low aflatoxin B1 contamination in the products under analysis. However, it is required to establish a broad monitoring program in order to obtain additional data and check up on the actual extension of contamination. PMID:24031973

Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton.

PubMed

Lee, Jeongyeo; Jung, Jaeeun; Son, Seung-Hyun; Kim, Hyun-Bi; Noh, Young-Hee; Min, Sung Ran; Park, Kun-Hyang; Kim, Dae-Soo; Park, Sang Un; Lee, Haeng-Soon; Kim, Cha Young; Kim, Hyun-Soon; Lee, Hyeong-Kyu; Kim, HyeRan

2018-01-01

Sophorae Radix ( Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p -coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.

Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton

PubMed Central

Son, Seung-Hyun; Kim, Hyun-Bi; Noh, Young-Hee; Min, Sung Ran; Park, Kun-Hyang; Kim, Dae-Soo; Lee, Haeng-Soon; Kim, Cha Young; Lee, Hyeong-Kyu

2018-01-01

Sophorae Radix (Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally. PMID:29686587

7 CFR 996.11 - Negative aflatoxin content.

Code of Federal Regulations, 2011 CFR

2011-01-01

... STANDARDS FOR DOMESTIC AND IMPORTED PEANUTS MARKETED IN THE UNITED STATES Definitions § 996.11 Negative aflatoxin content. Negative aflatoxin content means 15 parts per billion (ppb) or less for peanuts that have...

7 CFR 996.11 - Negative aflatoxin content.

Code of Federal Regulations, 2010 CFR

2010-01-01

... STANDARDS FOR DOMESTIC AND IMPORTED PEANUTS MARKETED IN THE UNITED STATES Definitions § 996.11 Negative aflatoxin content. Negative aflatoxin content means 15 parts per billion (ppb) or less for peanuts that have...

Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

PubMed

Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

2016-05-02

Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids. Copyright © 2016 Elsevier B.V. All rights reserved.

Gibepyrone Biosynthesis in the Rice Pathogen Fusarium fujikuroi Is Facilitated by a Small Polyketide Synthase Gene Cluster*

PubMed Central

Janevska, Slavica; Arndt, Birgit; Niehaus, Eva-Maria; Burkhardt, Immo; Rösler, Sarah M.; Brock, Nelson L.; Humpf, Hans-Ulrich; Dickschat, Jeroen S.; Tudzynski, Bettina

2016-01-01

The 2H-pyran-2-one gibepyrone A and its oxidized derivatives gibepyrones B–F have been isolated from the rice pathogenic fungus Fusarium fujikuroi already more than 20 years ago. However, these products have not been linked to the respective biosynthetic genes, and therefore, their biosynthesis has not yet been analyzed on a molecular level. Feeding experiments with isotopically labeled precursors clearly supported a polyketide origin for the formal monoterpenoid gibepyrone A, whereas the terpenoid pathway could be excluded. Targeted gene deletion verified that the F. fujikuroi polyketide synthase PKS13, designated Gpy1, is responsible for gibepyrone A biosynthesis. Next to Gpy1, the ATP-binding cassette transporter Gpy2 is encoded by the gibepyrone gene cluster. Gpy2 was shown to have only a minor impact on the actual efflux of gibepyrone A out of the cell. Instead, we obtained evidence that Gpy2 is involved in gene regulation as it represses GPY1 gene expression. Thus, GPY1 was up-regulated and gibepyrone A production was enhanced both extra- and intracellularly in Δgpy2 mutants. Furthermore, expression of GPY genes is strictly repressed by members of the fungus-specific velvet complex, Vel1, Vel2, and Lae1, whereas Sge1, a major regulator of secondary metabolism in F. fujikuroi, affects gibepyrone biosynthesis in a positive manner. The gibepyrone A derivatives gibepyrones B and D were shown to be produced by cluster-independent P450 monooxygenases, probably to protect the fungus from the toxic product. In contrast, the formation of gibepyrones E and F from gibepyrone A is a spontaneous process and independent of enzymatic activity. PMID:27856636

454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides.

PubMed

Zagrobelny, Mika; Scheibye-Alsing, Karsten; Jensen, Niels Bjerg; Møller, Birger Lindberg; Gorodkin, Jan; Bak, Søren

2009-12-02

An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

PubMed Central

Arias, Renée S.; Dang, Phat M.; Sobolev, Victor S.

2015-01-01

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p≤0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem.

PubMed

Arias, Renée S; Dang, Phat M; Sobolev, Victor S

2015-12-21

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p ≤ 0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng · g(-1) of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng · g(-1). This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other

Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

PubMed

Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

2014-05-01

The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes.

Aflatoxin regulations and global pistachio trade: insights from social network analysis.

PubMed

Bui-Klimke, Travis R; Guclu, Hasan; Kensler, Thomas W; Yuan, Jian-Min; Wu, Felicia

2014-01-01

Aflatoxins, carcinogenic toxins produced by Aspergillus fungi, contaminate maize, peanuts, and tree nuts in many regions of the world. Pistachios are the main source of human dietary aflatoxins from tree nuts worldwide. Over 120 countries have regulations for maximum allowable aflatoxin levels in food commodities. We developed social network models to analyze the association between nations' aflatoxin regulations and global trade patterns of pistachios from 1996-2010. The main pistachio producing countries are Iran and the United States (US), which together contribute to nearly 75% of the total global pistachio market. Over this time period, during which many nations developed or changed their aflatoxin regulations in pistachios, global pistachio trade patterns changed; with the US increasingly exporting to countries with stricter aflatoxin standards. The US pistachio crop has had consistently lower levels of aflatoxin than the Iranian crop over this same time period. As similar trading patterns have also been documented in maize, public health may be affected if countries without aflatoxin regulations, or with more relaxed regulations, continually import crops with higher aflatoxin contamination. Unlike the previous studies on maize, this analysis includes a dynamic element, examining how trade patterns change over time with introduction or adjustment of aflatoxin regulations.

Aflatoxin Regulations and Global Pistachio Trade: Insights from Social Network Analysis

PubMed Central

Bui-Klimke, Travis R.; Guclu, Hasan; Kensler, Thomas W.; Yuan, Jian-Min; Wu, Felicia

2014-01-01

Aflatoxins, carcinogenic toxins produced by Aspergillus fungi, contaminate maize, peanuts, and tree nuts in many regions of the world. Pistachios are the main source of human dietary aflatoxins from tree nuts worldwide. Over 120 countries have regulations for maximum allowable aflatoxin levels in food commodities. We developed social network models to analyze the association between nations’ aflatoxin regulations and global trade patterns of pistachios from 1996–2010. The main pistachio producing countries are Iran and the United States (US), which together contribute to nearly 75% of the total global pistachio market. Over this time period, during which many nations developed or changed their aflatoxin regulations in pistachios, global pistachio trade patterns changed; with the US increasingly exporting to countries with stricter aflatoxin standards. The US pistachio crop has had consistently lower levels of aflatoxin than the Iranian crop over this same time period. As similar trading patterns have also been documented in maize, public health may be affected if countries without aflatoxin regulations, or with more relaxed regulations, continually import crops with higher aflatoxin contamination. Unlike the previous studies on maize, this analysis includes a dynamic element, examining how trade patterns change over time with introduction or adjustment of aflatoxin regulations. PMID:24670581

The Genome Sequence of the Cyanobacterium Oscillatoria sp. PCC 6506 Reveals Several Gene Clusters Responsible for the Biosynthesis of Toxins and Secondary Metabolites▿

PubMed Central

Méjean, Annick; Mazmouz, Rabia; Mann, Stéphane; Calteau, Alexandra; Médigue, Claudine; Ploux, Olivier

2010-01-01

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites. PMID:20675499

Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins

PubMed Central

Siciliano, Ilenia; Spadaro, Davide; Prelle, Ambra; Vallauri, Dario; Cavallero, Maria Chiara; Garibaldi, Angelo; Gullino, Maria Lodovica

2016-01-01

Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N2, 0.1% O2 and 1% O2, 21% O2), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB1 of over 70% was obtained. Aflatoxins B1 and G1 were more sensitive to plasma treatments compared to aflatoxins B2 and G2, respectively. Under plasma treatment, aflatoxin B1 was more sensitive compared to aflatoxin G1. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics. PMID:27128939

SNP in starch biosynthesis genes associated with nutritional and functional properties of rice

PubMed Central

Kharabian-Masouleh, Ardashir; Waters, Daniel L. E.; Reinke, Russell F.; Ward, Rachelle; Henry, Robert J.

2012-01-01

Starch is a major component of human diets. The relative contribution of variation in the genes of starch biosynthesis to the nutritional and functional properties of the rice was evaluated in a rice breeding population. Sequencing 18 genes involved in starch synthesis in a population of 233 rice breeding lines discovered 66 functional SNPs in exonic regions. Five genes, AGPS2b, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Association analysis found 31 of the SNP were associated with differences in pasting and cooking quality properties of the rice lines. Two genes appear to be the major loci controlling traits under human selection in rice, GBSSI (waxy gene) and SSIIa. GBSSI influenced amylose content and retrogradation. Other genes contributing to retrogradation were GPT1, SSI, BEI and SSIIIa. SSIIa explained much of the variation in cooking characteristics. Other genes had relatively small effects. PMID:22870386

Aflatoxin contamination of developing corn kernels.

PubMed

Amer, M A

2005-01-01

Preharvest of corn and its contamination with aflatoxin is a serious problem. Some environmental and cultural factors responsible for infection and subsequent aflatoxin production were investigated in this study. Stage of growth and location of kernels on corn ears were found to be one of the important factors in the process of kernel infection with A. flavus & A. parasiticus. The results showed positive correlation between the stage of growth and kernel infection. Treatment of corn with aflatoxin reduced germination, protein and total nitrogen contents. Total and reducing soluble sugar was increase in corn kernels as response to infection. Sucrose and protein content were reduced in case of both pathogens. Shoot system length, seeding fresh weigh and seedling dry weigh was also affected. Both pathogens induced reduction of starch content. Healthy corn seedlings treated with aflatoxin solution were badly affected. Their leaves became yellow then, turned brown with further incubation. Moreover, their total chlorophyll and protein contents showed pronounced decrease. On the other hand, total phenolic compounds were increased. Histopathological studies indicated that A. flavus & A. parasiticus could colonize corn silks and invade developing kernels. Germination of A. flavus spores was occurred and hyphae spread rapidly across the silk, producing extensive growth and lateral branching. Conidiophores and conidia had formed in and on the corn silk. Temperature and relative humidity greatly influenced the growth of A. flavus & A. parasiticus and aflatoxin production.

Coconut as a Medium for the Experimental Production of Aflatoxin

PubMed Central

Arseculeratne, S. N.; De Silva, L. M.; Wijesundera, S.; Bandunatha, C. H. S. R.

1969-01-01

Fresh, grated coconut has been found to be an excellent medium for aflatoxin production by Aspergillus flavus. Under optimal conditions, yields of 8 mg of total aflatoxin per g of substrate were obtained. Continuous agitation of the growth medium under moist conditions at 24 C produced highest yields. Aflatoxin was assayed both biologically and chromatographically. The aflatoxin content of cultures varied biphasically with the duration of incubation. It is suggested that this pattern could result from the sequential operation of factors promoting aflatoxin formation on the one hand and a detoxifying mechanism on the other. Images PMID:5803632

Isolation and Characterization of Canthaxanthin Biosynthesis Genes from the Photosynthetic Bacterium Bradyrhizobium sp. Strain ORS278

PubMed Central

Hannibal, Laure; Lorquin, Jean; D'Ortoli, Nicolas Angles; Garcia, Nelly; Chaintreuil, Clemence; Masson-Boivin, Catherine; Dreyfus, Bernard; Giraud, Eric

2000-01-01

A carotenoid biosynthesis gene cluster involved in canthaxanthin production was isolated from the photosynthetic Bradyrhizobium sp. strain ORS278. This cluster includes five genes identified as crtE, crtY, crtI, crtB, and crtW that are organized in at least two operons. The functional assignment of each open reading frame was confirmed by complementation studies. PMID:10851005

Aflatoxin B1 in common Egyptian foods.

PubMed

Selim, M I; Popendorf, W; Ibrahim, M S; el Sharkawy, S; el Kashory, E S

1996-01-01

Samples of common Egyptian foods (17 nuts and seeds, 10 spices, 31 herbs and medicinal plants, 12 dried vegetables, and 28 cereal grains) were collected from markets in Cairo and Giza. A portion of each sample was extracted with chloroform, and the concentrated extract was cleaned by passing through a silica gel column. Aflatoxin B1 was determined by reversed-phase liquid chromatography with UV detection. The highest prevalence of aflatoxin B1 was in nuts and seeds (82%), followed by spices (40%), herbs and medicinal plants (29%), dried vegetables (25%), and cereal grains (21%). The highest mean concentration of aflatoxin B1 was in herb and medicinal plants (49 ppb), followed by cereals (36 ppb), spices (25 ppb), nuts and seeds (24 ppb), and dried vegetables (20 ppb). Among nuts and seeds, the prevalence of aflatoxin B1 was highest (100%) in watermelon seeds, inshell peanuts, and unshelled peanuts. The lowest prevalence and concentrations were in hommos (garbanzo beans). The highest concentrations of aflatoxin B1 were detected in foods that had no potential for field contamination but required drying during processing and storage, such as pomegranate peel, watermelon seeds, and molokhia.

High pressure liquid chromatographic determination of aflatoxins in spices.

PubMed

Awe, M J; Schranz, J L

1981-11-01

High pressure liquid chromatography with fluorescence detection is used to determine aflatoxin in 5 common spices. A 10 micrometer microparticulate silica gel column is used with a dichloromethane-cyclohexane-acetonitrile solvent system to resolve aflatoxins B1, G1, B2, and G2. The fluorescence detector contained a silica gel-packed flowcell. Samples of black, white, and red pepper, ginger, and nutmeg were extracted according to a previously published method. Recoveries from aflatoxin-free samples of white pepper, ginger, and red pepper spiked with 1-50 micrograms aflatoxin/kg ranged from 64 to 92%.

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

PubMed Central

Cheng, Yi-Qiang; Tang, Gong-Li; Shen, Ben

2002-01-01

Leinamycin (LNM), produced by Streptomyces atroolivaceus, is a thiazole-containing hybrid peptide-polyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from S. atroolivaceus S-140 that harbors the lnm biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, lnmG, lnmH, and lnmI, and the deduced product of lnmI is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the lnmI gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for l-Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis. PMID:12446651

High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

PubMed

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

2013-01-01

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

PubMed Central

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

2013-01-01

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

PubMed Central

Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R.

2014-01-01

In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria. PMID

The threats to food safety and biocontrol of aflatoxins

USDA-ARS?s Scientific Manuscript database

Aflatoxins are a serious food safety concern for human and animal health. Great attention should be paid to the dietary exposure to these toxins in order to reduce the risk of aflatoxin contamination in the food chain. Although the research on aflatoxins was started more than 50 years ago, it is sti...

The Molecular Epidemiology of Chronic Aflatoxin Driven Impaired Child Growth

PubMed Central

Turner, Paul Craig

2013-01-01

Aflatoxins are toxic secondary fungal metabolites that contaminate dietary staples in tropical regions; chronic high levels of exposure are common for many of the poorest populations. Observations in animals indicate that growth and/or food utilization are adversely affected by aflatoxins. This review highlights the development of validated exposure biomarkers and their use here to assess the role of aflatoxins in early life growth retardation. Aflatoxin exposure occurs in utero and continues in early infancy as weaning foods are introduced. Using aflatoxin-albumin exposure biomarkers, five major studies clearly demonstrate strong dose response relationships between exposure in utero and/or early infancy and growth retardation, identified by reduced birth weight and/or low HAZ and WAZ scores. The epidemiological studies include cross-sectional and longitudinal surveys, though aflatoxin reduction intervention studies are now required to further support these data and guide sustainable options to reduce the burden of exposure. The use of aflatoxin exposure biomarkers was essential in understanding the observational data reviewed and will likely be a critical monitor of the effectiveness of interventions to restrict aflatoxin exposure. Given that an estimated 4.5 billion individuals live in regions at risk of dietary contamination the public health concern cannot be over stated. PMID:24455429

Aflatoxin B1 and M1: Biological Properties and Their Involvement in Cancer Development.

PubMed

Marchese, Silvia; Polo, Andrea; Ariano, Andrea; Velotto, Salvatore; Costantini, Susan; Severino, Lorella

2018-05-24

Aflatoxins are fungal metabolites found in feeds and foods. When the ruminants eat feedstuffs containing Aflatoxin B1 (AFB1), this toxin is metabolized and Aflatoxin M1 (AFM1) is excreted in milk. International Agency for Research on Cancer (IARC) classified AFB1 and AFM1 as human carcinogens belonging to Group 1 and Group 2B, respectively, with the formation of DNA adducts. In the last years, some epidemiological studies were conducted on cancer patients aimed to evaluate the effects of AFB1 and AFM1 exposure on cancer cells in order to verify the correlation between toxin exposure and cancer cell proliferation and invasion. In this review, we summarize the activation pathways of AFB1 and AFM1 and the data already reported in literature about their correlation with cancer development and progression. Moreover, considering that few data are still reported about what genes/proteins/miRNAs can be used as damage markers due to AFB1 and AFM1 exposure, we performed a bioinformatic analysis based on interaction network and miRNA predictions to identify a panel of genes/proteins/miRNAs that can be used as targets in further studies for evaluating the effects of the damages induced by AFB1 and AFM1 and their capacity to induce cancer initiation.

Horizontally Acquired Biosynthesis Genes Boost Coxiella burnetii's Physiology.

PubMed

Moses, Abraham S; Millar, Jess A; Bonazzi, Matteo; Beare, Paul A; Raghavan, Rahul

2017-01-01

Coxiella burnetii , the etiologic agent of acute Q fever and chronic endocarditis, has a unique biphasic life cycle, which includes a metabolically active intracellular form that occupies a large lysosome-derived acidic vacuole. C. burnetii is the only bacterium known to thrive within such an hostile intracellular niche, and this ability is fundamental to its pathogenicity; however, very little is known about genes that facilitate Coxiella 's intracellular growth. Recent studies indicate that C. burnetii evolved from a tick-associated ancestor and that the metabolic capabilities of C. burnetii are different from that of Coxiella -like bacteria found in ticks. Horizontally acquired genes that allow C. burnetii to infect and grow within mammalian cells likely facilitated the host shift; however, because of its obligate intracellular replication, C. burnetii would have lost most genes that have been rendered redundant due to the availability of metabolites within the host cell. Based on these observations, we reasoned that horizontally derived biosynthetic genes that have been retained in the reduced genome of C. burnetii are ideal candidates to begin to uncover its intracellular metabolic requirements. Our analyses identified a large number of putative foreign-origin genes in C. burnetii , including tRNA Glu 2 that is potentially required for heme biosynthesis, and genes involved in the production of lipopolysaccharide-a virulence factor, and of critical metabolites such as fatty acids and biotin. In comparison to wild-type C. burnetii , a strain that lacks tRNA Glu 2 exhibited reduced growth, indicating its importance to Coxiella 's physiology. Additionally, by using chemical agents that block heme and biotin biosyntheses, we show that these pathways are promising targets for the development of new anti- Coxiella therapies.

A mini review on aflatoxin exposure in Malaysia: past, present and future.

PubMed

Mohd-Redzwan, Sabran; Jamaluddin, Rosita; Abd-Mutalib, Mohd Sokhini; Ahmad, Zuraini

2013-11-13

This mini review article described the exposure of aflatoxin in Malaysia, including its presence in the foodstuffs and the detection of aflatoxin biomarkers in human biological samples. Historically, the exposure of aflatoxin in Malaysia can be dated in 1960s where an outbreak of disease in pig farms caused severe liver damage to the animals. Later, an aflatoxicosis case in Perak in 1988 was reported and caused death to 13 children, as up to 3 mg of aflatoxin was present in a single serving of contaminated noodles. Since then, extensive research on aflatoxin has been conducted in Malaysia. The food commodities such as peanuts, cereals, spices, and their products are the main commodities commonly found to be contaminated with aflatoxin. Surprisingly, some of the contaminated foods had levels greater than the permissible limit adopted by the Malaysian Food Regulation 1985. Besides, exposure assessment through the measurement of aflatoxin biomarkers in human biological samples is still in its infancy stage. Nevertheless, some studies had reported the presence of these biomarkers. In fact, it is postulated that Malaysians are moderately exposed to aflatoxin compared to those high risk populations, where aflatoxin contamination in the diets is prevalent. Since the ingestion of aflatoxin could be the integral to the development of liver cancer, the incidence of cancer attributable by dietary aflatoxin exposure in Malaysia has also been reported and published in the literatures. Regardless of these findings, the more important task is to monitor and control humans from being exposed to aflatoxin. The enforcement of law is insufficient to minimize human exposure to aflatoxin. Preventive strategies include agricultural, dietary, and clinical measures should be implemented. With the current research on aflatoxin in Malaysia, a global networking for research collaboration is needed to expand the knowledge and disseminate the information to the global scientific community.

A mini review on aflatoxin exposure in Malaysia: past, present and future

PubMed Central

Mohd-Redzwan, Sabran; Jamaluddin, Rosita; Abd.-Mutalib, Mohd Sokhini; Ahmad, Zuraini

2013-01-01

This mini review article described the exposure of aflatoxin in Malaysia, including its presence in the foodstuffs and the detection of aflatoxin biomarkers in human biological samples. Historically, the exposure of aflatoxin in Malaysia can be dated in 1960s where an outbreak of disease in pig farms caused severe liver damage to the animals. Later, an aflatoxicosis case in Perak in 1988 was reported and caused death to 13 children, as up to 3 mg of aflatoxin was present in a single serving of contaminated noodles. Since then, extensive research on aflatoxin has been conducted in Malaysia. The food commodities such as peanuts, cereals, spices, and their products are the main commodities commonly found to be contaminated with aflatoxin. Surprisingly, some of the contaminated foods had levels greater than the permissible limit adopted by the Malaysian Food Regulation 1985. Besides, exposure assessment through the measurement of aflatoxin biomarkers in human biological samples is still in its infancy stage. Nevertheless, some studies had reported the presence of these biomarkers. In fact, it is postulated that Malaysians are moderately exposed to aflatoxin compared to those high risk populations, where aflatoxin contamination in the diets is prevalent. Since the ingestion of aflatoxin could be the integral to the development of liver cancer, the incidence of cancer attributable by dietary aflatoxin exposure in Malaysia has also been reported and published in the literatures. Regardless of these findings, the more important task is to monitor and control humans from being exposed to aflatoxin. The enforcement of law is insufficient to minimize human exposure to aflatoxin. Preventive strategies include agricultural, dietary, and clinical measures should be implemented. With the current research on aflatoxin in Malaysia, a global networking for research collaboration is needed to expand the knowledge and disseminate the information to the global scientific community

[Appearance of aflatoxin M1 during the manufacture of Camembert cheese].

PubMed

Frémy, J M; Roiland, J C

1979-01-01

Several classic cheese making of camembert are made from raw milk spiked with Aflatoxin M1. Three Aflatoxin levels 7.5 microgram/l, 3 microgram/l are used. In respective curds 35.6, 47.1 and 57.7% of Aflatoxin M1 are recovered and 64.4, 52.9 and 42.3% in respective whey. During the first 15 days of storage the Aflatoxin M1 content of different cheeses decrease respectively 25, 55, 75%. A similar experience is made with a milk contamined in Aflatoxin M1 C14 labelled. Same results are recovered, except about behaviour of Aflatoxin M1 in cheese: a same C14 activity is recovered during storage for 30 days.

Aflatoxin biosynthesis control produced by Aspergillus flavus in layer hens feed during storage period of six months.

PubMed

Hassan, S M; Sultana, B; Iqbal, M

2017-06-01

Aflatoxins (AFTs) are a group of closely related toxins that are produced by different fungus species. Food and feed contamination with AFT is a worldwide health-related problem. As a result of fungal attack, the food and feed resulted in a principal socioeconomic loss and toxins produced in feed and food items harm the humans and animals in different ways. The anti-aflatoxigenic effect Psidium guajava, Ficus benghalensis, Gardenia radicans, Punica granatum and Ziziphus jujuba leaves were evaluated against aflatoxins (AFTs), produced by Aspergillus flavus in layer feed during storage. Among the investigated medicinal plant leaves, P. granatum showed highly promising anti-aflatoxigenic activity and completely inhibited the AFTs (B1 and B2) production over storage period without compromising the nutritive quality of feed (ash, protein, fat, fiber, Fe, Ca, P and K contents). Leaves of F. benghalensis and Z. jujuba were also effective however, higher concentration (15%) inhibited the AFTs production up to 99% and also maintained nutritive quality of feed. G. radicans was found least effective in controlling the AFTs production. Results revealed that all plant leaves were effective in controlling AFTs production in layer feed over the storage period of six months and these plants are potential candidate to replace the fungicides used to protect feed and other agricultural commodities from AFTs production during storage. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis.

PubMed

Shockey, Jay; Regmi, Anushobha; Cotton, Kimberly; Adhikari, Neil; Browse, John; Bates, Philip D

2016-01-01

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Purification of aflatoxin B1 antibody for the development of aflatoxin biosensor

NASA Astrophysics Data System (ADS)

Prihantoro, E. A. B.; Saepudin, E.; Ivandini, T. A.

2017-07-01

Aflatoxin B1 (AFB1) is produced from agricultural products especially peanuts overgrown with aspergillus flavus during the post-harvest process. Aflatoxin is classified as a highly toxic and carcinogenic substance to humans by the International Agency for Research on Cancer (IARC), WHO. This research was conducted to develop the AFB1 biosensor using antibody that specifically binds to aflatoxin B1. This antibody was produced by injecting an AFB1 hapten-protein (immunogen) to a rabbit. Antibody was obtained from rabbit's blood serum and purified using Protein A affinity chromatography and precipitation at the isoelectric point. The result showed that purification using protein A contains antibody of 4.0 mg/mL, whereas purification using precipitation at isoelectric pH contains antibody of 0.3 mg/mL. The pure antibody was tested for its specificity against AFB1, tetrahydrofuran (THF), dimethyl formamide (DMF), bovine serum albumin (BSA), and ethanol. The result revealed that THF, BSA, and ethanol were bound to antibody, while DMF showed no interaction. It was concluded that the polyclonal antibody which have been successfully purified from rabbit's blood serum using protein A affinity chromatography and precipitation methods showed an unspecific identification.

Monoclonal IgA Antibodies for Aflatoxin Immunoassays

PubMed Central

Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

2016-01-01

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

Insights into the Biosynthesis of the Benzoquinone Ansamycins Geldanamycin and Herbimycin, Obtained by Gene Sequencing and Disruption†

PubMed Central

Rascher, Andreas; Hu, Zhihao; Buchanan, Greg O.; Reid, Ralph; Hutchinson, C. Richard

2005-01-01

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized. PMID:16085885

Single Aflatoxin Contaminated Corn Kernel Analysis with Fluorescence Hyperspectral Image

USDA-ARS?s Scientific Manuscript database

Aflatoxins are toxic secondary metabolites of the fungi Aspergillus flavus and Aspergillus parasiticus, among others. Aflatoxin contaminated corn is toxic to domestic animals when ingested in feed and is a known carcinogen associated with liver and lung cancer in humans. Consequently, aflatoxin leve...

Isolation and purification of a new kalimantacin/batumin-related polyketide antibiotic and elucidation of its biosynthesis gene cluster.

PubMed

Mattheus, Wesley; Gao, Ling-Jie; Herdewijn, Piet; Landuyt, Bart; Verhaegen, Jan; Masschelein, Joleen; Volckaert, Guido; Lavigne, Rob

2010-02-26

Kal/bat, a polyketide, isolated to high purity (>95%) is characterized by strong and selective antibacterial activity against Staphylococcus species (minimum inhibitory concentration, 0.05 microg/mL), and no resistance was observed in strains already resistant to commonly used antibiotics. The kal/bat biosynthesis gene cluster was determined to a 62 kb genomic region of Pseudomonas fluorescens BCCM_ID9359. The kal/bat gene cluster consists of 16 open reading frames (ORF), encoding a hybrid PKS-NRPS system, extended with trans-acting tailoring functions. A full model for kal/bat biosynthesis is postulated and experimentally tested by gene inactivation, structural confirmation (using NMR spectroscopy), and complementation. The structural and microbiological study of biosynthetic kal/bat analogs revealed the importance of the carbamoyl group and 17-keto group for antibacterial activity. The mechanism of self-resistance lies within the production of an inactive intermediate, which is activated in a one-step enzymatic oxidation upon export. The genetic basis and biochemical elucidation of the biosynthesis pathway of this antibiotic will facilitate rational engineering for the design of novel structures with improved activities. This makes it a promising new therapeutic option to cope with multidrug-resistant clinical infections. Copyright 2010 Elsevier Ltd. All rights reserved.

Epidermal expression of a sterol biosynthesis gene regulates root growth by a non-cell-autonomous mechanism in Arabidopsis.

PubMed

Short, Eleri; Leighton, Margaret; Imriz, Gul; Liu, Dongbin; Cope-Selby, Naomi; Hetherington, Flora; Smertenko, Andrei; Hussey, Patrick J; Topping, Jennifer F; Lindsey, Keith

2018-05-15

The epidermis is hypothesized to play a signalling role during plant development. One class of mutants showing defects in signal transduction and radial patterning are those in sterol biosynthesis. The expectation is that living cells require sterols, but it is not clear that all cell types express sterol biosynthesis genes. The HYDRA1 ( HYD1 ) gene of Arabidopsis encodes sterol Δ8-Δ7 isomerase, and although hyd1 seedlings are defective in radial patterning across several tissues, we show that the HYD1 gene is expressed most strongly in the root epidermis. Transgenic activation of HYD1 transcription in the epidermis of hyd1 null mutants reveals a major role in root patterning and growth. HYD1 expression in the vascular tissues and root meristem, though not endodermis or pericycle, also leads to some phenotypic rescue. Phenotypic rescue is associated with rescued patterning of the PIN1 and PIN2 auxin efflux carriers. The importance of the epidermis in controlling root growth and development is proposed to be, in part, due to its role as a site for sterol biosynthesis, and auxin is a candidate for the non-cell-autonomous signal. © 2018. Published by The Company of Biologists Ltd.

Identification of a trichothecene gene cluster and description of the harzianum A biosynthesis pathway in the fungus Trichoderma arundinaceum

USDA-ARS?s Scientific Manuscript database

Trichothecenes are sesquiterpenes that act like mycotoxins. Their biosynthesis has been mainly studied in the fungal genera Fusarium, where most of the biosynthetic genes (tri) are grouped in a cluster regulated by ambient conditions and regulatory genes. Unexpectedly, few studies are available abou...

Quantitative Scrutinization of Aflatoxins in Different Spices from Pakistan

PubMed Central

Kashif, Aiza; Kanwal, Kinza; Khan, Abdul Muqeet; Abbas, Mateen

2016-01-01

The current research work aimed to access the contamination level of aflatoxins B1, B2, G1, and G2 in the household spices that are widely consumed in huge amounts. 200 different spice samples, 100 packed and 100 unpacked, were analyzed for the aflatoxins profile by HPLC with an incidence of 61.5% contamination out of which 53.66% samples exceed the EU limit. The results disclosed that the unpacked samples are more contaminated as compared to the packed samples except for white cumin seeds. Among packed and unpacked samples of spices, the maximum value of aflatoxins was detected in fennel, that is, 27.93 μg/kg and 67.04 μg/kg, respectively. The lowest concentration of aflatoxin was detected in cinnamon in packed form (0.79 μg/kg) and in the unpacked samples of white cumin seeds which is 1.75 μg/kg. Caraway seeds and coriander in its unpacked form showed positive results whereas black pepper (packed and unpacked) was found free from aflatoxins. This is the first report on the occurrence of aflatoxins in packed and unpacked samples of spices from Pakistan. To ensure safe consumption of spices, there should be constant monitoring of aflatoxin and more studies need to be executed with the intention of preventing mycotoxin accretion in this commodity. PMID:27781067

Reduction of aflatoxins by Korean soybean paste and its effect on cytotoxicity and reproductive toxicity--Part 3. Inhibitory effects of Korean soybean paste (doen-jang) on aflatoxin toxicity in laying hens and aflatoxin accumulation in their eggs.

PubMed

Kim, Jong-Gyu; Lee, Yong-Wook; Kim, Pan-Gyi; Roh, Woo-Sup; Shintani, Hideharu

2003-05-01

This study was conducted to determine the effects of Korean soybean paste (doen-jang [dwen-jahng]) (at concentrations of 0.5, 1, and 5%) on the toxicity of 500 ppb of aflatoxin in the diets of 60 laying hens (Isa Brown) divided into five groups and treated from week 15 to week 67. The aflatoxin-treated hens exhibited many deleterious effects, including reduced body weight; increased relative organ weights; decreased egg production; aflatoxin accumulation in eggs; decreased serum calcium, phosphorus, and alanino amonotransferase (ALT) levels; increased serum gammaglutamil transferase and lactic dehydrogenase levels; and, most significantly, severely altered cell foci and sinusoid dilatation in the liver, relative to control hens. The feeding of 1% soybean paste to hens reduced the adverse effects of aflatoxin on body weight, relative organ weights, egg production, and aflatoxin accumulation in eggs and improved serum calcium and ALT levels and the histopathological lesions of the liver. The feeding of 5% soybean paste to hens resulted in higher levels of the same types of improvements, especially with regard to the histopathological findings for the liver. On the basis of these results, it was suggested that a diet including 5% (and in some cases only 1%) Korean soybean paste protected laying hens and their eggs from the major deleterious effects of 500 microg of aflatoxin per kg of diet and from aflatoxin accumulation. These results indicate that dietary supplementation with Korean soybean paste reduces aflatoxin toxicity in laying hens that ultimately produce human foods such as eggs and poultry.

Molecular Characterization of Atoxigenic Strains for Biological Control of Aflatoxins in Nigeria

USDA-ARS?s Scientific Manuscript database

Aflatoxins are highly toxic, carcinogens produced by several species in Aspergillus section Flavi. Strains of A. flavus that do not produce aflatoxins, called atoxigenic strains, have been used commercially in North America as tools for limiting aflatoxin contamination. A similar aflatoxin manage...

Behavior of 14C aflatoxin M1 during camembert cheese making.

PubMed

Fremy, J M; Roiland, J C; Gaymard, A

1990-01-01

Camembert cheeses are made from raw milk spiked with aflatoxin M1. Three aflatoxin M1 levels (7.5 micrograms/L, 3 micrograms/L, and 0.3 micrograms/L) are used. In curds 35.6, 47.1, and 57.7% of aflatoxin M1, respectively, are recovered, and in wheys 64.4, 52.9, and 42.3%, respectively, are recovered. During the first 15 days of storage, the aflatoxin M1 content of different cheeses decreases 25, 55, and 75%, respectively. A similar experiment is made with milk contaminated with 14C labeled aflatoxin M1. The same results are obtained, except for the behavior of aflatoxin M1 in cheese; the same 14C activity is recovered during storage for 30 days.

A functional gene cluster for toxoflavin biosynthesis in the genome of the soil bacterium Pseudomonas protegens Pf-5

USDA-ARS?s Scientific Manuscript database

Toxoflavin is a broad-spectrum toxin best known for its role in virulence of Burkholderia glumae, which causes panicle blight of rice. A gene cluster containing homologs of toxoflavin biosynthesis genes (toxA-E) of B. glumae is present in the genome of Pseudomonas protegens Pf-5, a biological contr...

Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water

PubMed Central

Jardon-Xicotencatl, Samantha; Díaz-Torres, Roberto; Marroquín-Cardona, Alicia; Villarreal-Barajas, Tania; Méndez-Albores, Abraham

2015-01-01

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize. PMID:26512692

Multiclonal plastic antibodies for selective aflatoxin extraction from food samples.

PubMed

Bayram, Engin; Yılmaz, Erkut; Uzun, Lokman; Say, Rıdvan; Denizli, Adil

2017-04-15

Herein, we focused on developing a new generation of monolithic columns for extracting aflatoxin from real food samples by combining the superior features of molecularly imprinted polymers and cryogels. To accomplish this, we designed multiclonal plastic antibodies through simultaneous imprinting of aflatoxin subtypes B1, B2, G1, and G2. We applied Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and spectrofluorimetry to characterize the materials, and conducted selectivity studies using ochratoxin A and aflatoxin M1 (a metabolite of aflatoxin B1), as well as other aflatoxins, under competitive conditions. We determined optimal aflatoxin extraction conditions in terms of concentration, flow rate, temperature, and embedded particle amount as up to 25ng/mL for each species, 0.43mL/min, 7.0, 30°C, and 200mg, respectively. These multiclonal plastic antibodies showed imprinting efficiencies against ochratoxin A and aflatoxin M1 of 1.84 and 26.39, respectively, even under competitive conditions. Finally, we tested reusability, repeatability, reproducibility, and robustness of columns throughout inter- and intra-column variation studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of bacterial biocontrol agents on aflatoxin biosynthetic genes, aflD and aflR expression, and phenotypic aflatoxin B₁ production by Aspergillus flavus under different environmental and nutritional regimes.

PubMed

Al-Saad, Labeed A; Al-Badran, Adnan I; Al-Jumayli, Sami A; Magan, Naresh; Rodríguez, Alicia

2016-01-18

The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B1 (AFB1) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (aw) on nutrient medium; 0.995, 0.98 aw on maize meal agar medium) at 35°C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway (aflD and aflR respectively) and the production of AFB1. These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 aw on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 aw and some at 0.995 aw. However, the results for AFB1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB1 production on both nutrient and maize-based media regardless of aw. However, some bacterial treatments did inhibit AFB1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB1. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of Oil Biosynthesis in Algae

DTIC Science & Technology

2008-06-25

for future engineering purposes 3. Biochemical analysis of diacylglycerol acyltransferases ( DGATs ). These are key enzymes of oil biosynthesis...catalyzing the assembly of triacylglycerol in many organisms. 5 Genes predicted to encode DGATs and their role in triacylglycerol biosynthesis were identified

Survey of aflatoxins in maize tortillas from Mexico City.

PubMed

Castillo-Urueta, Pável; Carvajal, Magda; Méndez, Ignacio; Meza, Florencia; Gálvez, Amanda

2011-01-01

In Mexico, maize tortillas are consumed on a daily basis, leading to possible aflatoxin exposure. In a survey of 396 2-kg samples, taken over four sampling days in 2006 and 2007 from tortilla shops and supermarkets in Mexico City, aflatoxin levels were quantified by HPLC. In Mexico, the regulatory limit is 12 µg kg⁻¹ total aflatoxins for maize tortillas. In this survey, 17% of tortillas contained aflatoxins at levels of 3-385 µg kg⁻¹ or values below the limit of quantification (12 µg kg⁻¹ and 87% were below the regulatory limit. Average aflatoxin concentrations in 56 contaminated samples were: AFB1 (12.1 µg kg⁻¹); AFB2 (2.7 µg kg⁻¹); AFG1 (64.1 µg kg⁻¹) and AFG2 (3.7 µg kg⁻¹), and total AF (20.3 µg kg⁻¹).

Occurrence of aflatoxins in human foodstuffs in South Africa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loetter, L.H.; Kroehm, H.J.

1988-02-01

Aflatoxins are toxic metabolites of Aspergillus spp and have been reported as contaminants in a number of foodstuffs, namely corn, rice, peanuts, and cereals. In the Republic of South Africa, aflatoxin levels in human foodstuffs are limited to a maximum of 10 ..mu..g/kg for the total and 5 ..mu..g/kg for aflatoxin B/sub 1/. During 1985 and 1986, samples of sorghum beer, sorghum cereal, peanuts, peanut butter and maize meal were purchased from supermarkets in Johannesburg and analyzed for aflatoxins. A total of 414 samples were analyzed during the survey. In 1985, roughly a third of the samples were contaminated withmore » aflatoxins, with no levels in excess of the legal limit. In 1986 the percentage of contaminated samples rose significantly, but the levels of contamination remained low, with only one sample exceeding the legal maximum.« less

Aflatoxins in spices marketed in Portugal.

PubMed

Martins, M L; Martins, H M; Bernardo, F

2001-04-01

Seventy-nine prepackaged samples of 12 different types of spice powders (five cardamom, five cayenne pepper, eight chilli, five cloves, seven cumin, five curry) powder, five ginger, five mustard, 10 nutmeg, 12 paprika, five saffron and seven white pepper) were selected from supermarkets and ethnic shops in Lisbon (Portugal) for estimation of aflatoxins by immunoaffinity column clean-up followed by HPLC. Aflatoxin B1 (AFB1) was detected in 34 samples of prepackaged spices (43.0%). All of the cayenne pepper samples were contaminated with levels ranging from 2 to 32 microg AFB1/kg. Three nutmeg samples contained levels ranging from 1 to 5 microg/kg, three samples had levels ranging from 6 to 20 microg/kg, and there were two with 54 microg/kg and 58 microg/ kg. Paprika contained levels of aflatoxin B1 ranging from 1 to 20 microg/kg. Chilli, cumin, curry powder, saffron and white pepper samples had levels ranging from 1 to 5 microg/kg. Aflotoxins were not detected in cardamon, cloves, ginger and mustard. None of the samples analysed contained aflatoxins B2, G1 and G2.

Survey of aflatoxin concentrations in wild bird seed purchased in Texas.

PubMed

Henke, S E; Gallardo, V C; Martinez, B; Balley, R

2001-10-01

The use of backyard feeders to attract avian wildlife is a common practice throughout the United States. However, feeding wildlife may create a problem due to aflatoxin, a harmful fungal metabolite, which can affect wildlife that are fed contaminated grain. Our study was initiated to determine if songbirds were being exposed to aflatoxin-contaminated feed throughout Texas. Bags of wild bird seed (n = 142) were purchased from grain cooperatives, grocery stores, and pet shops located in the panhandle, central, south, east, and west regions of Texas during spring and summer 1999. Aflatoxin concentrations in bird seed ranged from non-detectable to 2,780 micrograms/kg. Overall, 17% of samples had aflatoxin concentrations greater than 100 micrograms/kg, of which 83% contained corn as an ingredient. Retail establishment effects were noted in the southern and western regions of Texas, with average concentrations of aflatoxin greater from bags of bird seed purchased from grain cooperatives, followed by pet shops, then grocery stores. Regional differences in aflatoxin levels were not apparent from bags of seed purchased at pet shops: however, regional differences were noted in aflatoxin levels from seeds obtained at grocery stores and grain cooperatives. Average aflatoxin concentration from seed purchased at grocery stores was greatest in the panhandle region, followed by the remaining regions. Within grain cooperatives, the panhandle, south, and west regions of Texas exhibited higher levels of aflatoxin-contaminated bird seed than cooperatives within the east and central regions of Texas. Granivorous songbirds in Texas are exposed to aflatoxins at backyard feeders, which may be a significant morbidity and mortality factor.

Aflatoxin in detannin coffee and tea and its destruction.

PubMed

Hasan, H A H

2002-05-01

The aflatoxins produced byAspergillus parasiticus var. globosus IMI 12090 in detannin-caffeinated coffee and black tea were five times more concentrated than in regular coffee and tea. The activity of caffeine and tannin on the fungus growth and aflatoxin production in liquid broth was tested at three levels: viz. 0.1, 0.3, and 0.6%. Tannin and caffeine induced 95% inhibition in aflatoxins at 0.3% and 0.6%, respectively. The antiaflatoxigenic properties of regular coffee and tea appear to be due to tannin, followed by caffeine. The roasting of contaminated coffee beans at 200 degrees C for 20 min is effective in the destruction of aflatoxins.

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

PubMed

Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

2011-07-01

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Integrated database for identifying candidate genes for Aspergillus flavus resistance in maize

PubMed Central

2010-01-01

Background Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. Results In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. Conclusions CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query

Integrated database for identifying candidate genes for Aspergillus flavus resistance in maize.

PubMed

Kelley, Rowena Y; Gresham, Cathy; Harper, Jonathan; Bridges, Susan M; Warburton, Marilyn L; Hawkins, Leigh K; Pechanova, Olga; Peethambaran, Bela; Pechan, Tibor; Luthe, Dawn S; Mylroie, J E; Ankala, Arunkanth; Ozkan, Seval; Henry, W B; Williams, W P

2010-10-07

Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database

Aflatoxin contamination in foods and foodstuffs in Tokyo: 1986-1990.

PubMed

Tabata, S; Kamimura, H; Ibe, A; Hashimoto, H; Iida, M; Tamura, Y; Nishima, T

1993-01-01

Aflatoxins were determined in 3054 samples of foods or foodstuffs, including cereals, nuts, beans, spices, dairy products, dry fruits, and edible oil. Samples were collected in Tokyo from 1986 to 1990. Aflatoxins were found in rice products, adlay, corn, crude sugar, peanut products, pistachio nuts, brazil nuts, sesame products, butter beans, white pepper, red pepper, paprika, nutmeg, and mixed spices. The highest incidence of aflatoxin contamination was observed in nutmeg (80%), and the highest level of aflatoxin B1 was observed in pistachio nuts (1382 ppb).

Workflow to study genetic biodiversity of aflatoxigenic Aspergillus spp. in Georgia, USA

USDA-ARS?s Scientific Manuscript database

Peanut seeds were sampled from the entire state of Georgia in 2014. More than 600 isolates of Aspergillus spp. were collected using modified-dichloran rose Bengal (MDRB) medium, 240 of those isolates were fingerprinted with 25 InDel markers within the aflatoxin-biosynthesis gene cluster (ABC). Clust...

Ameliorating Effects of Bacillus subtilis ANSB060 on Growth Performance, Antioxidant Functions, and Aflatoxin Residues in Ducks Fed Diets Contaminated with Aflatoxins.

PubMed

Zhang, Liyuan; Ma, Qiugang; Ma, Shanshan; Zhang, Jianyun; Jia, Ru; Ji, Cheng; Zhao, Lihong

2016-12-22

Bacillus subtilis ANSB060 isolated from fish gut is very effective in detoxifying aflatoxins in feed and feed ingredients. The purpose of this research was to investigate the effects of B. subtilis ANSB060 on growth performance, body antioxidant functions, and aflatoxin residues in ducks fed moldy maize naturally contaminated with aflatoxins. A total of 1500 18-d-old male Cherry Valley ducks with similar body weight were randomly assigned to five treatments with six replicates of 50 ducks per repeat. The experiment design consisted of five dietary treatments labeled as C0 (basal diet containing 60% normal maize), M0 (basal diet containing 60% moldy maize contaminated with aflatoxins substituted for normal maize), M500, M1000, and M2000 (M0 +500, 1000 or 2000 g/t aflatoxin biodegradation preparation mainly consisted of B. subtilis ANSB060). The results showed that ducks fed 22.44 ± 2.46 μg/kg of AFB₁ (M0) exhibited a decreasing tendency in average daily gain (ADG) and total superoxide dismutase (T-SOD) activity in serum, and T-SOD and glutathione peroxidase (GSH-Px) activities in the liver significantly decreased along with the appearance of AFB₁ and AFM₁ compared with those in Group C0. The supplementation of B. subtilis ANSB060 into aflatoxin-contaminated diets increased the ADG of ducks ( p > 0.05), significantly improved antioxidant enzyme activities, and reduced aflatoxin accumulation in duck liver. In conclusion, Bacillus subtilis ANSB060 in diets showed an ameliorating effect to duck aflatoxicosis and may be a promising feed additive.

Ameliorating Effects of Bacillus subtilis ANSB060 on Growth Performance, Antioxidant Functions, and Aflatoxin Residues in Ducks Fed Diets Contaminated with Aflatoxins

PubMed Central

Zhang, Liyuan; Ma, Qiugang; Ma, Shanshan; Zhang, Jianyun; Jia, Ru; Ji, Cheng; Zhao, Lihong

2016-01-01

Bacillus subtilis ANSB060 isolated from fish gut is very effective in detoxifying aflatoxins in feed and feed ingredients. The purpose of this research was to investigate the effects of B. subtilis ANSB060 on growth performance, body antioxidant functions, and aflatoxin residues in ducks fed moldy maize naturally contaminated with aflatoxins. A total of 1500 18-d-old male Cherry Valley ducks with similar body weight were randomly assigned to five treatments with six replicates of 50 ducks per repeat. The experiment design consisted of five dietary treatments labeled as C0 (basal diet containing 60% normal maize), M0 (basal diet containing 60% moldy maize contaminated with aflatoxins substituted for normal maize), M500, M1000, and M2000 (M0 +500, 1000 or 2000 g/t aflatoxin biodegradation preparation mainly consisted of B. subtilis ANSB060). The results showed that ducks fed 22.44 ± 2.46 μg/kg of AFB1 (M0) exhibited a decreasing tendency in average daily gain (ADG) and total superoxide dismutase (T-SOD) activity in serum, and T-SOD and glutathione peroxidase (GSH-Px) activities in the liver significantly decreased along with the appearance of AFB1 and AFM1 compared with those in Group C0. The supplementation of B. subtilis ANSB060 into aflatoxin-contaminated diets increased the ADG of ducks (p > 0.05), significantly improved antioxidant enzyme activities, and reduced aflatoxin accumulation in duck liver. In conclusion, Bacillus subtilis ANSB060 in diets showed an ameliorating effect to duck aflatoxicosis and may be a promising feed additive. PMID:28025501

Cyclo(l-Leucyl-l-Prolyl) Produced by Achromobacter xylosoxidans Inhibits Aflatoxin Production by Aspergillus parasiticus

PubMed Central

Yan, Pei-Sheng; Song, Yuan; Sakuno, Emi; Nakajima, Hiromitsu; Nakagawa, Hiroyuki; Yabe, Kimiko

2004-01-01

Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(l-leucyl-l-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml−1, as determined by the tip culture method. High concentrations (more than 6.0 mg ml−1) of cyclo(l-leucyl-l-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(d-leucyl-d-prolyl) and cyclo(l-valyl-l-prolyl), whereas cyclo(d-prolyl-l-leucyl) and cyclo(l-prolyl-d-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(l-leucyl-l-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production. PMID:15574949

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

PubMed

Wang, Da-Zhi; Zhang, Shu-Fei; Zhang, Yong; Lin, Lin

2016-03-01

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in

Effect of temperature on aflatoxin production in Mucuna pruriens seeds.

PubMed Central

Roy, A K; Chourasia, H K

1989-01-01

This paper describes the effect of temperature on the level of aflatoxin production in Mucuna pruriens seeds. The highest level of aflatoxin B1 (1.75 micrograms/g) was detected in the samples incubated at 25 degrees C for three weeks. At 20, 30, and 35 degrees C, aflatoxin levels were 0.30 to 0.56, 0.37 to 1.20, and 0.26 to 0.65 micrograms/g, respectively. The lowest concentration of aflatoxin B1 (0.10 to 0.29 microgram/g) was produced at 15 degrees C. PMID:2719482

Gene PA2449 Is Essential for Glycine Metabolism and Pyocyanin Biosynthesis in Pseudomonas aeruginosa PAO1

PubMed Central

Lundgren, Benjamin R.; Thornton, William; Dornan, Mark H.; Villegas-Peñaranda, Luis Roberto; Boddy, Christopher N.

2013-01-01

Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria. PMID:23457254

Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

PubMed

Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

2017-06-01

The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome (GT) was carried out. In addition to the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes, about 210 and 196 different terpene synthases (TPSs) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (three from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. © 2017 Scandinavian Plant Physiology Society.

[Survey of aflatoxins contamination of foodstuffs and edible oil in Shenzhen].

PubMed

Li, Ke; Qiu, Fen; Yang, Mei; Liang, Zhaohai; Zhou, Haitao

2013-07-01

To identify the aflatoxins contamination of foodstuffs and edible oil sold in Shenzhen. As research subjects stratified random sampling of 238 foodstuffs and edible oil, and applied with immuno-affinity column clean-up plus UPLC to determine the content of aflatoxin B1, B2, G1, and G2. Positive ratio of aflatoxin in rice, rice products, wheat flour, corn flour, edible oil were 35.3%, 33.8%, 13.9%, 46.7% and 24.5%,respectively. There were statistical differences between the positive ratio of aflatoxin in stereotypes packaged rice (26.5%) and bulk rice (56.3%) (chi2 = 11.6, P < 0.05). There were statistical differences between the positive ratio of aflatoxin in the rice produced in the area north of the Yangtze River (27.3%) and in the rice (41.4%) produced in the area south of the Yangtze River (chi2 = 7.257, P < 0.05). Aflatoxin B1 and B2 were detected in rice products, wheat flour, corn flour. Positive ratio of aflatoxin B1, B2, G1, and G2 were 24.5%, 24.5%, 11.3% and 3.8% in the edible oil,respectively. The over standard rate of aflatoxin B1 was 5.66%, excessive samples were producted bulk and self-pressed peanut oil from unlicensed workshop. All the four kinds of aflatoxin were detected, while subtype B1 and B2 dominated aflatoxin contamination in the rice and edible oil samples. There are differences between in the northern and southern rice, and the same as in the stereotypes packaged and bulk rice sold at Shenzhen.

Genome-Wide Characterization of bHLH Genes in Grape and Analysis of their Potential Relevance to Abiotic Stress Tolerance and Secondary Metabolite Biosynthesis

PubMed Central

Wang, Pengfei; Su, Ling; Gao, Huanhuan; Jiang, Xilong; Wu, Xinying; Li, Yi; Zhang, Qianqian; Wang, Yongmei; Ren, Fengshan

2018-01-01

Basic helix-loop-helix (bHLH) transcription factors are involved in many abiotic stress responses as well as flavonol and anthocyanin biosynthesis. In grapes (Vitis vinifera L.), flavonols including anthocyanins and condensed tannins are most abundant in the skins of the berries. Flavonols are important phytochemicals for viticulture and enology, but grape bHLH genes have rarely been examined. We identified 94 grape bHLH genes in a genome-wide analysis and performed Nr and GO function analyses for these genes. Phylogenetic analyses placed the genes into 15 clades, with some remaining orphans. 41 duplicate gene pairs were found in the grape bHLH gene family, and all of these duplicate gene pairs underwent purifying selection. Nine triplicate gene groups were found in the grape bHLH gene family and all of these triplicate gene groups underwent purifying selection. Twenty-two grape bHLH genes could be induced by PEG treatment and 17 grape bHLH genes could be induced by cold stress treatment including a homologous form of MYC2, VvbHLH007. Based on the GO or Nr function annotations, we found three other genes that are potentially related to anthocyanin or flavonol biosynthesis: VvbHLH003, VvbHLH007, and VvbHLH010. We also performed a cis-acting regulatory element analysis on some genes involved in flavonoid or anthocyanin biosynthesis and our results showed that most of these gene promoters contained G-box or E-box elements that could be recognized by bHLH family members. PMID:29449854

Effect of methionine and lactic acid bacteria as aflatoxin binder on broiler performance

NASA Astrophysics Data System (ADS)

Istiqomah, Lusty; Damayanti, Ema; Julendra, Hardi; Suryani, Ade Erma; Sakti, Awistaros Angger; Anggraeni, Ayu Septi

2017-06-01

The use of aflatoxin binder product based amino acids, lacic acid bacteria, and natural product gived the opportunity to be an alternative biological decontamination of aflatoxins. A study was conducted to determine the efficacy of aflatoxin binder administration (amino acid methionine and lactic acid bacteria (Lactobacillus plantarum G7)) as feed additive on broiler performance. In this study, 75 Lohmann unsexed day old chicks were distributed randomly into 5 units of cages, each filled with 15 broilers. Five cages were assigned into 5 treatments groups and fed with feed contained aflatoxin. The treatments as follow: P1 (aflatoxin feed without aflatoxin binder), P3 (aflatoxin feed + 0.8% of methionine + 1% of LAB), P4 (aflatoxin feed + 1.2% of methionine + 1% of LAB), P5 (aflatoxin feed + 1% of LAB), and K0 (commercial feed). The measurement of aflatoxin content in feed was performed by Enzyme Linked Immunosorbent Assay method using AgraQuant® Total Aflatoxin Assay Romer Labs procedure. The experimental period was 35 days with feeding and drinking ad libitum. LAB was administered into drinking water, while methionine into feed. Vaccination program of Newcastle Disease (ND) was using active vaccine at 4 and 18 day old, while Infectious Bursal Disease (IBD) was given at 8 day old. Parameter of body weight was observed weekly, while feed consumption noted daily. The result showed that aflatoxin in feed for 35 days period did not significantly affect the body weight gain and feed conversion. The lowest percentage of organ damage at 21 day old was found in P5 treatment (55%), while at 35day old was found in P4 treatment (64%). It could be concluded that technological process of detoxifying aflatoxin could be applied in an attempt to reduce the effect on the toxicity of aflatoxin in poultry feed.

Structural characteristics of ScBx genes controlling the biosynthesis of hydroxamic acids in rye (Secale cereale L.).

PubMed

Bakera, Beata; Makowska, Bogna; Groszyk, Jolanta; Niziołek, Michał; Orczyk, Wacław; Bolibok-Brągoszewska, Hanna; Hromada-Judycka, Aneta; Rakoczy-Trojanowska, Monika

2015-08-01

Benzoxazinoids (BX) are major secondary metabolites of gramineous plants that play an important role in disease resistance and allelopathy. They also have many other unique properties including anti-bacterial and anti-fungal activity, and the ability to reduce alfa-amylase activity. The biosynthesis and modification of BX are controlled by the genes Bx1 ÷ Bx10, GT and glu, and the majority of these Bx genes have been mapped in maize, wheat and rye. However, the genetic basis of BX biosynthesis remains largely uncharacterized apart from some data from maize and wheat. The aim of this study was to isolate, sequence and characterize five genes (ScBx1, ScBx2, ScBx3, ScBx4 and ScBx5) encoding enzymes involved in the synthesis of DIBOA, an important defense compound of rye. Using a modified 3D procedure of BAC library screening, seven BAC clones containing all of the ScBx genes were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of these genes, including their promoters, introns and 3'UTRs. Comparative analysis showed that the ScBx genes are similar to those of other Poaceae species, especially to the TaBx genes. The polymorphisms present both in the coding sequences and non-coding regions of ScBx in relation to other Bx genes are predicted to have an impact on the expression, structure and properties of the encoded proteins.

RNA-Seq mediated root transcriptome analysis of Chlorophytum borivilianum for identification of genes involved in saponin biosynthesis.

PubMed

Kumar, Sunil; Kalra, Shikha; Singh, Baljinder; Kumar, Avneesh; Kaur, Jagdeep; Singh, Kashmir

2016-01-01

Chlorophytum borivilianum is an important species of liliaceae family, owing to its vital medicinal properties. Plant roots are used for aphrodisiac, adaptogen, anti-aging, health-restorative and health-promoting purposes. Saponins, are considered to be the principal bioactive components responsible for the wide variety of pharmacological properties of this plant. In the present study, we have performed de novo root transcriptome sequencing of C. borivilianum using Illumina Hiseq 2000 platform, to gain molecular insight into saponins biosynthesis. A total of 33,963,356 high-quality reads were obtained after quality filtration. Sequences were assembled using various programs which generated 97,344 transcripts with a size range of 100-5,216 bp and N50 value of 342. Data was analyzed against non-redundant proteins, gene ontology (GO), and enzyme commission (EC) databases. All the genes involved in saponins biosynthesis along with five full-length genes namely farnesyl pyrophosphate synthase, cycloartenol synthase, β-amyrin synthase, cytochrome p450, and sterol-3-glucosyltransferase were identified. Read per exon kilobase per million (RPKM)-based comparative expression profiling was done to study the differential regulation of the genes. In silico expression analysis of seven selected genes of saponin biosynthetic pathway was validated by qRT-PCR.

A Comparative Study on Aflatoxin B1 Metabolism in Mice and Rats

PubMed Central

Steyn, M.; Pitout, M. J.; Purchase, I. F. H.

1971-01-01

In vivo metabolic studies on rats and mice revealed a marked difference in the fluorescent compounds produced after ingestion of aflatoxin B1. The mouse converted aflatoxin B1 to three unknown fluorescent compounds, designated x1, x2 and x3 and the known aflatoxin M1, while the rat was only capable of producing aflatoxin M1. The results suggested that metabolites x1, x2, x3 and aflatoxin M1 were not part of a major metabolic pathway, but produced independently. These unknown yellowish-green fluorescent compounds did not seem to be conjugated with sulphate or glucuronic acid. In vitro incubations of various mouse liver cell fractions with aflatoxin B1 showed that metabolites x1, x2, x3 and aflatoxin M1, could only be produced by the microsomal fraction and that NADPH was needed as a co-factor. The differences in aflatoxin metabolism by mice and rats are discussed in relation to the apparent resistance of the mouse to the carcinogenic effects of this toxin. PMID:4398926

De novo assembly and analysis of the Artemisia argyi transcriptome and identification of genes involved in terpenoid biosynthesis.

PubMed

Liu, Miaomiao; Zhu, Jinhang; Wu, Shengbing; Wang, Chenkai; Guo, Xingyi; Wu, Jiawen; Zhou, Meiqi

2018-04-11

Artemisia argyi Lev. et Vant. (A. argyi) is widely utilized for moxibustion in Chinese medicine, and the mechanism underlying terpenoid biosynthesis in its leaves is suggested to play an important role in its medicinal use. However, the A. argyi transcriptome has not been sequenced. Herein, we performed RNA sequencing for A. argyi leaf, root and stem tissues to identify as many as possible of the transcribed genes. In total, 99,807 unigenes were assembled by analysing the expression profiles generated from the three tissue types, and 67,446 of those unigenes were annotated in public databases. We further performed differential gene expression analysis to compare leaf tissue with the other two tissue types and identified numerous genes that were specifically expressed or up-regulated in leaf tissue. Specifically, we identified multiple genes encoding significant enzymes or transcription factors related to terpenoid synthesis. This study serves as a valuable resource for transcriptome information, as many transcribed genes related to terpenoid biosynthesis were identified in the A. argyi transcriptome, providing a functional genomic basis for additional studies on molecular mechanisms underlying the medicinal use of A. argyi.

Sampling hazelnuts for aflatoxin: uncertainty associated with sampling, sample preparation, and analysis.

PubMed

Ozay, Guner; Seyhan, Ferda; Yilmaz, Aysun; Whitaker, Thomas B; Slate, Andrew B; Giesbrecht, Francis

2006-01-01

The variability associated with the aflatoxin test procedure used to estimate aflatoxin levels in bulk shipments of hazelnuts was investigated. Sixteen 10 kg samples of shelled hazelnuts were taken from each of 20 lots that were suspected of aflatoxin contamination. The total variance associated with testing shelled hazelnuts was estimated and partitioned into sampling, sample preparation, and analytical variance components. Each variance component increased as aflatoxin concentration (either B1 or total) increased. With the use of regression analysis, mathematical expressions were developed to model the relationship between aflatoxin concentration and the total, sampling, sample preparation, and analytical variances. The expressions for these relationships were used to estimate the variance for any sample size, subsample size, and number of analyses for a specific aflatoxin concentration. The sampling, sample preparation, and analytical variances associated with estimating aflatoxin in a hazelnut lot at a total aflatoxin level of 10 ng/g and using a 10 kg sample, a 50 g subsample, dry comminution with a Robot Coupe mill, and a high-performance liquid chromatographic analytical method are 174.40, 0.74, and 0.27, respectively. The sampling, sample preparation, and analytical steps of the aflatoxin test procedure accounted for 99.4, 0.4, and 0.2% of the total variability, respectively.

Gene Identification of Pheromone Gland Genes Involved in Type II Sex Pheromone Biosynthesis and Transportation in Female Tea Pest Ectropis grisescens

PubMed Central

Li, Zhao-Qun; Ma, Long; Yin, Qian; Cai, Xiao-Ming; Luo, Zong-Xiu; Bian, Lei; Xin, Zhao-Jun; He, Peng; Chen, Zong-Mao

2018-01-01

Moths can biosynthesize sex pheromones in the female sex pheromone glands (PGs) and can distinguish species-specific sex pheromones using their antennae. However, the biosynthesis and transportation mechanism for Type II sex pheromone components has rarely been documented in moths. In this study, we constructed a massive PG transcriptome database (14.72 Gb) from a moth species, Ectropis grisescens, which uses type II sex pheromones and is a major tea pest in China. We further identified putative sex pheromone biosynthesis and transportation-related unigenes: 111 cytochrome P450 monooxygenases (CYPs), 25 odorant-binding proteins (OBPs), and 20 chemosensory proteins (CSPs). Tissue expression and phylogenetic tree analyses showed that one CYP (EgriCYP341-fragment3), one OBP (EgriOBP4), and one CSP (EgriCSP10) gene displayed an enriched expression in the PGs, and that EgriOBP2, 3, and 25 are clustered in the moth pheromone-binding protein clade. We considered these our candidate genes. Our results yielded large-scale PG sequence information for further functional studies. PMID:29317471

Characterization and engineering of the biosynthesis gene cluster for antitumor macrolides PM100117 and PM100118 from a marine actinobacteria: generation of a novel improved derivative.

PubMed

Salcedo, Raúl García; Olano, Carlos; Gómez, Cristina; Fernández, Rogelio; Braña, Alfredo F; Méndez, Carmen; de la Calle, Fernando; Salas, José A

2016-02-22

PM100117 and PM100118 are glycosylated polyketides with remarkable antitumor activity, which derive from the marine symbiotic actinobacteria Streptomyces caniferus GUA-06-05-006A. Structurally, PM100117 and PM100118 are composed of a macrocyclic lactone, three deoxysugar units and a naphthoquinone (NQ) chromophore that shows a clear structural similarity to menaquinone. Whole-genome sequencing of S. caniferus GUA-06-05-006A has enabled the identification of PM100117 and PM100118 biosynthesis gene cluster, which has been characterized on the basis of bioinformatics and genetic engineering data. The product of four genes shows high identity to proteins involved in the biosynthesis of menaquinone via futalosine. Deletion of one of these genes led to a decay in PM100117 and PM100118 production, and to the accumulation of several derivatives lacking NQ. Likewise, five additional genes have been genetically characterized to be involved in the biosynthesis of this moiety. Moreover, the generation of a mutant in a gene coding for a putative cytochrome P450 has led to the production of PM100117 and PM100118 structural analogues showing an enhanced in vitro cytotoxic activity relative to the parental products. Although a number of compounds structurally related to PM100117 and PM100118 has been discovered, this is, to our knowledge, the first insight reported into their biosynthesis. The structural resemblance of the NQ moiety to menaquinone, and the presence in the cluster of four putative menaquinone biosynthetic genes, suggests a connection between the biosynthesis pathways of both compounds. The availability of the PM100117 and PM100118 biosynthetic gene cluster will surely pave a way to the combinatorial engineering of more derivatives.

Identification of additive, dominant, and epistatic variation conferred by key genes in cellulose biosynthesis pathway in Populus tomentosa†

PubMed Central

Du, Qingzhang; Tian, Jiaxing; Yang, Xiaohui; Pan, Wei; Xu, Baohua; Li, Bailian; Ingvarsson, Pär K.; Zhang, Deqiang

2015-01-01

Economically important traits in many species generally show polygenic, quantitative inheritance. The components of genetic variation (additive, dominant and epistatic effects) of these traits conferred by multiple genes in shared biological pathways remain to be defined. Here, we investigated 11 full-length genes in cellulose biosynthesis, on 10 growth and wood-property traits, within a population of 460 unrelated Populus tomentosa individuals, via multi-gene association. To validate positive associations, we conducted single-marker analysis in a linkage population of 1,200 individuals. We identified 118, 121, and 43 associations (P< 0.01) corresponding to additive, dominant, and epistatic effects, respectively, with low to moderate proportions of phenotypic variance (R2). Epistatic interaction models uncovered a combination of three non-synonymous sites from three unique genes, representing a significant epistasis for diameter at breast height and stem volume. Single-marker analysis validated 61 associations (false discovery rate, Q ≤ 0.10), representing 38 SNPs from nine genes, and its average effect (R2 = 3.8%) nearly 2-fold higher than that identified with multi-gene association, suggesting that multi-gene association can capture smaller individual variants. Moreover, a structural gene–gene network based on tissue-specific transcript abundances provides a better understanding of the multi-gene pathway affecting tree growth and lignocellulose biosynthesis. Our study highlights the importance of pathway-based multiple gene associations to uncover the nature of genetic variance for quantitative traits and may drive novel progress in molecular breeding. PMID:25428896

Molecular identification of a pancreatic lipase-like gene involved in sex pheromone biosynthesis of Bombyx mori.

PubMed

Zhang, Song-Dou; Li, Xun; Bin, Zhu; Du, Meng-Fang; Yin, Xin-Ming; An, Shi-Heng

2014-08-01

Cytoplasmic lipid droplet (LD) lipolysis is regulated by pheromone biosynthesis activating neuropeptide (PBAN) in Bombyx mori. To elucidate the molecular mechanism of cytoplasm LD lipolysis, the pancreatic lipase-like gene in B. mori pheromone glands (PGs), designated as B. mori pancreatic lipase-like gene (BmPLLG), was identified in this study. Spatial expression analysis revealed that BmPLLG is a ubiquitous gene present in all studied tissues, such as PGs, brain, epidermis, egg, midgut, flight muscle and fat body. Temporal expression analysis showed that the BmPLLG transcript begins to express 96 h before eclosion (-96 h), continues to increase, peaks in newly emerged females and steadily decreases after eclosion. Translational expression analysis of BmPLLG using a prepared antiserum demonstrated that BmPLLG was expressed in an age-dependent pattern at different development stages in B. mori. This finding was similar to the transcript expression pattern. Further RNA interference-mediated knockdown of BmPLLG significantly inhibited bombykol production. Overall, these results demonstrated that BmPLLG is involved in PBAN-induced sex pheromone biosynthesis and release. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Potential economic losses to the USA corn industry from aflatoxin contamination

PubMed Central

Mitchell, N.J.; Bowers, E.; Hurburgh, C.; Wu, F.

2016-01-01

Mycotoxins, toxins produced by fungi that colonize food crops, can pose a heavy economic burden to the United States corn industry. In terms of economic burden, aflatoxins are the most problematic mycotoxins in US agriculture. Estimates of their market impacts are important in determining the benefits of implementing mitigation strategies within the US corn industry, and the value of strategies to mitigate mycotoxin problems. Additionally, climate change may cause increases in aflatoxin contamination in corn, greatly affecting the economy of the US Midwest and all sectors in the US and worldwide that rely upon its corn production. We propose two separate models for estimating the potential market loss to the corn industry from aflatoxin contamination, in the case of potential near-future climate scenarios (based on aflatoxin levels in Midwest corn in warm summers in the last decade). One model uses probability of acceptance based on operating characteristic (OC) curves for aflatoxin sampling and testing, while the other employs partial equilibrium economic analysis, assuming no Type 1 or Type 2 errors, to estimate losses due to proportions of lots above the US Food and Drug Administration (FDA) aflatoxin action levels. We estimate that aflatoxin contamination could cause losses to the corn industry ranging from $52.1 million to $1.68 billion annually in the United States, if climate change causes more regular aflatoxin contamination in the Corn Belt as was experienced in years such as 2012. The wide range represents the natural variability in aflatoxin contamination from year to year in US corn, with higher losses representative of warmer years. PMID:26807606

Potential economic losses to the US corn industry from aflatoxin contamination.

PubMed

Mitchell, Nicole J; Bowers, Erin; Hurburgh, Charles; Wu, Felicia

2016-01-01

Mycotoxins, toxins produced by fungi that colonise food crops, can pose a heavy economic burden to the US corn industry. In terms of economic burden, aflatoxins are the most problematic mycotoxins in US agriculture. Estimates of their market impacts are important in determining the benefits of implementing mitigation strategies within the US corn industry, and the value of strategies to mitigate mycotoxin problems. Additionally, climate change may cause increases in aflatoxin contamination in corn, greatly affecting the economy of the US Midwest and all sectors in the United States and worldwide that rely upon its corn production. We propose two separate models for estimating the potential market loss to the corn industry from aflatoxin contamination, in the case of potential near-future climate scenarios (based on aflatoxin levels in Midwest corn in warm summers in the last decade). One model uses the probability of acceptance based on operating characteristic (OC) curves for aflatoxin sampling and testing, while the other employs partial equilibrium economic analysis, assuming no Type 1 or Type 2 errors, to estimate losses due to proportions of lots above the US Food and Drug Administration (USFDA) aflatoxin action levels. We estimate that aflatoxin contamination could cause losses to the corn industry ranging from US$52.1 million to US$1.68 billion annually in the United States, if climate change causes more regular aflatoxin contamination in the Corn Belt as was experienced in years such as 2012. The wide range represents the natural variability in aflatoxin contamination from year to year in US corn, with higher losses representative of warmer years.

Cost-Effectiveness of Aflatoxin Control Methods: Economic Incentives

USDA-ARS?s Scientific Manuscript database

Multiple sectors in U.S. crop industries – growers, elevators, handlers/shellers, processors, distributors, and consumers – are affected by aflatoxin contamination of commodities, and have the potential to control it. Aflatoxin control methods at both preharvest and postharvest levels have been dev...

Discovery and characterization of proteins associated with aflatoxin-resistance: evaluating their potential as breeding markers.

PubMed

Brown, Robert L; Chen, Zhi-Yuan; Warburton, Marilyn; Luo, Meng; Menkir, Abebe; Fakhoury, Ahmad; Bhatnagar, Deepak

2010-04-01

Host resistance has become a viable approach to eliminating aflatoxin contamination of maize since the discovery of several maize lines with natural resistance. However, to derive commercial benefit from this resistance and develop lines that can aid growers, markers need to be identified to facilitate the transfer of resistance into commercially useful genetic backgrounds without transfer of unwanted traits. To accomplish this, research efforts have focused on the identification of kernel resistance-associated proteins (RAPs) including the employment of comparative proteomics to investigate closely-related maize lines that vary in aflatoxin accumulation. RAPs have been identified and several further characterized through physiological and biochemical investigations to determine their causal role in resistance and, therefore, their suitability as breeding markers. Three RAPs, a 14 kDa trypsin inhibitor, pathogenesis-related protein 10 and glyoxalase I are being investigated using RNAi gene silencing and plant transformation. Several resistant lines have been subjected to QTL mapping to identify loci associated with the aflatoxin-resistance phenotype. Results of proteome and characterization studies are discussed.

The Rickettsia Endosymbiont of Ixodes pacificus Contains All the Genes of De Novo Folate Biosynthesis

PubMed Central

Bodnar, James; Mortazavi, Bobak; Laurent, Timothy; Deason, Jeff; Thephavongsa, Khanhkeo; Zhong, Jianmin

2015-01-01

Ticks and other arthropods often are hosts to nutrient providing bacterial endosymbionts, which contribute to their host’s fitness by supplying nutrients such as vitamins and amino acids. It has been detected, in our lab, that Ixodes pacificus is host to Rickettsia species phylotype G021. This endosymbiont is predominantly present, and 100% maternally transmitted in I. pacificus. To study roles of phylotype G021 in I. pacificus, bioinformatic and molecular approaches were carried out. MUMmer genome alignments of whole genome sequence of I. scapularis, a close relative to I. pacificus, against completely sequenced genomes of R. bellii OSU85-389, R. conorii, and R. felis, identified 8,190 unique sequences that are homologous to Rickettsia sequences in the NCBI Trace Archive. MetaCyc metabolic reconstructions revealed that all folate gene orthologues (folA, folC, folE, folKP, ptpS) required for de novo folate biosynthesis are present in the genome of Rickettsia buchneri in I. scapularis. To examine the metabolic capability of phylotype G021 in I. pacificus, genes of the folate biosynthesis pathway of the bacterium were PCR amplified using degenerate primers. BLAST searches identified that nucleotide sequences of the folA, folC, folE, folKP, and ptpS genes possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to the corresponding genes of Rickettsia buchneri. Phylogenetic tree constructions show that the folate genes of phylotype G021 and homologous genes from various Rickettsia species are monophyletic. This study has shown that all folate genes exist in the genome of Rickettsia species phylotype G021 and that this bacterium has the genetic capability for de novo folate synthesis. PMID:26650541

Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei.

PubMed

Hackbart, H C S; Machado, A R; Christ-Ribeiro, A; Prietto, L; Badiale-Furlong, E

2014-08-01

This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B1, B2, G1, G2, and M1. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B1, B2, and G1 in the 96 h and aflatoxins M1 and G2 in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B1, B2, and M1 in the 96 h and aflatoxin G1 in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture.

Reduction of Aflatoxins in Apricot Kernels by Electronic and Manual Color Sorting.

PubMed

Zivoli, Rosanna; Gambacorta, Lucia; Piemontese, Luca; Solfrizzo, Michele

2016-01-19

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B₁ and B₂ were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB₁ + AFB₂, whereas AFG₁ and AFG₂ were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%-19.9% of total peeled kernels) removed 97.3%-99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a feasible strategy to remove 97%-99% of aflatoxins accumulated in naturally-contaminated samples. Electronic optical sorter gave highly variable results since the amount of AFB₁ + AFB₂ measured in rejected fractions (15%-18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01-0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB₁ and from 0.06 to 1.79 μg/kg for total aflatoxins.

Reduction of Aflatoxins in Apricot Kernels by Electronic and Manual Color Sorting

PubMed Central

Zivoli, Rosanna; Gambacorta, Lucia; Piemontese, Luca; Solfrizzo, Michele

2016-01-01

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B1 and B2 were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB1 + AFB2, whereas AFG1 and AFG2 were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%–19.9% of total peeled kernels) removed 97.3%–99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a feasible strategy to remove 97%–99% of aflatoxins accumulated in naturally-contaminated samples. Electronic optical sorter gave highly variable results since the amount of AFB1 + AFB2 measured in rejected fractions (15%–18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01–0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB1 and from 0.06 to 1.79 μg/kg for total aflatoxins. PMID:26797635

Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideae-infective genotypes of Erwinia amylovora.

PubMed

Rezzonico, Fabio; Braun-Kiewnick, Andrea; Mann, Rachel A; Rodoni, Brendan; Goesmann, Alexander; Duffy, Brion; Smits, Theo H M

2012-10-01

Comparative genomic analysis revealed differences in the lipopolysaccharide (LPS) biosynthesis gene cluster between the Rubus-infecting strain ATCC BAA-2158 and the Spiraeoideae-infecting strain CFBP 1430 of Erwinia amylovora. These differences corroborate rpoB-based phylogenetic clustering of E. amylovora into four different groups and enable the discrimination of Spiraeoideae- and Rubus-infecting strains. The structure of the differences between the two groups supports the hypothesis that adaptation to Rubus spp. took place after species separation of E. amylovora and E. pyrifoliae that contrasts with a recently proposed scenario, based on CRISPR data, in which the shift to domesticated apple would have caused an evolutionary bottleneck in the Spiraeoideae-infecting strains of E. amylovora which would be a much earlier event. In the core region of the LPS biosynthetic gene cluster, Spiraeoideae-infecting strains encode three glycosyltransferases and an LPS ligase (Spiraeoideae-type waaL), whereas Rubus-infecting strains encode two glycosyltransferases and a different LPS ligase (Rubus-type waaL). These coding domains share little to no homology at the amino acid level between Rubus- and Spiraeoideae-infecting strains, and this genotypic difference was confirmed by polymerase chain reaction analysis of the associated DNA region in 31 Rubus- and Spiraeoideae-infecting strains. The LPS biosynthesis gene cluster may thus be used as a molecular marker to distinguish between Rubus- and Spiraeoideae-infecting strains of E. amylovora using primers designed in this study. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

[Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

PubMed

Petrova, L P; Prilipov, A G; Katsy, E I

2017-01-01

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18more » cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.« less

Behavior of sup 14 C aflatoxin M1 during camembert cheese making

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fremy, J.M.; Roiland, J.C.; Gaymard, A.

Camembert cheeses are made from raw milk spiked with aflatoxin M1. Three aflatoxin M1 levels (7.5 micrograms/L, 3 micrograms/L, and 0.3 micrograms/L) are used. In curds 35.6, 47.1, and 57.7% of aflatoxin M1, respectively, are recovered, and in wheys 64.4, 52.9, and 42.3%, respectively, are recovered. During the first 15 days of storage, the aflatoxin M1 content of different cheeses decreases 25, 55, and 75%, respectively. A similar experiment is made with milk contaminated with {sup 14}C labeled aflatoxin M1. The same results are obtained, except for the behavior of aflatoxin M1 in cheese; the same 14C activity is recoveredmore » during storage for 30 days.« less

Susceptibility to aflatoxin contamination among maize landraces from Mexico.

PubMed

Ortega-Beltran, Alejandro; Guerrero-Herrera, Manuel D J; Ortega-Corona, Alejandro; Vidal-Martinez, Victor A; Cotty, Peter J

2014-09-01

Maize, the critical staple food for billions of people, was domesticated in Mexico about 9,000 YBP. Today, a great array of maize landraces (MLRs) across rural Mexico is harbored in a living library that has been passed among generations since before the establishment of the modern state. MLRs have been selected over hundreds of generations by ethnic groups for adaptation to diverse environmental settings. The genetic diversity of MLRs in Mexico is an outstanding resource for development of maize cultivars with beneficial traits. Maize is frequently contaminated with aflatoxins by Aspergillus flavus, and resistance to accumulation of these potent carcinogens has been sought for over three decades. However, MLRs from Mexico have not been evaluated as potential sources of resistance. Variation in susceptibility to both A. flavus reproduction and aflatoxin contamination was evaluated on viable maize kernels in laboratory experiments that included 74 MLR accessions collected from 2006 to 2008 in the central west and northwest regions of Mexico. Resistant and susceptible MLR accessions were detected in both regions. The most resistant accessions accumulated over 99 % less aflatoxin B1 than did the commercial hybrid control Pioneer P33B50. Accessions supporting lower aflatoxin accumulation also supported reduced A. flavus sporulation. Sporulation on the MLRs was positively correlated with aflatoxin accumulation (R = 0.5336, P < 0.0001), suggesting that resistance to fungal reproduction is associated with MLR aflatoxin resistance. Results of the current study indicate that MLRs from Mexico are potentially important sources of aflatoxin resistance that may contribute to the breeding of commercially acceptable and safe maize hybrids and/or open pollinated cultivars for human and animal consumption.

Assay for Aflatoxin Production by the Genera Aspergillus and Penicillium1

PubMed Central

Mislivec, Philip B.; Hunter, J. H.; Tuite, John

1968-01-01

A total of 260 isolates, including 43 species of Penicillium and 7 species of Aspergillus, were screened for their ability to produce aflatoxin on rice. Chloroform extracts were analyzed by thin-layer chromatography. None of the isolates produced aflatoxin. Certain species of Penicillium produced fluorescent substances that either were similar in RF or were of similar color to B and G aflatoxins. These substances were subsequently proved not to be aflatoxin by two-dimensional chromatography, by reaction with iodine fumes, or by both methods. PMID:5664121

Discovering the role of the apolipoprotein gene and the genes in the putative pullulan biosynthesis pathway on the synthesis of pullulan, heavy oil and melanin in Aureobasidium pullulans.

PubMed

Guo, Jian; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

2017-12-18

Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.

The inhibitory effect of Bacillus megaterium on aflatoxin biosynthetic pathway gene expression in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Aspergillus flavus is one of the major fungal mold that colonize peanut in the field and during storage. The impacts to human and animal health and to economy in agriculture and commerce are significant since this mould produces the most potent natural toxins, aflatoxins, which are carcinogenic, mut...

Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes.

PubMed

Xiao, Yun-yi; Chen, Jian-ye; Kuang, Jiang-fei; Shan, Wei; Xie, Hui; Jiang, Yue-ming; Lu, Wang-jin

2013-05-01

The involvement of ethylene response factor (ERF) transcription factor (TF) in the transcriptional regulation of ethylene biosynthesis genes during fruit ripening remains largely unclear. In this study, 15 ERF genes, designated as MaERF1-MaERF15, were isolated and characterized from banana fruit. These MaERFs were classified into seven of the 12 known ERF families. Subcellular localization showed that MaERF proteins of five different subfamilies preferentially localized to the nucleus. The 15 MaERF genes displayed differential expression patterns and levels in peel and pulp of banana fruit, in association with four different ripening treatments caused by natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and combined 1-MCP and ethylene treatments. MaERF9 was upregulated while MaERF11 was downregulated in peel and pulp of banana fruit during ripening or after treatment with ethylene. Furthermore, yeast-one hybrid (Y1H) and transient expression assays showed that the potential repressor MaERF11 bound to MaACS1 and MaACO1 promoters to suppress their activities and that MaERF9 activated MaACO1 promoter activity. Interestingly, protein-protein interaction analysis revealed that MaERF9 and -11 physically interacted with MaACO1. Taken together, these results suggest that MaERFs are involved in banana fruit ripening via transcriptional regulation of or interaction with ethylene biosynthesis genes.

Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes

PubMed Central

Xiao, Yun-yi; Chen, Jian-ye; Kuang, Jiang-fei; Shan, Wei; Xie, Hui; Jiang, Yue-ming; Lu, Wang-jin

2013-01-01

The involvement of ethylene response factor (ERF) transcription factor (TF) in the transcriptional regulation of ethylene biosynthesis genes during fruit ripening remains largely unclear. In this study, 15 ERF genes, designated as MaERF1–MaERF15, were isolated and characterized from banana fruit. These MaERFs were classified into seven of the 12 known ERF families. Subcellular localization showed that MaERF proteins of five different subfamilies preferentially localized to the nucleus. The 15 MaERF genes displayed differential expression patterns and levels in peel and pulp of banana fruit, in association with four different ripening treatments caused by natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and combined 1-MCP and ethylene treatments. MaERF9 was upregulated while MaERF11 was downregulated in peel and pulp of banana fruit during ripening or after treatment with ethylene. Furthermore, yeast-one hybrid (Y1H) and transient expression assays showed that the potential repressor MaERF11 bound to MaACS1 and MaACO1 promoters to suppress their activities and that MaERF9 activated MaACO1 promoter activity. Interestingly, protein–protein interaction analysis revealed that MaERF9 and -11 physically interacted with MaACO1. Taken together, these results suggest that MaERFs are involved in banana fruit ripening via transcriptional regulation of or interaction with ethylene biosynthesis genes. PMID:23599278

Reduced Graphene Oxide-Gold Nanoparticle Nanoframework as a Highly Selective Separation Material for Aflatoxins.

PubMed

Guo, Wenbo; Wu, Lidong; Fan, Kai; Nie, Dongxia; He, Weijing; Yang, Junhua; Zhao, Zhihui; Han, Zheng

2017-11-03

Graphene-based materials have been studied in many applications, owing to the excellent electrical, mechanical, and thermal properties of graphene. In the current study, an environmentally friendly approach to the preparation of a reduced graphene oxide-gold nanoparticle (rGO-AuNP) nanocomposite was developed by using L-cysteine and vitamin C as reductants under mild reaction conditions. The rGO-AuNP material showed a highly selective separation ability for 6 naturally occurring aflatoxins, which are easily adsorbed onto traditional graphene materials but are difficult to be desorbed. The specificity of the nanocomposite was evaluated in the separation of 6 aflatoxin congeners (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1 and aflatoxin M2) from 23 other biotoxins (including, ochratoxin A, citrinin, and deoxynivalenol). The results indicated that this material was specific for separating aflatoxin congeners. The synthesized material was further validated by determining the recovery (77.6-105.0%), sensitivity (limit of detection in the range of 0.05-0.21 μg kg -1 ), and precision (1.5-11.8%), and was then successfully applied to the separation of aflatoxins from real-world maize, wheat and rice samples.

Effect of almond processing on levels and distribution of aflatoxins in finished products and byproducts.

PubMed

Zivoli, Rosanna; Gambacorta, Lucia; Perrone, Giancarlo; Solfrizzo, Michele

2014-06-18

The fate of aflatoxins during processing of contaminated almonds into nougat, pastries, and almond syrup was evaluated by testing the effect of each processing step (blanching, peeling, roasting, caramelization, cooking, and water infusion) on the distribution and levels of aflatoxins. Blanching and peeling did not reduce total aflatoxins that were distributed between peeled almonds (90-93%) and skins (7-10%). Roasting of peeled almonds reduced up to 50% of aflatoxins. Up to 70% reduction of aflatoxins was observed during preparation and cooking of almond nougat in caramelized sugar. Aflatoxins were substantially stable during preparation and cooking of almond pastries. The whole process of almond syrup preparation produced a marked increase of total aflatoxins (up to 270%) that were distributed between syrup (18-25%) and spent almonds (75-82%). The increase of total aflatoxins was probably due to the activation of almond enzymes during the infusion step that released free aflatoxins from masked aflatoxins.

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

DOE PAGES

Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan; ...

2016-04-18

We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wildmore » type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.« less

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan

We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wildmore » type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.« less

Aflatoxin contamination of groundnut and maize in Zambia: observed and potential concentrations.

PubMed

Kachapulula, P W; Akello, J; Bandyopadhyay, R; Cotty, P J

2017-06-01

The aims of the study were to quantify aflatoxins, the potent carcinogens associated with stunting and immune suppression, in maize and groundnut across Zambia's three agroecologies and to determine the vulnerability to aflatoxin increases after purchase. Aflatoxin concentrations were determined for 334 maize and groundnut samples from 27 districts using lateral-flow immunochromatography. Seventeen per cent of crops from markets contained aflatoxin concentrations above allowable levels in Zambia (10 μg kg -1 ). Proportions of crops unsafe for human consumption differed significantly (P < 0·001) among agroecologies with more contamination (38%) in the warmest (Agroecology I) and the least (8%) in cool, wet Agroecology III. Aflatoxin in groundnut (39 μg kg -1 ) and maize (16 μg kg -1 ) differed (P = 0·032). Poor storage (31°C, 100% RH, 1 week) increased aflatoxin in safe crops by over 1000-fold in both maize and groundnut. The L morphotype of Aspergillus flavus was negatively correlated with postharvest increases in groundnut. Aflatoxins are common in Zambia's food staples with proportions of unsafe crops dependent on agroecology. Fungal community structure influences contamination suggesting Zambia would benefit from biocontrol with atoxigenic A. flavus. Aflatoxin contamination across the three agroecologies of Zambia is detailed and the case for aflatoxin management with atoxigenic biocontrol agents provided. The first method for evaluating the potential for aflatoxin increase after purchase is presented. Published 2017. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.

Dietary exposure to aflatoxin in human male infertility in Benin City, Nigeria.

PubMed

Ibeh, I N; Uraih, N; Ogonar, J I

1994-01-01

To discover the relationship between aflatoxin levels, if any, in serum of infertile men in comparison with random controls from the community. In a parallel experiment, adult male rats were given an aflatoxin-contaminated diet. 100 adult males, yielding 50 semen samples, from men attending Infertility Clinics at a university teaching hospital and 50 normal men in the same community. The staple foods of the men were assayed for aflatoxin content. The rats were given the aflatoxin-rich diet, and their spermatozoa were examined and their ability to reproduce assessed. A random sampling of semen from 100 adult males comprising 50 samples drawn from infertile men and 50 drawn from normal individuals within the same community revealed the presence of aflatoxins in 20 semen samples from the infertile group (40.0%) and four samples from the fertile group (8.0%). The mean aflatoxin concentrations were 1.660 +/- 0.04 micrograms/mL (infertile men) and 1.041 +/- 0.01 micrograms/mL (fertile men). Infertile men with aflatoxin in their semen showed a higher percentage of spermatozoal abnormality (50.0%) than the fertile men (10.0-15.0%). Dietary exposure of adult male Albino rats to aflatoxin (8.5 micrograms AF1/g of Guinea growers feed for 14 days) produced deleterious effects on the spermatozoa of the affected rats, producing features that resemble those seen in semen of infertile men exposed to aflatoxin.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

PubMed Central

Garin, Intza; Edghill, Emma L.; Akerman, Ildem; Rubio-Cabezas, Oscar; Rica, Itxaso; Locke, Jonathan M.; Maestro, Miguel Angel; Alshaikh, Adnan; Bundak, Ruveyde; del Castillo, Gabriel; Deeb, Asma; Deiss, Dorothee; Fernandez, Juan M.; Godbole, Koumudi; Hussain, Khalid; O’Connell, Michele; Klupa, Thomasz; Kolouskova, Stanislava; Mohsin, Fauzia; Perlman, Kusiel; Sumnik, Zdenek; Rial, Jose M.; Ugarte, Estibaliz; Vasanthi, Thiruvengadam; Johnstone, Karen; Flanagan, Sarah E.; Martínez, Rosa; Castaño, Carlos; Patch, Ann-Marie; Fernández-Rebollo, Eduardo; Raile, Klemens; Morgan, Noel; Harries, Lorna W.; Castaño, Luis; Ellard, Sian; Ferrer, Jorge; de Nanclares, Guiomar Perez; Hattersley, Andrew T.

2010-01-01

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man. PMID:20133622

Aflatoxins--experimental studies.

PubMed

Tulpule, P G

1981-01-01

The susceptibility of animals to both chronic and acute aflatoxicosis is variable between species and depends upon not only the dose of the toxin and the duration of exposure but also upon the age, sex, and nutritional status of the animal. In general, acute toxicity is manifested by necrosis and cirrhosis, and chronic toxicity by carcinoma of the liver. Current research using both in vivo and in vitro studies has shown that the differences in response to aflatoxin in different animals can be attributed to their differential metabolism. The rates of metabolism and intermediate products formed are important factors in determining the type of toxic action of aflatoxin B1. According to these criteria, monkey and man are more susceptible to acute aflatoxicosis and relatively resistant to carcinogenic effects. On the other hand, animals, such as sheep and rat, are more susceptible to carcinogenic effects.

Identification of the pheromone biosynthesis genes from the sex pheromone gland transcriptome of the diamondback moth, Plutella xylostella.

PubMed

Chen, Da-Song; Dai, Jian-Qing; Han, Shi-Chou

2017-11-24

The diamondback moth was estimated to increase costs to the global agricultural economy as the global area increase of Brassica vegetable crops and oilseed rape. Sex pheromones traps are outstanding tools available in Integrated Pest Management for many years and provides an effective approach for DBM population monitoring and control. The ratio of two major sex pheromone compounds shows geographical variations. However, the limitation of our information in the DBM pheromone biosynthesis dampens our understanding of the ratio diversity of pheromone compounds. Here, we constructed a transcriptomic library from the DBM pheromone gland and identified genes putatively involved in the fatty acid biosynthesis, pheromones functional group transfer, and β-oxidation enzymes. In addition, odorant binding protein, chemosensory protein and pheromone binding protein genes encoded in the pheromone gland transcriptome, suggest that female DBM moths may receive odors or pheromone compounds via their pheromone gland and ovipositor system. Tissue expression profiles further revealed that two ALR, three DES and one FAR5 genes were pheromone gland tissue biased, while some chemoreception genes expressed extensively in PG, pupa, antenna and legs tissues. Finally, the candidate genes from large-scale transcriptome information may be useful for characterizing a presumed biosynthetic pathway of the DBM sex pheromone.

De novo transcriptome assembly in chili pepper (Capsicum frutescens) to identify genes involved in the biosynthesis of capsaicinoids.

PubMed

Liu, Shaoqun; Li, Wanshun; Wu, Yimin; Chen, Changming; Lei, Jianjun

2013-01-01

The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (Capsicum frutescens L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for de novo assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (C. annuum) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in C. frutescens and C. annuum sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of de novo transcriptome assembly is applicable to a wide range of similar studies.

De Novo Transcriptome Assembly in Chili Pepper (Capsicum frutescens) to Identify Genes Involved in the Biosynthesis of Capsaicinoids

PubMed Central

Liu, Shaoqun; Li, Wanshun; Wu, Yimin; Chen, Changming; Lei, Jianjun

2013-01-01

The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (Capsicum frutescens L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for de novo assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (C. annuum) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in C. frutescens and C. annuum sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of de novo transcriptome assembly is applicable to a wide range of similar studies. PMID:23349661

Correlation of Zn2+ content with aflatoxin content of corn.

PubMed Central

Failla, L J; Lynn, D; Niehaus, W G

1986-01-01

Forty-nine samples from the 1983 Virginia corn harvest were analyzed for aflatoxin, zinc, copper, iron, and manganese content. Values (mean +/- standard deviation) were as follows: aflatoxin, 117 +/- 360 micrograms/kg; zinc, 22.5 +/- 3.4 mg/kg; copper, 2.27 +/- 0.56 mg/kg; iron, 40.8 +/- 18.7 mg/kg; and manganese, 5.1 +/- 1.1 mg/kg. Aflatoxin levels positively correlated with zinc (Spearman correlation coefficient, 0.385; P less than 0.006) and copper levels (Spearman correlation coefficient, 0.573; P less than 0.0001). Based on biochemical data in the literature, we believe that the correlation with zinc is important and that there may be a cause-and-effect relationship between zinc levels in corn and aflatoxin levels which are produced upon infection with Aspergillus flavus or A. parasiticus. Control of aflatoxin contamination in field corn by decreasing the zinc levels may be feasible, but no methods to decrease zinc levels are currently available. PMID:3729406

Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov.

PubMed

Frisvad, Jens C; Skouboe, Pernille; Samson, Robert A

2005-07-01

Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis1[OPEN

PubMed Central

Browse, John

2016-01-01

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. PMID:26586834

Banana peel: an effective biosorbent for aflatoxins.

PubMed

Shar, Zahid Hussain; Fletcher, Mary T; Sumbal, Gul Amer; Sherazi, Syed Tufail Hussain; Giles, Cindy; Bhanger, Muhammad Iqbal; Nizamani, Shafi Muhammad

2016-05-01

This work reports the application of banana peel as a novel bioadsorbent for in vitro removal of five mycotoxins (aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A). The effect of operational parameters including initial pH, adsorbent dose, contact time and temperature were studied in batch adsorption experiments. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and point of zero charge (pHpzc) analysis were used to characterise the adsorbent material. Aflatoxins' adsorption equilibrium was achieved in 15 min, with highest adsorption at alkaline pH (6-8), while ochratoxin has not shown any significant adsorption due to surface charge repulsion. The experimental equilibrium data were tested by Langmuir, Freundlich and Hill isotherms. The Langmuir isotherm was found to be the best fitted model for aflatoxins, and the maximum monolayer coverage (Q0) was determined to be 8.4, 9.5, 0.4 and 1.1 ng mg(-1) for AFB1, AFB2, AFG1 and AFG2 respectively. Thermodynamic parameters including changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were determined for the four aflatoxins. Free energy change and enthalpy change demonstrated that the adsorption process was exothermic and spontaneous. Adsorption and desorption study at different pH further demonstrated that the sorption of toxins was strong enough to sustain pH changes that would be experienced in the gastrointestinal tract. This study suggests that biosorption of aflatoxins by dried banana peel may be an effective low-cost decontamination method for incorporation in animal feed diets.

Post-genome research on the biosynthesis of ergot alkaloids.

PubMed

Li, Shu-Ming; Unsöld, Inge A

2006-10-01

Genome sequencing provides new opportunities and challenges for identifying genes for the biosynthesis of secondary metabolites. A putative biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine type, was identified in the genome sequence of ASPERGILLUS FUMIGATUS by a bioinformatic approach. This cluster spans 22 kb of genomic DNA and comprises at least 11 open reading frames (ORFs). Seven of them are orthologous to genes from the biosynthetic gene cluster of ergot alkaloids in CLAVICEPS PURPUREA. Experimental evidence of the identified cluster was provided by heterologous expression and biochemical characterization of two ORFs, FgaPT1 and FgaPT2, in the cluster of A. FUMIGATUS, which show remarkable similarities to dimethylallyltryptophan synthase from C. PURPUREA and function as prenyltransferases. FgaPT2 converts L-tryptophan to dimethylallyltryptophan and thereby catalyzes the first step of ergot alkaloid biosynthesis, whilst FgaPT1 catalyzes the last step of the fumigaclavine C biosynthesis, i. e., the prenylation of fumigaclavine A at C-2 position of the indole nucleus. In addition to information obtained from the gene cluster of ergot alkaloids from C. PURPUREA, the identification of the biosynthetic gene cluster of fumigaclavine C in A. FUMIGATUS opens an alternative way to study the biosynthesis of ergot alkaloids in fungi.

Aflatoxin contamination of groundnut and maize in Zambia: observed and potential concentrations

USDA-ARS?s Scientific Manuscript database

Maize and groundnut, important staples in Zambia, are susceptible to aflatoxin-producing fungi. Aflatoxins are potent human carcinogens also associated with stunting and immunosuppression. Although health and economic burdens of aflatoxins are well known, patterns of contamination in maize and grou...

The Thiamine Biosynthesis Gene THI1 Promotes Nodule Growth and Seed Maturation1

PubMed Central

Nagae, Miwa; Kawaguchi, Masayoshi; Takeda, Naoya

2016-01-01

Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculated leaves. THI2-knockdown plants and the transposon insertion mutant thiC had chlorotic leaves. This typical phenotype of thiamine deficiency was rescued by an exogenous supply of thiamine. In wild-type plants, THI1 was expressed mainly in roots and nodules, and the thi1 mutant had green leaves even in the absence of exogenous thiamine. THI1 was highly expressed in actively dividing cells of nodule primordia. The thi1 mutant had small nodules, and this phenotype was rescued by exogenous thiamine and by THI1 complementation. Exogenous thiamine increased nodule diameter, but the level of arbuscular mycorrhizal colonization was unaffected in the thi1 mutant or by exogenous thiamine. Expression of symbiotic marker genes was induced normally, implying that mainly nodule growth was delayed in the thi1 mutant. Furthermore, this mutant formed many immature seeds with reduced seed weight. These results indicate that thiamine biosynthesis mediated by THI1 enhances nodule enlargement and is required for seed development in L. japonicus. PMID:27702844

Biosynthesis of actinorhodin and related antibiotics: discovery of alternative routes for quinone formation encoded in the act gene cluster.

PubMed

Okamoto, Susumu; Taguchi, Takaaki; Ochi, Kozo; Ichinose, Koji

2009-02-27

All known benzoisochromanequinone (BIQ) biosynthetic gene clusters carry a set of genes encoding a two-component monooxygenase homologous to the ActVA-ORF5/ActVB system for actinorhodin biosynthesis in Streptomyces coelicolor A3(2). Here, we conducted molecular genetic and biochemical studies of this enzyme system. Inactivation of actVA-ORF5 yielded a shunt product, actinoperylone (ACPL), apparently derived from 6-deoxy-dihydrokalafungin. Similarly, deletion of actVB resulted in accumulation of ACPL, indicating a critical role for the monooxygenase system in C-6 oxygenation, a biosynthetic step common to all BIQ biosyntheses. Furthermore, in vitro, we showed a quinone-forming activity of the ActVA-ORF5/ActVB system in addition to that of a known C-6 monooxygenase, ActVA-ORF6, by using emodinanthrone as a model substrate. Our results demonstrate that the act gene cluster encodes two alternative routes for quinone formation by C-6 oxygenation in BIQ biosynthesis.

Analysis of cocoa products for ochratoxin A and aflatoxins.

PubMed

Turcotte, Anne-Marie; Scott, Peter M; Tague, Brett

2013-08-01

Eighty-five samples of cocoa products sampled in Canada were analysed for ochratoxin A (OTA) and aflatoxins in 2011-2012. Inclusion of the aflatoxins in this survey required additional method development. Chocolate was extracted with methanol-water plus NaCl, while for cocoa two successive extractions with methanol and methanol-water were made. Extracts were cleaned on an AflaOchra immunoaffinity column (IAC). Determination was by reversed phase high performance liquid chromatography (HPLC). Detection of the aflatoxins was with a post-column photochemical reactor and of OTA by fluorescence detection. Mean limits of quantification (LOQ) of chocolate and cocoa powders were 0.16 ng/g (OTA) and 0.07 ng/g (aflatoxin B1), respectively. Survey results showed that the incidences of OTA above the LOQ in natural cocoa were 15/15 (mean 1.17 ng/g), 20/21 for alkalized cocoa (mean 1.06 ng/g), 9/9 for baking chocolate (mean 0.49 ng/g), 20/20 for dark chocolate (mean 0.39 ng/g), 7/10 for milk chocolate (mean 0.19 ng/g), 5/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. These results confirm our previous work with OTA. In the same samples, incidences of aflatoxin B1 above the LOQ were 14/15 for natural cocoa (mean 0.86 ng/g), 20/21 for alkalized cocoa (mean 0.37 ng/g), 7/9 for baking chocolate (mean 0.22 ng/g), 16/20 for dark chocolate (mean 0.19 ng/g), 7/10 for milk chocolate (mean 0.09 ng/g), 4/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. Both aflatoxins and OTA were confirmed by HPLC-MS/MS when OTA or aflatoxin levels found were above 2 ng/g in cocoa.

Vitamin B1 diversity and characterization of biosynthesis genes in cassava.

PubMed

Mangel, Nathalie; Fudge, Jared B; Fitzpatrick, Teresa B; Gruissem, Wilhelm; Vanderschuren, Hervé

2017-06-15

Vitamin B1, which consists of the vitamers thiamin and its phosphorylated derivatives, is an essential micronutrient for all living organisms because it is required as a metabolic cofactor in several enzymatic reactions. Genetic diversity of vitamin B1 biosynthesis and accumulation has not been investigated in major crop species other than rice and potato. We analyzed cassava germplasm for accumulation of B1 vitamers. Vitamin B1 content in leaves and roots of 41 cassava accessions showed significant variation between accessions. HPLC analyses of B1 vitamers revealed distinct profiles in cassava leaves and storage roots, with nearly equal relative levels of thiamin pyrophosphate and thiamin monophosphate in leaves, but mostly thiamin pyrophosphate in storage roots. Unusually, the cassava genome has two genes encoding the 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, THIC (MeTHIC1 and MeTHIC2), both of which carry a riboswitch in the 3'-UTR, as well as the adenylated thiazole synthase, THI1 (MeTHI1a and MeTHI1b). The THIC and THI1 genes are expressed at very low levels in storage roots compared with the accumulation of vitamin B1, indicating only limited biosynthesis de novo therein. In leaves, vitamin B1 content is negatively correlated with THIC and THI1 expression levels, suggesting post-transcriptional regulation of THIC by the riboswitch present in the 3'-UTR of the THIC mRNA and regulation of THI1 by promoter activity or alternative post-transcriptional mechanisms. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata

PubMed Central

Hussain, Tajammul; Plunkett, Blue; Ejaz, Mahwish; Espley, Richard V.; Kayser, Oliver

2018-01-01

The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

Aflatoxin B1 albumin adducts in plasma and aflatoxin M1 in urine are associated with plasma concentrations of vitamins A and E

PubMed Central

Obuseh, Francis A.; Jolly, Pauline E.; Jiang, Yi; Shuaib, Faisal M. B.; Waterbor, John; Ellis, William O.; Piyathilake, Chandrika J.; Desmond, Renee A.; Afriyie-Gyawu, Evans; Phillips, Timothy D.

2011-01-01

Background Although aflatoxin exposure has been shown to be associated with micronutrient deficiency in animals, there are few investigations on the effects of aflatoxin exposure on micronutrient metabolism in humans. Objective To examine the relationship between aflatoxin B1 (AFB1) albumin adducts (AF-ALB) in plasma and the aflatoxin M1 (AFM1) metabolite in urine and plasma concentrations of retinol (vitamin A) and α-tocopherol (vitamin E) in Ghanaians. Methods A cross-sectional study of 147 adult participants was conducted. Blood and urine samples were tested for aflatoxin and vitamins A and E levels. Results Multivariable analysis showed that participants with high AF-ALB (≥ 0.80 pmol/mg albumin) had increased odds of having vitamin A deficiency compared to those with lower AF-ALB [Odds Ratio (OR) = 2.61; CI = 1.03 – 6.58; p=0.04]. Participants with high AF-ALB also showed increased odds of having vitamin E deficiency but this was not statistically significant (OR = 2.4; CI = 0.96–6.05; p = 0.06). Conversely, those with higher AFM1 values had a statistically nonsignificant reduced odds of having vitamin A deficiency (OR = 0.31; CI = 1.15–0.09; p=0.05) and statistically significant reduced odds of having vitamin E deficiency (OR = 0.31; CI = 0.10 – 0.97; p = 0.04). Participants with high AF-ALB or high AFM1 (≥ 437.95 pg/dL creatinine) were almost 6 times more likely to be hepatitis B virus surface antigen (HBsAg)- positive (OR = 5.88; CI = 1.71–20.14; p = 0.005) and (OR = 5.84; CI = 1.15–29.54; p = 0.03) respectively. Conclusions These data indicate that aflatoxin may modify plasma micronutrient status. Thus, preventing aflatoxin exposure may greatly reduce vitamins A and E deficiencies. PMID:21792816

Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

PubMed

Lowe, A M; Deresiewicz, R L

1999-01-01

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma▿†

PubMed Central

Cardoza, R. E.; Malmierca, M. G.; Hermosa, M. R.; Alexander, N. J.; McCormick, S. P.; Proctor, R. H.; Tijerino, A. M.; Rumbero, A.; Monte, E.; Gutiérrez, S.

2011-01-01

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function. PMID:21642405

De novo leaf and root transcriptome analysis to identify putative genes involved in triterpenoid saponins biosynthesis in Hedera helix L.

PubMed Central

Li, Fang; Xu, Zijian; Sun, Mengli; Cong, Hanqing; Qiao, Fei; Zhong, Xiaohong

2017-01-01

Hedera helix L. is an important traditional medicinal plant in Europe. The main active components are triterpenoid saponins, but none of the potential enzymes involved in triterpenoid saponins biosynthesis have been discovered and annotated. Here is reported the first study of global transcriptome analyses using the Illumina HiSeq™ 2500 platform for H. helix. In total, over 24 million clean reads were produced and 96,333 unigenes were assembled, with an average length of 1385 nt; more than 79,085 unigenes had at least one significant match to an existing gene model. Differentially Expressed Gene analysis identified 6,222 and 7,012 unigenes which were expressed either higher or lower in leaf samples when compared with roots. After functional annotation and classification, two pathways and 410 unigenes related to triterpenoid saponins biosynthesis were discovered. The accuracy of these de novo sequences was validated by RT-qPCR analysis and a RACE clone. These data will enrich our knowledge of triterpenoid saponin biosynthesis and provide a theoretical foundation for molecular research on H. helix. PMID:28771546

(-)-Menthol biosynthesis and molecular genetics

NASA Astrophysics Data System (ADS)

Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

2005-12-01

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

Transcriptional Regulator LsrB of Sinorhizobium meliloti Positively Regulates the Expression of Genes Involved in Lipopolysaccharide Biosynthesis

PubMed Central

Tang, Guirong; Wang, Ying

2014-01-01

Rhizobia induce nitrogen-fixing nodules on host legumes, which is important in agriculture and ecology. Lipopolysaccharide (LPS) produced by rhizobia is required for infection or bacteroid survival in host cells. Genes required for LPS biosynthesis have been identified in several Rhizobium species. However, the regulation of their expression is not well understood. Here, Sinorhizobium meliloti LsrB, a member of the LysR family of transcriptional regulators, was found to be involved in LPS biosynthesis by positively regulating the expression of the lrp3-lpsCDE operon. An lsrB in-frame deletion mutant displayed growth deficiency, sensitivity to the detergent sodium dodecyl sulfate, and acidic pH compared to the parent strain. This mutant produced slightly less LPS due to lower expression of the lrp3 operon. Analysis of the transcriptional start sites of the lrp3 and lpsCDE gene suggested that they constitute one operon. The expression of lsrB was positively autoregulated. The promoter region of lrp3 was specifically precipitated by anti-LsrB antibodies in vivo. The promoter DNA fragment containing TN11A motifs was bound by the purified LsrB protein in vitro. These new findings suggest that S. meliloti LsrB is associated with LPS biosynthesis, which is required for symbiotic nitrogen fixation on some ecotypes of alfalfa plants. PMID:24951786

The case for aflatoxins in the causal chain of gallbladder cancer.

PubMed

Foerster, Claudia; Koshiol, Jill; Guerrero, Ariel R; Kogan, Marcelo J; Ferreccio, Catterina

2016-01-01

Chronic aflatoxin exposure has long been related to hepatocellular carcinoma (HCC). Recently, its association with gallbladder cancer (GBC) was postulated. Here we present the data supporting this hypothesis in Chile, the country with the highest GBC mortality worldwide with age-standardized mortality rates (ASMR) of 10.3 in women and 5.04 in men. The highest GBC rates occur in Southern Chile (ASMR=18), characterized by: high Amerindian ancestry, associated with high bile acid synthesis and gallstones; high poverty and high cereal agriculture, both associated with aflatoxin exposure. Aflatoxins have been detected in imported and locally grown foods items. We estimated population dietary exposure ranging from 0.25 to 35.0 ng/kg-body weight/day. The only report on human exposure in Chile found significantly more aflatoxin biomarkers in GBC than in controls (Odds Ratio=13.0). The hypothesis of aflatoxin-GBC causal link in the Chilean population is supported by: genetically-determined rapid cholesterol excretion and high gallstones prevalence (49.4%); low prevalence of HCC (ASMR=4.9) and low HBV infection (0.15%) the main co-factor of aflatoxins in HCC risk. If the association between aflatoxins and GBC were confirmed, public health interventions based on food regulation could have a substantial public health impact. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras.

PubMed Central

Wei, C; Macy, J M; Hsieh, D P

1981-01-01

Rat cecal microflora from high- and low-fiber-fed animals hydrolyzed aflatoxin conjugates to metabolites indistinguishable from aflatoxin B1 and aflatoxin P1, but aflatoxicol was not a transformation product. PMID:6263185

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain.

PubMed Central

Moores, J C; Magazin, M; Ditta, G S; Leong, J

1984-01-01

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin. Images PMID:6690426

Some high-performance liquid-chromatographic studies of the metabolism of aflatoxins by rat liver microsomal preparations.

PubMed Central

Neal, G E; Colley, P J

1978-01-01

The metabolism of aflatoxin B1 in vitro was examined in rat liver microsomal preparations. 2. H.p.l.c. (high-performance liquid-chromatographic) systems were used. A silica column was used to separate non-polar metabolites. A system utilizing a reversed-phase column which separates both poar and non-polar metabolites was also developed. 3. The principal metabolites of aflatoxin B1 found were aflatoxin M1, aflatoxin Q1 and a compound which co-chromatographed with a degradation product of aflatoxin B1 2,3-dihydrodiol. 4. The time course of metabolism of aflatoxin B1 by microsomal preparations isolated from control and phenobarbitone-pretreated rats was examined. The rate and extent of metabolism was greater with microsomal preparations from the latter. The formation of aflatoxin Q1 was enhanced 4--5-fold by phenobarbitone pretreatment, whereas the production of aflatoxin M1 was only increased 1--2-fold. The formation of the degradation product of aflatoxin B1 2,3-dihydrodiol was increased 4--5-fold by the pretreatment with phenobarbitone. 5. The microsomal metabolism of aflatoxins M1, P1 and Q1 was examined. Aflatoxin M1 apparently underwent very limited microsomal metabolism to more polar compounds. Aflatoxin P1 was not metabolized. The situation with aflatoxin Q1 was complicated in that it was metabolized in the absence of NADPH to an unidentified metabolite. Aflatoxin B1 appeared as a metabolite of aflatoxin Q1 only when NADPH was present, and the formation of more polar metabolites was also then observed. PMID:728090

Crop stress and aflatoxin contamination: perspectives and prevention strategies.

USDA-ARS?s Scientific Manuscript database

The fungal metabolites called aflatoxins are potent naturally occurring carcinogens, produced primarily by Aspergillus flavus and A. parasiticus. A. flavus affects many agricultural crops such as maize, cotton, peanuts, and tree nuts. It can contaminate these crops with aflatoxins in the field befor...

Aflatoxin: A 50-Year Odyssey of Mechanistic and Translational Toxicology

PubMed Central

Kensler, Thomas W.; Roebuck, Bill D.; Wogan, Gerald N.; Groopman, John D.

2011-01-01

Since their discovery 50 years ago, the aflatoxins have become recognized as ubiquitous contaminants of the human food supply throughout the economically developing world. The adverse toxicological consequences of these compounds in populations are quite varied because of a wide range of exposures leading to acute effects, including rapid death, and chronic outcomes such as hepatocellular carcinoma. Furthermore, emerging studies describe a variety of general adverse health effects associated with aflatoxin, such as impaired growth in children. Aflatoxin exposures have also been demonstrated to multiplicatively increase the risk of liver cancer in people chronically infected with hepatitis B virus (HBV) illustrating the deleterious impact that even low toxin levels in the diet can pose for human health. The public health impact of aflatoxin exposure is pervasive. Aflatoxin biomarkers of internal and biologically effective doses have been integral to the establishment of the etiologic role of this toxin in human disease through better estimates of exposure, expanded knowledge of the mechanisms of disease pathogenesis, and as tools for implementing and evaluating preventive interventions. PMID:20881231

[Adsorption of aflatoxin on montmorillonite modified by low-molecular-weight humic acids].

PubMed

Yao, Jia-Jia; Kang, Fu-Xing; Gao, Yan-Zheng

2012-03-01

The adsorption of a typical biogenic toxin aflatoxin B1 on montmorillonite modified by low-molecular-weight humic acids (M(r) < 3 500) was investigated. The montmorillonite rapidly adsorbed the aflatoxin B1 until amounting to the maximal capacity, and then the adsorbed aflatoxin B1 slowly released into solution and reached the sorption equilibrium state after 12 h. The sorption isotherm of aflatoxin B1 by montmorillonite could be well described by Langmiur model, while the sorption isotherm by humic acid-modified montmorillonite was well fitted by using the Freundlich model. The modification of the montmorillonite with humic acids obviously enhanced its adsorption capacity for aflatoxin B1, and the amounts of aflatoxin adsorbed by modified montmorillonite were obviously higher than those by montmorillonite. The sorption enhancement by humic acid modification was attributed to (1) the enlarged adsorption sites which owed to the surface collapse of crystal layers induced by organic acids, and (2) the binding of aflatoxin with the humic acid sorbed on mineral surface. In addition, the adsorption amounts of aflatoxin by montmorillonite and modified montmorillonite increased with the increase of pH values in solution, and more significant enhancement was observed for the latter than the former, which attributed to the release of humic acids from the modified montmorillonite with the high pH values in solution. This indicates that increasing the pH values resulted in the enhanced hydrophilic property and the release of the organic acids presented in modified montmorillonite, and more sorption sites were available for aflatoxin on the modified montmorillonite. Results of this work would strengthen our understanding of the behavior and fate of biological contaminants in the environment.

Starch Biosynthesis during Pollen Maturation Is Associated with Altered Patterns of Gene Expression in Maize1

PubMed Central

Datta, Rupali; Chamusco, Karen C.; Chourey, Prem S.

2002-01-01

Starch biosynthesis during pollen maturation is not well understood in terms of genes/proteins and intracellular controls that regulate it in developing pollen. We have studied two specific developmental stages: “early,” characterized by the lack of starch, before or during pollen mitosis I; and “late,” an actively starch-filling post-pollen mitosis I phase in S-type cytoplasmic male-sterile (S-CMS) and two related male-fertile genotypes. The male-fertile starch-positive, but not the CMS starch-deficient, genotypes showed changes in the expression patterns of a large number of genes during this metabolic transition. In addition to a battery of housekeeping genes of carbohydrate metabolism, we observed changes in hexose transporter, plasma membrane H+-ATPase, ZmMADS1, and 14-3-3 proteins. Reduction or deficiency in 14-3-3 protein levels in all three major cellular sites (amyloplasts [starch], mitochondria, and cytosol) in male-sterile relative to male-fertile genotypes are of potential interest because of interorganellar communication in this CMS system. Further, the levels of hexose sugars were significantly reduced in male-sterile as compared with male-fertile tissues, not only at “early” and “late” stages but also at an earlier point during meiosis. Collectively, these data suggest that combined effects of both reduced sugars and their reduced flux in starch biosynthesis along with a strong possibility for altered redox passage may lead to the observed temporal changes in gene expressions, and ultimately pollen sterility. PMID:12481048

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

PubMed

Eklund, D Magnus; Thelander, Mattias; Landberg, Katarina; Ståldal, Veronika; Nilsson, Anders; Johansson, Monika; Valsecchi, Isabel; Pederson, Eric R A; Kowalczyk, Mariusz; Ljung, Karin; Ronne, Hans; Sundberg, Eva

2010-04-01

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

Hepatitis B virus x gene and cyanobacterial toxins promote aflatoxin B1-induced hepatotumorigenesis in mice

PubMed Central

Lian, Min; Liu, Ying; Yu, Shun-Zhang; Qian, Geng-Sun; Wan, Shu-Guang; Dixon, Kenneth R

2006-01-01

AIM: To assess the combinative role of aflatoxin B1 (AFB1), cyanobacterial toxins (cyanotoxins), and hepatitis B virus (HBV) x gene in hepatotumorigenicity. METHODS: One-week-old animals carrying HBV x gene and their wild-type littermates were intraperitoneally (ip) injected with either single-dose AFB1 [6 mg/kg body weight (bw)], repeated-dose cyanotoxins (microcystin-LR or nodularin, 10 μg/kg bw once a week for 15 wk), DMSO (vehicle control) alone, or AFB1 followed by cyanotoxins a week later, and were sacrificed at 24 and 52 wk post-treatment. RESULTS: AFB1 induced liver tumors in 13 of 29 (44.8%) transgenic mice at 52 wk post-treatment, significantly more frequent than in wild-type mice (13.3%). This significant difference was not shown in the 24-wk study. Compared with AFB1 exposure alone, MC-LR and nodularin yielded approximately 3-fold and 6-fold increases in the incidence of AFB1-induced liver tumors in wild-type animals at 24 wk, respectively. HBV x gene did not further elevate the risk associated with co-exposure to AFB1 and cyanotoxins. With the exception of an MC-LR-dosed wild-type mouse, no liver tumor was observed in mice treated with cyanotoxins alone at 24 wk. Neither DMSO-treated transgenic mice nor their wild-type littermates had pathologic alterations relevant to hepatotumorigenesis in even up to 52 wk. CONCLUSION: HBV x gene and nodularin promote the development of AFB1-induced liver tumors. Co-exposure to AFB1 and MC-LR tends to elevate the risk of liver tumors at 24 wk relative to exposure to one of them. The combinative effect of AFB1, cyanotoxins and HBVx on hepatotumorigenesis is weak at 24 wk. PMID:16718789

Two fatty acyl reductases involved in moth pheromone biosynthesis

PubMed Central

Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

2016-01-01

Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

The expanding universe of alkaloid biosynthesis.

PubMed

De Luca, V; Laflamme, P

2001-06-01

Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

Aflatoxin and outcome from acute lower respiratory infection in children in The Philippines.

PubMed

Denning, D W; Quiepo, S C; Altman, D G; Makarananda, K; Neal, G E; Camallere, E L; Morgan, M R; Tupasi, T E

1995-09-01

Aflatoxin is immunosuppressive in experimental conditions. This study addressed its potentially contributory role in the poor outcome of acute lower respiratory infections (ALRI) in children in The Philippines. The catchment area included peri-urban slums and middle-class housing. One hundred and fifteen children (mean age 2.1, range 0.08-12 years) were enrolled and their serum and urine obtained at presentation with ALRI. Aflatoxins in serum and aflatoxin metabolites in urine were measured by previously validated ELISA tests. Using the 1986 WHO criteria for the severity of ALRI, 31% had mild, 12% moderate, 49% severe and 8% severe complicated pneumonia. Eighty of 97 (82%) chest radiographs were abnormal. Ninety per cent of the children were below average weight for age, using Filipino standards, with a mean of 79% (range 27-157%). Thirteen (11%) children died. Aflatoxin in their serum, reflecting recent ingestion, was detected in 33%, with a mean positive value of 462 pg/ml. Aflatoxin metabolites (reflecting chronic ingestion) were detected in 64 of 65 urines collected, with a mean value of 0.1-4.77ng/ml. None of the children with detectable serum aflatoxin died. Anorexia and impaired consciousness were strongly associated with a poor outcome (prolonged fever or death). There was a strong association between undetectable serum aflatoxin concentrations and death (p = 0.004), perhaps reflecting anorexia. There was no relationship between the concentration of urinary aflatoxin metabolites and outcome. Serum was also obtained from 29 mothers on admission and none contained detectable aflatoxin. As virtually all the children had evidence of exposure to aflatoxin, a potentially immunosuppressive role in the context of pneumonia cannot be excluded.

Morphological characteristics, anatomical structure, and gene expression: novel insights into gibberellin biosynthesis and perception during carrot growth and development.

PubMed

Wang, Guang-Long; Xiong, Fei; Que, Feng; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

2015-01-01

Gibberellins (GAs) are considered potentially important regulators of cell elongation and expansion in plants. Carrot undergoes significant alteration in organ size during its growth and development. However, the molecular mechanisms underlying gibberellin accumulation and perception during carrot growth and development remain unclear. In this study, five stages of carrot growth and development were investigated using morphological and anatomical structural techniques. Gibberellin levels in leaf, petiole, and taproot tissues were also investigated for all five stages. Gibberellin levels in the roots initially increased and then decreased, but these levels were lower than those in the petioles and leaves. Genes involved in gibberellin biosynthesis and signaling were identified from the carrotDB, and their expression was analyzed. All of the genes were evidently responsive to carrot growth and development, and some of them showed tissue-specific expression. The results suggested that gibberellin level may play a vital role in carrot elongation and expansion. The relative transcription levels of gibberellin pathway-related genes may be the main cause of the different bioactive GAs levels, thus exerting influences on gibberellin perception and signals. Carrot growth and development may be regulated by modification of the genes involved in gibberellin biosynthesis, catabolism, and perception.

Regulation of Oil Biosynthesis in Algae

DTIC Science & Technology

2011-03-14

transportation fuels can potentially be addressed by exploring oil (triacylglycerol) biosynthesis in microalgae . Many microalgae , including Chlamydomonas...biosynthesis in microalgae have not been studied at the molecular level. Chlamydomonas is being used as a microalgal model to identify genes and regulatory...of this phenomenon will shed light on the physiological significance of oil production in microalgae . A first paper describing this interesting

The Ethylene Biosynthesis Gene CitACS4 Regulates Monoecy/Andromonoecy in Watermelon (Citrullus lanatus)

PubMed Central

Manzano, Susana; Aguado, Encarnación; Martínez, Cecilia; Megías, Zoraida; García, Alicia; Jamilena, Manuel

2016-01-01

Monoecious and andromonoecious cultivars of watermelon are characterised by the production of male and female flower or male and hermaphrodite flowers, respectively. The segregation analysis in the offspring of crosses between monoecious and andromonoecious lines has demonstrated that this trait is controlled by a single gene pair, being the monoecious allele M semi-dominant to the andromonoecious allele A. The two studied F1 hybrids (MA) had a predominantly monoecious phenotype since both produced not only female flowers, but also bisexual flowers with incomplete stamens, and hermaphrodite flowers with pollen. Given that in other cucurbit species andromonoecy is conferred by mutations in the ethylene biosynthesis genes CmACS7, CsACS2 and CpACS27A we have cloned and characterised CitACS4, the watermelon gene showing the highest similarity with the formers. CitACS4 encoded for a type ACS type III enzyme that is predominantly expressed in pistillate flowers of watermelon. In the andromonoecious line we have detected a missense mutation in a very conserved residue of CitACS4 (C364W) that cosegregates with the andromonoecious phenotype in two independent F2 populations, concomitantly with a reduction in ethylene production in the floral buds that will develop as hermaphrodite flowers. The gene does not however co-segregates with other sex expression traits regulated by ethylene in this species, including pistillate flowering transition and the number of pistillate flowers per plant. These data indicate that CitAC4 is likely to be involved in the biosynthesis of the ethylene required for stamen arrest during the development of female flowers. The C364W mutation would reduce the production of ethylene in pistillate floral buds, promoting the conversion of female into hermaphrodite flowers, and therefore of monoecy into andromonoecy. PMID:27149159

Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

PubMed

Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

2015-02-01

Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis. © 2014 Scandinavian Plant Physiology Society.

Aspergillus section Flavi community structure in Zambia influences aflatoxin contamination of maize and groundnut.

PubMed

Kachapulula, Paul W; Akello, Juliet; Bandyopadhyay, Ranajit; Cotty, Peter J

2017-11-16

Aflatoxins are cancer-causing, immuno-suppressive mycotoxins that frequently contaminate important staples in Zambia including maize and groundnut. Several species within Aspergillus section Flavi have been implicated as causal agents of aflatoxin contamination in Africa. However, Aspergillus populations associated with aflatoxin contamination in Zambia have not been adequately detailed. Most of Zambia's arable land is non-cultivated and Aspergillus communities in crops may originate in non-cultivated soil. However, relationships between Aspergillus populations on crops and those resident in non-cultivated soils have not been explored. Because characterization of similar fungal populations outside of Zambia have resulted in strategies to prevent aflatoxins, the current study sought to improve understanding of fungal communities in cultivated and non-cultivated soils and in crops. Crops (n=412) and soils from cultivated (n=160) and non-cultivated land (n=60) were assayed for Aspergillus section Flavi from 2012 to 2016. The L-strain morphotype of Aspergillus flavus and A. parasiticus were dominant on maize and groundnut (60% and 42% of Aspergillus section Flavi, respectively). Incidences of A. flavus L-morphotype were negatively correlated with aflatoxin in groundnut (log y=2.4990935-0.09966x, R 2 =0.79, P=0.001) but not in maize. Incidences of A. parasiticus partially explained groundnut aflatoxin concentrations in all agroecologies and maize aflatoxin in agroecology III (log y=0.1956034+0.510379x, R 2 =0.57, P<0.001) supporting A. parasiticus as the dominant etiologic agent of aflatoxin contamination in Zambia. Communities in both non-cultivated and cultivated soils were dominated by A. parasiticus (69% and 58%, respectively). Aspergillus parasiticus from cultivated and non-cultivated land produced statistically similar concentrations of aflatoxins. Aflatoxin-producers causing contamination of crops in Zambia may be native and, originate from non-cultivated areas

Altered expression of genes involved in progesterone biosynthesis, metabolism and action in endometrial cancer.

PubMed

Sinreih, Maša; Hevir, Neli; Rižner, Tea Lanišnik

2013-02-25

Endometrial cancer (EC) is one of the most common gynecological malignancies worldwide. It is associated with prolonged exposure to estrogens that is unopposed by the protective effects of progesterone, which suggests that altered progesterone biosynthesis, metabolism and actions might be implicated in the development of EC. Our aim was to evaluate these processes through quantitative real-time PCR expression analysis in up to 47 pairs of EC tissue and adjacent control endometrium. First, we examined the expression of genes encoding proteins associated with progesterone biosynthesis: steroidogenic acute regulatory protein (STAR); a side chain cleavage enzyme (CYP11A1); and 3β-hydroxysteroid dehydrogenase/ketosteroid isomerase (HSD3B). There were 1.9- and 10.0-fold decreased expression of STAR and CYP11A1, respectively, in EC versus adjacent control endometrium, with no significant differences in the expression of HSD3B1 and HSD3B2. Next, we examined expression of genes encoding five progesterone metabolizing enzymes: the 3-keto and 20-ketosteroid reductases (AKR1C1-AKR1C3) and 5α-reductases (SRD5A1 and SRD5A2); and the opposing 20α-hydroxysteroid dehydrogenase (HSD17B2). These genes are expressed in EC and adjacent control endometrium. No statistically significant differences were seen in mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1. Expression of HSD17B2 was 3.0-fold increased, and expression of SRD5A2 was 3.7-fold decreased, in EC versus adjacent control endometrium. We also examined mRNA levels of progesterone receptors A and B (PGR), and separately the expression of progesterone receptor B (PR-B). Here we saw 1.8- and 2.0-fold lower mRNA levels of PGR and PR-B, respectively, in EC versus adjacent control endometrium. This down-regulation of STAR, CYP11A1 and PGR in endometrial cancer may lead to decreased progesterone biosynthesis and actions although the effects on progesterone levels should be further studied. Copyright © 2012 Elsevier Ireland Ltd

Assessing airborne aflatoxin B1 during on-farm grain handling activities.

PubMed

Selim, M I; Juchems, A M; Popendorf, W

1998-04-01

The presence of aflatoxin in corn and corn dust during relatively normal years and the increased risk of Aspergillus flavus infestation during drought conditions suggest that airborne agricultural exposures should be of considerable concern. Liquid extraction, thin layer chromatography, and high pressure liquid chromatography were used for the analysis of aflatoxin B1 in grain dust and bulk corn samples. A total of 24 samples of airborne dust were collected from 8 farms during harvest, 22 samples from 9 farms during animal feeding, and 14 sets of Andersen samples from 11 farms during bin cleaning. A total of 14 samples of settled dust and 18 samples of bulk corn were also collected and analyzed. The airborne concentration of aflatoxin B1 found in dust collected during harvest and grain unloading ranged from 0.04 to 92 ng/m3. Higher levels of aflatoxin B1 were found in the airborne dust samples collected from enclosed animal feeding buildings (5-421 ng/m3) and during bin cleaning (124-4849 ng/m3). Aflatoxin B1 up to 5100 ng/g were detected in settled dust collected from an enclosed animal feeding building; however, no apparent correlation was found between the airborne concentration of aflatoxin B1 and its concentration in settled dust or bulk corn. The data demonstrate that farmers and farm workers may be exposed to potentially hazardous concentrations of aflatoxin B1, particularly during bin cleaning and animal feeding in enclosed buildings.

Determination of aflatoxins in by-products of industrial processing of cocoa beans.

PubMed

Copetti, Marina V; Iamanaka, Beatriz T; Pereira, José Luiz; Lemes, Daniel P; Nakano, Felipe; Taniwaki, Marta H

2012-01-01

This study has examined the occurrence of aflatoxins in 168 samples of different fractions obtained during the processing of cocoa in manufacturing plants (shell, nibs, mass, butter, cake and powder) using an optimised methodology for cocoa by-products. The method validation was based on selectivity, linearity, limit of detection and recovery. The method was shown to be adequate for use in quantifying the contamination of cocoa by aflatoxins B(1), B(2), G(1) and G(2). Furthermore, the method was easier to use than other methods available in the literature. For aflatoxin extraction from cocoa samples, a methanol-water solution was used, and then immunoaffinity columns were employed for clean-up before the determination by high-performance liquid chromatography. A survey demonstrated a widespread occurrence of aflatoxins in cocoa by-products, although in general the levels of aflatoxins present in the fractions from industrial processing of cocoa were low. A maximum aflatoxin contamination of 13.3 ng g(-1) was found in a nib sample. The lowest contamination levels were found in cocoa butter. Continued monitoring of aflatoxins in cocoa by-products is nevertheless necessary because these toxins have a high toxicity to humans and cocoa is widely consumed by children through cocoa-containing products, like candies.

A Genomics Approach to Deciphering Lignin Biosynthesis in Switchgrass[W

PubMed Central

Shen, Hui; Mazarei, Mitra; Hisano, Hiroshi; Escamilla-Trevino, Luis; Fu, Chunxiang; Pu, Yunqiao; Rudis, Mary R.; Tang, Yuhong; Xiao, Xirong; Jackson, Lisa; Li, Guifen; Hernandez, Tim; Chen, Fang; Ragauskas, Arthur J.; Stewart, C. Neal; Wang, Zeng-Yu; Dixon, Richard A.

2013-01-01

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species. PMID:24285795

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana.

PubMed

Mphande, Fingani A; Siame, Bupe A; Taylor, Joanne E

2004-01-01

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.

Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA

PubMed Central

Enami, Kazuhiko; Ozawa, Tomoki; Motohashi, Noriko; Nakamura, Masayuki; Tanaka, Kan; Hanaoka, Mitsumasa

2011-01-01

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. PMID:21771917

Origin of the Allyl Group in FK506 Biosynthesis*

PubMed Central

Goranovič, Dušan; Kosec, Gregor; Mrak, Peter; Fujs, Štefan; Horvat, Jaka; Kuščer, Enej; Kopitar, Gregor; Petković, Hrvoje

2010-01-01

FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex. PMID:20194504

Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

PubMed Central

Paget, Vincent; Lechevrel, Mathilde; André, Véronique; Le Goff, Jérémie; Pottier, Didier; Billet, Sylvain; Garçon, Guillaume; Shirali, Pirouz; Sichel, François

2012-01-01

Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B1 exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B1 and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers. PMID:22319594

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development.

PubMed

Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L; Vega-Sánchez, Miguel E; Williams, Brian; Chiniquy, Dawn M; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G; Willats, William G T; Scheller, Henrik V; Ronald, Pamela C; Bartley, Laura E

2016-10-01

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Aflatoxin contamination in corn sold for wildlife feed in texas.

PubMed

Dunham, Nicholas R; Peper, Steven T; Downing, Carson D; Kendall, Ronald J

2017-05-01

Supplemental feeding with corn to attract and manage deer is a common practice throughout Texas. Other species, including northern bobwhites (Colinus virginianus), are commonly seen feeding around supplemental deer feeders. In many cases, supplemental feeding continues year-round so feed supply stores always have supplemental corn in stock. Fluctuating weather and improper storage of corn can lead to and/or amplify aflatoxin contamination. Due to the recent decline of bobwhites throughout the Rolling Plains ecoregion of Texas, there has been interest in finding factors such as toxins that could be linked to their decline. In this study, we purchased and sampled supplemental corn from 19 locations throughout this ecoregion to determine if aflatoxin contamination was present in individual bags prior to being dispersed to wildlife. Of the 57 bags sampled, 33 bags (approximately 58%) contained aflatoxin with a bag range between 0.0-19.91 parts per billion (ppb). Additionally, three metal and three polypropylene supplemental feeders were each filled with 45.4 kg of triple cleaned corn and placed in an open field to study long-term aflatoxin buildup. Feeders were sampled every 3 months from November 2013-November 2014. Average concentration of aflatoxin over the year was 4.08 ± 2.53 ppb (±SE) in metal feeders, and 1.43 ± 0.89 ppb (±SE) in polypropylene feeders. The concentration of aflatoxins is not affected by the type of feeder (metal vs polypropylene), the season corn was sampled, and the location in the feeder (top, middle, bottom) where corn is sampled. It is unlikely that corn used in supplemental feeders is contributing to the bobwhite decline due to the low levels of aflatoxin found in purchased corn and long-term storage of corn used in supplemental feeders.

Prevalence of Aflatoxin Contamination in Herbs and Spices in Different Regions of Iran

PubMed Central

KHAZAELI, Payam; MEHRABANI, Mitra; HEIDARI, Mahmoud Reza; ASADIKARAM, Gholamreza; LARI NAJAFI, Moslem

2017-01-01

Background: Mycotoxins are natural toxins, produced by several fungal species and are associated with morbidity or even mortality in animals, plants, and humans. In this study, 120 samples of herbs and spices in both bulk and packaged forms were prepared in order to measure aflatoxin level in different regions of Iran Methods: The aflatoxin was extracted during Mar to May 2015, using 80% methanol and then purified via immunoaffinity column. Measurements were performed, using high-performance liquid chromatography, equipped with a fluorescence detection system at excitation and emission wavelengths of 365 and 435 nm, respectively. Results: The highest prevalence of aflatoxin contamination in food products was attributed to aflatoxin B1 (30.8%). In addition, the highest prevalence of aflatoxin contamination was reported in red pepper (100%). Examination of effective factors indicated the substantial impact of moisture on aflatoxin level (P=0.046). Conclusion: Even at low levels of aflatoxin, contamination could be a serious threat, given the prevalent use of spices (either raw or not) as ingredients in food preparation. Therefore, regular monitoring of spices, especially chili pepper, is highly recommended. PMID:29167773

Global population structure and adaptive evolution of aflatoxin-producing fungi

USDA-ARS?s Scientific Manuscript database

We employed interspecific principal component analyses for six different categories (geography, species, precipitation, temperature, aflatoxin chemotype profile, and mating type) and inferred maximum likelihood phylogenies for six combined loci, including two aflatoxin cluster regions (aflM/alfN and...

Fatty acid cosubstrates provide β-oxidation precursors for rhamnolipid biosynthesis in Pseudomonas aeruginosa, as evidenced by isotope tracing and gene expression assays.

PubMed

Zhang, Lin; Veres-Schalnat, Tracey A; Somogyi, Arpad; Pemberton, Jeanne E; Maier, Raina M

2012-12-01

Rhamnolipids have multiple potential applications as "green" surfactants for industry, remediation, and medicine. As a result, they have been intensively investigated to add to our understanding of their biosynthesis and improve yields. Several studies have noted that the addition of a fatty acid cosubstrate increases rhamnolipid yields, but a metabolic explanation has not been offered, partly because biosynthesis studies to date have used sugar or sugar derivatives as the carbon source. The objective of this study was to investigate the role of fatty acid cosubstrates in improving rhamnolipid biosynthesis. A combination of stable isotope tracing and gene expression assays was used to identify lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis when fatty acid cosubstrates are present. To this end, we compared the rhamnolipids produced and their yields using either glucose alone or glucose and octadecanoic acid-d(35) as cosubstrates. Using a combination of sugar and fatty acids, the rhamnolipid yield was significantly higher (i.e., doubled) than when glucose was used alone. Two patterns of deuterium incorporation (either 1 or 15 deuterium atoms) in a single Rha-C(10) lipid chain were observed for octadecanoic acid-d(35) treatment, indicating that in the presence of a fatty acid cosubstrate, both de novo fatty acid synthesis and β-oxidation are used to provide lipid precursors for rhamnolipids. Gene expression assays showed a 200- to 600-fold increase in the expression of rhlA and rhlB rhamnolipid biosynthesis genes and a more modest increase of 3- to 4-fold of the fadA β-oxidation pathway gene when octadecanoic acid was present. Taken together, these results suggest that the simultaneous use of de novo fatty acid synthesis and β-oxidation pathways allows for higher production of lipid precursors, resulting in increased rhamnolipid yields.

[Cloning and identification of the priming glycosyltransferase gene involved in exopolysaccharide 139A biosynthesis in Streptomyces].

PubMed

Wang, Ling-Yan; Li, Shi-Tao; Guo, Lian-Hong; Jiang, Rong; Li, Yuan

2003-08-01

Recently in our laboratory, Streptomyces sp. 139 has been identified to produce a new exopolysaccharide designated EPS 139A that shows anti-rheumatic arthritis activity. The strategy of studying EPS 139A biosynthesis is to clone the key gene in the EPS biosynthesis pathway, i.e. the priming glycosyltransferase gene catalyzing the first step of nucleotide sugar transfer. Degenerate primers-based PCR approach was adopted to isolate the putative priming glycosyltransferase gene in Streptomyces sp. 139. According to the genes encoding the priming glycosyltransferases that have been identified in several microorganisms, a multiple alignment of the amino acid sequences of these genes was used to identify regions conserved between all genes. To clone the priming glycosyltransferase gene in Streptomyces sp. 139, degenerate primers were designed from these conserved regions taking into account information on Streptomyces codon usage to amplify an internal DNA fragment of this gene. A distinctive PCR product with the expected size of 0.3 kb was amplified from Streptomyces sp. 139 total genomic DNA. Sequence analysis showed that it is part of a putative priming glycosyltransferase gene and contains the predicted conserved domain B. To isolate the complete priming glycosyltransferase gene, a Streptomyces sp. 139 genomic library was constructed in the E. coli--Streptomyces shuttle vector pOJ446. Using the 0.3 kb PCR product of priming glycosyltransferase gene as a probe, 17 positive colonies were isolated by colony hybridization. A 4.0 kb BamHI fragment from all positive cosmids that hybridized to this probe was sequenced, which revealed the complete priming glycosyltransferase gene. The priming glycosyltransferase gene ste5 (GenBank under accession number AY131229) most likely begins with GTG, preceded by a probable ribosome binding site (RBS), GGGGA. It encodes a 492-amino-acid protein with molecular weight of 54 kDa and isoelectric point of 10.6. The G + C content of ste5 is