Structure and Chemical Analysis of Major Specialized Metabolites Produced by the Lichen Evernia prunastri.

We performed comparative profiling of four specialized metabolites in the lichen Evernia prunastri, collected at three different geographic locations, California and Maine, USA, and Yoshkar Ola, Mari El, Russia. Among the compounds produced at high concentrations that were identified in all three specimens, evernic acid, usnic acid, lecanoric acid and chloroatranorin, evernic acid was the most abundant. Two depsidones, salazinic acid and physodic acid, were detected in the Yoshkar-Ola collection only. The crystalline structure of evernic acid (2-hydroxy-4-[(2-hydroxy-4-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoate) (hmb) revealed two crystallographically and conformationally distinct hmb anions, along with two monovalent sodium atoms. One hmb moiety contained an exotetradentate binding mode to sodium, whereas the other exhibited an exohexadentate binding mode to sodium. Embedded edge-sharing {Na2 O8 }n sodium-oxygen chains connected the hmb anions into the full three-dimensional crystal structure of the title compound. The crystal used for single-crystal X-ray diffraction exhibited non-merohedral twinning. The data suggest the importance of the acetyl-polymalonyl pathway products to processes of maintaining integrity of the lichen holobiont community.

RNA Seq analysis of the role of calcium chloride stress and electron transport in mitochondria for malachite green decolorization by Aspergillus niger.

Aspergillus niger was previously demonstrated to decolorize the commercial dye malachite green (MG) and this process was enhanced under calcium chloride (CaCl2) treatment. Previous data also suggested that the decolorization process is related to mitochondrial cytochrome c. In the current work, we analyzed in depth the specific relationship between CaCl2 treatment and MG decolorization. Gene expression analysis (RNA Seq) using Next Generation Sequencing (NGS) revealed up-regulation of 28 genes that are directly or indirectly associated with stress response functions as early as 30min of CaCl2 treatment; these data further strengthen our previous findings that CaCl2 treatment induces a stress response in A. niger which enhances the ability to decolorize MG. A significant increase in fluorescence observed by MitoTracker dye suggests that CaCl2 treatment also increased mitochondrial membrane potential. Isolated mitochondrial membrane protein fractions obtained from A. niger grown under standard growth conditions decolorized MG in the presence of NADH and decolorization was enhanced in samples isolated from CaCl2-treated A. niger cultures. Treatment of whole mitochondrial fraction with KCN which inhibits electron transport by cytochrome c oxidase and Triton-X 100 which disrupts mitochondrial membrane integrity suggests that cyanide sensitive cytochrome c oxidase activity is a key biochemical step in MG decolorization. This suggestion was confirmed by the addition of palladium α-lipoic acid complex (PLAC) which resulted in an initial increase in decolorization. Although the role of cytochrome c and cytochrome c oxidase was confirmed at the biochemical level, changes in levels of transcripts encoding these enzymes after CaCl2 treatment were not found to be statistically significant in RNA Seq analysis. These data suggest that the regulation of cytochrome c enzymes occur predominantly at the post-transcriptional level under CaCl2 stress. Thus, using global transcriptomics and biochemical approaches, our study provides a molecular association between fungal mitochondrial electron transfer systems and MG decolorization.

Aspergillus parasiticus SU-1 genome sequence, predicted chromosome structure, and comparative gene expression under aflatoxin-inducing conditions: evidence that differential expression contributes to species phenotype.

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus produce the carcinogenic secondary metabolite aflatoxin on susceptible crops. These species differ in the quantity of aflatoxins B1, B2, G1, and G2 produced in culture, in the ability to produce the mycotoxin cyclopiazonic acid, and in morphology of mycelia and conidiospores. To understand the genetic basis for differences in biochemistry and morphology, we conducted next-generation sequence (NGS) analysis of the A. parasiticus strain SU-1 genome and comparative gene expression (RNA sequence analysis [RNA Seq]) analysis of A. parasiticus SU-1 and A. flavus strain NRRL 3357 (3357) grown under aflatoxin-inducing and -noninducing culture conditions. Although A. parasiticus SU-1 and A. flavus 3357 are highly similar in genome structure and gene organization, we observed differences in the presence of specific mycotoxin gene clusters and differential expression of specific mycotoxin genes and gene clusters that help explain differences in the type and quantity of mycotoxins synthesized. Using computer-aided analysis of secondary metabolite clusters (antiSMASH), we demonstrated that A. parasiticus SU-1 and A. flavus 3357 may carry up to 93 secondary metabolite gene clusters, and surprisingly, up to 10% of the genome appears to be dedicated to secondary metabolite synthesis. The data also suggest that fungus-specific zinc binuclear cluster (C6) transcription factors play an important role in regulation of secondary metabolite cluster expression. Finally, we identified uniquely expressed genes in A. parasiticus SU-1 that encode C6 transcription factors and genes involved in secondary metabolism and stress response/cellular defense. Future work will focus on these differentially expressed A. parasiticus SU-1 loci to reveal their role in determining distinct species characteristics.

VeA is associated with the response to oxidative stress in the aflatoxin producer Aspergillus flavus.

Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms to counteract and survive the stress in the presence of ROS. In many fungi, the HOG signaling pathway is crucial for the oxidative stress response as well as for osmotic stress response. This study revealed that while the osmotic stress response is only slightly affected by the master regulator veA, this gene, also known to control morphological development and secondary metabolism in numerous fungal species, has a profound effect on the oxidative stress response in the aflatoxin-producing fungus Aspergillus flavus. We found that the expression of A. flavus homolog genes involved in the HOG signaling pathway is regulated by veA. Deletion of veA resulted in a reduction in transcription levels of oxidative stress response genes after exposure to hydrogen peroxide. Furthermore, analyses of the effect of VeA on the promoters of cat1 and trxB indicate that the presence of VeA alters DNA-protein complex formation. This is particularly notable in the cat1 promoter, where the absence of VeA results in abnormally stronger complex formation with reduced cat1 expression and more sensitivity to ROS in a veA deletion mutant, suggesting that VeA might prevent binding of negative transcription regulators to the cat1 promoter. Our study also revealed that veA positively influences the expression of the transcription factor gene atfB and that normal formation of DNA-protein complexes in the cat1 promoter is dependent on AtfB.

A volatile relationship: profiling an inter-kingdom dialogue between two plant pathogens, Ralstonia Solanacearum and Aspergillus Flavus.

Microbes in the rhizosphere have a suite of extracellular compounds, both primary and secondary, that communicate with other organisms in their immediate environment. Here, we describe a two-way volatile interaction between two widespread and economically important soil-borne pathogens of peanut, Aspergillus flavus and Ralstonia solanacearum, a fungus and bacterium, respectively. In response to A. flavus volatiles, R. solanacearum reduced production of the major virulence factor extracellular polysaccharide (EPS). In parallel, A. flavus responded to R. solanacearum volatiles by reducing conidia production, both on plates and on peanut seeds and by increasing aflatoxin production on peanut. Volatile profiling of these organisms using solid-phase micro-extraction gas chromatography mass spectroscopy (SPME-GCMS) provided a first glimpse at the compounds that may drive these interactions.

A "successful allele" at Campylobacter jejuni contingency locus Cj0170 regulates motility; "successful alleles" at locus Cj0045 are strongly associated with mouse colonization.

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific "successful alleles" at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a "successful"Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the "unsuccessful"Cj0045 11G allele experienced phase variation to "successful" 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the "successful" 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice.

Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism in Aspergillus parasiticus.

Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus Aspergillus parasiticus carry enzymes that catalyze the final two steps in aflatoxin synthesis, and these structures also play a role in aflatoxin storage and export. We hypothesized that endosomes house a complete and functional aflatoxin biosynthetic pathway. To address this hypothesis, we purified a cellular fraction containing endosomes, transport vesicles, and vacuoles (V fraction) from A. parasiticus grown under aflatoxin inducing and noninducing conditions. We also added (fed) aflatoxin pathway intermediates to V fraction to test the functional status of aflatoxin pathway enzymes. High throughput LC-MS/MS analysis of proteins in V fraction detected 8 aflatoxin enzymes with high reliability and 8 additional enzymes at lower reliability, suggesting that most aflatoxin pathway enzymes are present. Purified V fraction synthesized aflatoxin and addition of the pathway intermediate versicolorin A increased aflatoxin synthesis, confirming that middle and late aflatoxin enzymes in V fraction are functional. Of particular significance, proteomic and biochemical analysis strongly suggested that additional secondary metabolic pathways as well as proteins involved in response to heat, osmotic, and oxidative stress are housed in V fraction.

Stress-related transcription factor AtfB integrates secondary metabolism with oxidative stress response in aspergilli.

In filamentous fungi, several lines of experimental evidence indicate that secondary metabolism is triggered by oxidative stress; however, the functional and molecular mechanisms that mediate this association are unclear. The basic leucine zipper (bZIP) transcription factor AtfB, a member of the bZIP/CREB family, helps regulate conidial tolerance to oxidative stress. In this work, we investigated the role of AtfB in the connection between oxidative stress response and secondary metabolism in the filamentous fungus Aspergillus parasiticus. This well characterized model organism synthesizes the secondary metabolite and carcinogen aflatoxin. Chromatin immunoprecipitation with specific anti-AtfB demonstrated AtfB binding at promoters of seven genes in the aflatoxin gene cluster that carry CREs. Promoters lacking CREs did not show AtfB binding. The binding of AtfB to the promoters occurred under aflatoxin-inducing but not under aflatoxin-noninducing conditions and correlated with activation of transcription of the aflatoxin genes. Deletion of veA, a global regulator of secondary metabolism and development, nearly eliminated this binding. Electrophoretic mobility shift analysis demonstrated that AtfB binds to the nor-1 (an early aflatoxin gene) promoter at a composite regulatory element that consists of highly similar, adjacent CRE1 and AP-1-like binding sites. The five nucleotides immediately upstream from CRE1, AGCC(G/C), are highly conserved in five aflatoxin promoters that demonstrate AtfB binding. We propose that AtfB is a key player in the regulatory circuit that integrates secondary metabolism and cellular response to oxidative stress.

Passage of Campylobacter jejuni through the chicken reservoir or mice promotes phase variation in contingency genes Cj0045 and Cj0170 that strongly associates with colonization and disease in a mouse model.

Human illness due to Camplyobacter jejuni infection is closely associated with consumption of poultry products. We previously demonstrated a 50 % shift in allele frequency (phase variation) in contingency gene Cj1139 (wlaN) during passage of C. jejuni NCTC11168 populations through Ross 308 broiler chickens. We hypothesized that phase variation in contingency genes during chicken passage could promote subsequent colonization and disease in humans. To test this hypothesis, we passaged C. jejuni strains NCTC11168, 33292, 81-176, KanR4 and CamR2 through broiler chickens and analysed the ability of passaged and non-passaged populations to colonize C57BL6 IL-10-deficient mice, our model for human colonization and disease. We utilized fragment analysis and nucleotide sequence analysis to measure phase variation in contingency genes. Passage through the chicken reservoir promoted phase variation in five specific contingency genes, and these 'successful' populations colonized mice. When phase variation did not occur in these same five contingency genes during chicken passage, these 'unsuccessful' populations failed to colonize mice. Phase variation during chicken passage generated small insertions or deletions (indels) in the homopolymeric tract (HT) in contingency genes. Single-colony isolates of C. jejuni strain KanR4 carrying an allele of contingency gene Cj0170 with a10G HT colonized mice at high frequency and caused disease symptoms, whereas single-colony isolates carrying the 9G allele failed to colonize mice. Supporting results were observed for the successful 9G allele of Cj0045 in strain 33292. These data suggest that phase variation in Cj0170 and Cj0045 is strongly associated with mouse colonization and disease, and that the chicken reservoir can play an active role in natural selection, phase variation and disease.

A key role for vesicles in fungal secondary metabolism.

Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B(1); these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.

Compartmentalization and molecular traffic in secondary metabolism: a new understanding of established cellular processes.

Great progress has been made in understanding the regulation of expression of genes involved in secondary metabolism. Less is known about the mechanisms that govern the spatial distribution of the enzymes, cofactors, and substrates that mediate catalysis of secondary metabolites within the cell. Filamentous fungi in the genus Aspergillus synthesize an array of secondary metabolites and provide useful systems to analyze the mechanisms that mediate the temporal and spatial regulation of secondary metabolism in eukaryotes. For example, aflatoxin biosynthesis in Aspergillus parasiticus has been studied intensively because this mycotoxin is highly toxic, mutagenic, and carcinogenic in humans and animals. Using aflatoxin synthesis to illustrate key concepts, this review focuses on the mechanisms by which sub-cellular compartmentalization and intra-cellular molecular traffic contribute to the initiation and completion of secondary metabolism within the cell. We discuss the recent discovery of aflatoxisomes, specialized trafficking vesicles that participate in the compartmentalization of aflatoxin synthesis and export of the toxin to the cell exterior; this work provides a new and clearer understanding of how cells integrate secondary metabolism into basic cellular metabolism via the intra-cellular trafficking machinery.

Willow volatiles influence growth, development, and secondary metabolism in Aspergillus parasiticus.

Aflatoxin is a mycotoxin and the most potent naturally occurring carcinogen in many animals. Aflatoxin contamination of food and feed crops causes a significant global burden on human and animal health. However, available methods to eliminate aflatoxin from food and feed are not fully effective. Our goal is to discover novel, efficient, and practical methods to control aflatoxin contamination in crops during storage. In the present study, we tested the effect of volatiles produced by willow (Salix acutifolia and Salix babylonica) and maple (Acer saccharinum) bark on fungal growth, development, and aflatoxin production by the fungus Aspergillus parasiticus, one economically important aflatoxin producer. S. acutifolia bark volatiles nearly eliminated aflatoxin accumulation (>90% reduction) by A. parasiticus grown on a minimal agar medium. The decrease in aflatoxin accumulation correlated with a twofold reduction in ver-1 (encodes a middle aflatoxin pathway enzyme) transcript level. Expression data also indicate that one histone H4 acetyltransferase, MYST3, may play a role in epigenetic control of aflatoxin gene transcription in response to volatile exposure. Volatiles derived from wood bark samples also increased fungal growth up to 20% and/or enhanced conidiospore development. Solid-phase microextraction-gas chromatographic-mass spectrometric analysis of bark samples identified sets of shared and unique volatile compounds that may mediate the observed regulatory effects on growth, development, and aflatoxin synthesis. This work provides an experimental basis for the use of willow industry by-products to control aflatoxin contamination in food and feed crops.

A possible role for exocytosis in aflatoxin export in Aspergillus parasiticus.

Filamentous fungi synthesize bioactive secondary metabolites with major human health and economic impacts. Little is known about the mechanisms that mediate the export of these metabolites to the cell exterior. Aspergillus parasiticus synthesizes aflatoxin, a secondary metabolite that is one of the most potent naturally occurring carcinogens known. We previously demonstrated that aflatoxin is synthesized and compartmentalized in specialized vesicles called aflatoxisomes and that these subcellular organelles also play a role in the export process. In the current study, we tested the hypothesis that aflatoxisomes fuse with the cytoplasmic membrane to facilitate the release of aflatoxin into the growth environment. Microscopic analysis of A. parasiticus grown under aflatoxin-inducing and non-aflatoxin-inducing conditions generated several lines of experimental evidence that supported the hypothesis. On the basis of the evidence, we propose that export of the mycotoxin aflatoxin in Aspergillus parasiticus occurs by exocytosis, and we present a model to illustrate this export mechanism.

Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice.

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism.

BACKGROUND: Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. RESULTS: Volatile compounds were examined using solid phase microextraction--gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. CONCLUSIONS: 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and ß-oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.

Prevalence and patterns of antimicrobial resistance in Campylobacter spp isolated from pigs reared under antimicrobial-free and conventional production methods in eight states in the Midwestern United States.

OBJECTIVE: To compare apparent prevalence and patterns of antimicrobial resistance in Campylobacter spp in feces collected from pigs reared with antimicrobial-free versus conventional production methods in 8 states in the Midwestern United States. DESIGN: Cross-sectional study. SAMPLE POPULATION: 95 swine farms that used antimicrobial-free (n = 35) or conventional (60) production methods. PROCEDURES: Fecal samples from 15 pigs/farm were collected. Biochemical and multiplex-PCR analyses were used to identify Campylobacter spp. The minimal inhibitory concentrations of erythromycin, azithromycin, ciprofloxacin, nalidixic acid, gentamicin, and tetracycline for these organisms were determined by use of a commercially available antimicrobial gradient strip. The data were analyzed by use of population-averaged statistical models. RESULTS: Campylobacter spp were isolated from 512 of 1,422 pigs. A subset (n = 464) of the 512 isolates was available for antimicrobial susceptibility testing. The apparent prevalence of Campylobacter spp isolates from pigs on conventional farms (35.8%) and antimicrobial-free farms (36.4%) did not differ significantly. Resistances to azithromycin, erythromycin, and tetracycline were significantly higher on conventional farms (70.0%, 68.3%, and 74.5%, respectively) than antimicrobial-free farms (20.1%, 21.3%, and 48.8%, respectively). Resistances to azithromycin, erythromycin, and tetracycline declined as the number of years that a farm was antimicrobial-free increased. CONCLUSIONS AND CLINICAL RELEVANCE: Production method did not affect the apparent prevalence of Campylobacter spp on swine farms. However, antimicrobial-free farms had a significantly lower prevalence of antimicrobial resistance. Although cessation of antimicrobial drug use will lower resistance over time, investigation of other interventions designed to reduce resistance levels is warranted.

Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus.

Aflatoxin biosynthesis in Aspergillus parasiticus requires at least 17 enzyme activities (from acetate). Although the activities of most aflatoxin biosynthetic enzymes have been established, the mechanisms that govern transport and sub-cellular localization of these enzymes are not clear. We developed plasmid constructs that express Nor-1 fused to a green fluorescent protein reporter (EGFP) to monitor transport and localization of this early pathway enzyme in real time in Aspergillus parasiticus. Plasmids expressing EGFP fused to Nor-1 were introduced into A. parasiticus B62 (carries non-functional Nor-1). Transformants were screened for increased aflatoxin accumulation (restored Nor-1 activity) on coconut agar medium and for EGFP expression using fluorescence microscopy. Increased aflatoxin accumulation was confirmed by TLC and ELISA. Nor-1 fused to EGFP at either the N- or C- terminus functionally complemented non-functional Nor-1 in B62 and increased aflatoxin synthesis to wild-type (N-terminus) or lower levels (C-terminus). We detected full-length Nor-1 fusion proteins in transformants with increased aflatoxin accumulation (Western blot) and determined that the expression plasmid integrated at the nor-1 locus in these cells (Southern blot). Confocal laser scanning microscopy (CLSM) demonstrated that Nor-1 fusion proteins localized in the cytoplasm and vacuoles of fungal hyphae grown on aflatoxin-inducing solid media for 48h; control EGFP (no Nor-1) did not localize to vacuoles until 72h. The highest rate of aflatoxin synthesis coincided with the highest rate of transport of Nor-1 fusion proteins to the vacuole strongly suggesting that Nor-1 is synthesized in the cytoplasm and transported to the vacuole to carry out an early step in aflatoxin synthesis.

Functional expression and subcellular localization of the aflatoxin pathway enzyme Ver-1 fused to enhanced green fluorescent protein.

Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.

Altered hydrophobicity and membrane composition in stress-adapted Listeria innocua.

Exposure of Listeria innocua to acid and starvation stress decreases sensitivity to the quaternary ammonium compound cetrimide, whereas exposure to cold and heat stress increases sensitivity to this compound. Changes in membrane lipids occur in response to certain types of stress, and these changes likely impact cell sensitivity to chemical sanitizers. The present study included an assessment of the effects of acid, starvation, cold, and heat stress on net cell hydrophobicity and fatty acid composition in L. innocua. Net cell hydrophobicity was determined by measuring absorbance of stress-adapted cell suspensions after partitioning with the nonpolar solvent n-hexadecane. Free fatty acids extracted from stress-adapted suspensions were analyzed by gas chromatography. Adaptation to acid and starvation increased net cell hydrophobicity and decreased membrane fluidity, which was correlated with reductions in anteiso fatty acids and in ratios of anteiso to iso fatty acids. Conversely, cold-stressed populations exhibited decreased net cell hydrophobicity and increased membrane fluidity with a corresponding increase in C15:C17 and anteiso:iso ratios and in C18 unsaturated fatty acids. No significant changes in net cell hydrophobicity or membrane fluidity were observed in heat-stressed cells, which exhibited increased sensitivity to cetrimide, suggesting another mechanism for altered cell sensitivity. These findings indicate that the efficacy of cetrimide against Listeria is partially dependent on the physiological state of the organism following exposure to various environmental stresses.

Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis.

A signal transduction pathway involving cAMP and protein kinase A (PKA) regulates aflatoxin accumulation and nor-1 and ver-1 (aflatoxin structural genes) promoter function in Aspergillus parasiticus by modulating expression of a key transcriptional activator, AflR. To understand the function of this pathway in greater detail we treated A. parasiticus in culture with wortmannin, a frequently used probe of phosphatidyl inositol (PI)-3 kinase activity. A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver-1::GUS reporter) were grown on a defined solid growth medium (GMS agar) under aflatoxin-inducing conditions. GMS containing wortmannin (1 microM) reduced aflatoxin B1 accumulation up to 15-fold accompanied by a similarly large decrease in ver-1 and nor-1 promoter activity. Wortmannin inhibited growth (colony diameter) and asexual sporulation but to a much smaller extent. Wortmannin treatment increased intracellular cAMP levels up to 25-fold; total PKA activity also increased within 10 min of wortmannin exposure. These data support a regulatory model in which PI-3 kinase activity modulates intracellular cAMP accumulation and PKA activity. This in turn regulates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation.