Production of sterigmatocystin by Aspergillus versicolor isolated from roughage

A total of 69 samples of hay and straw collected during the winter period of 1984/85 were surveyed for their contamination by Aspergillus versicolor. The percentage of A. versicolor-positive samples was 14.5%. Nineteen A. versicolor strains mainly isolated from roughage were tested for the production of sterigmatocystin. All of the isolates examined were capable of producing different levels of sterigmatocystin on a cracked corn substrate. The majority of these strains were highly toxigenic; 53% of the isolates produced more than 500 mg/kg of sterigmatocystin. These findings suggest that corn is a very suitable substrate for sterigmatocystin production and that particularly in the surface layers of feed stocks and corn silos such toxigenic strains of A. versicolor can produce considerable growth and possibly sterigmatocystin, too.     

Anthraquinones in the biosynthesis of sterigmatocystin by Aspergillus versicolor

14C-labeled averufin, versiconal hemiacetal acetate, and versicolorin A were efficiently converted to sterigmatocystin by Aspergillus versicolor, thus providing experimental evidence that these anthraquinones are biosynthetic precursors of sterigmatocystin, a xanthone.     

Anthraquinones in the biosynthesis of sterigmatocystin by Aspergillus versicolor.

14C-labeled averufin, versiconal hemiacetal acetate, and versicolorin A were efficiently converted to sterigmatocystin by Aspergillus versicolor, thus providing experimental evidence that these anthraquinones are biosynthetic precursors of sterigmatocystin, a xanthone.     

Production of sterigmatocystin by Aspergillus versicolor and Bipolaris sorokiniana on semisynthetic liquid and solid media.

Higher yields of sterigmatocystin were obtained with Aspergillus versicolor than with Bipolaris sorokiniana both in liquid and on solid media. The optimum temperature for sterigmatocystin production by A. versicolor was 27 to 29 degrees C and 23 degrees C for B. sorokiniana. In liquid shake cultures, production of sterigmatocystin by B. sorokiniana was negligible, whereas maximal production by A. versicolor was 210 mg/liter. On solid substrates, the highest yields (8 g/kg) were obtained with A. versicolor on still cultures of whole corn supplemented with Soytone.     

Metabolism of alkyl amines by the filamentous fungus Aspergillus versicolor

A variety of monoalkyl-substituted amines were able to act as nitrogen sources for heterotrophically growing cultures of Aspergillus versicolor. Only amines whose alkyl chains were at least five carbon atoms long were capable of supporting significant growth in the absence of a separate carbon substrate. However, biomass yields were significantly higher during growth on glucose-amine than on glucose-ammonia, indicating that some energy-generating dissimilation of the amine to CO2 took place.     

Metabolism of alkyl amines by the filamentous fungus Aspergillus versicolor.

A variety of monoalkyl-substituted amines were able to act as nitrogen sources for heterotrophically growing cultures of Aspergillus versicolor. Only amines whose alkyl chains were at least five carbon atoms long were capable of supporting significant growth in the absence of a separate carbon substrate. However, biomass yields were significantly higher during growth on glucose-amine than on glucose-ammonia, indicating that some energy-generating dissimilation of the amine to CO2 took place.     

Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments

Over the past decade, there has been growing concern regarding the role of toxigenic fungi in damp indoor environments; however, there is still a lack of field investigations on exposure to mycotoxins. The goal of our pilot study was to quantify the proportion of toxigenic Aspergillus versicolor isolates in native carpet dust from damp dwellings with mold problems and to determine whether sterigmatocystin can be detected in this matrix. Carpet dust samples (n = 11) contained from <2.5 x 10(1) to 3.6 x 10(5) (median, 3.1 x 10(4)) A. versicolor CFU/g of dust, and the median proportion of A. versicolor from total culturable fungi was 18%. Based on thin-layer chromatography detection of sterigmatocystin, 49 of 50 A. versicolor isolates (98%) were found to be toxigenic in vitro. By using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry, sterigmatocystin could be detected in low concentrations (2 to 4 ng/g of dust) in 2 of 11 native carpet dust samples. From this preliminary study, we conclude that most strains of A. versicolor isolated from carpet dust are able to produce sterigmatocystin in vitro and that sterigmatocystin may occasionally occur in carpet dust from damp indoor environments. Further research and systematic field investigation are needed to confirm our results and to provide an understanding of the health implications of mycotoxins in indoor environments.     

Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans

Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.     

New application of Aspergillus versicolor in promoting plant growth after suppressing sterigmatocystin production via genome mining and engineering

Aspergillus genus is a key component in fermentation and food processing. However, sterigmatocystin (STE)—a mycotoxin produced by several species of Aspergillus—limits the use of some Aspergillus species (such as Aspergillus versicolor, Aspergillus inflatus, and Aspergillus parasiticus) because of its toxicity and carcinogenicity. Here, we engineered an STE-free Aspergillus versicolor strain based on genome mining techniques. We sequenced and assembled the Aspergillus versicolor D5 genome (34.52 Mb), in which we identified 16 scaffolds and 54 biosynthetic gene clusters (BGCs). We silenced cytochrome P450 coding genes STC17 and STC27 by insertional inactivation. The production of STE in the Δstc17 mutant strain was increased by 282% but no STE was detected in the Δstc27 mutant. Metabolites of Δstc27 mutant exhibited growth-promoting effect on plants. Our study makes significant progress in improving the application of some Aspergillus strains by restricting their production of toxic and carcinogenic compounds.     

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).     

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).     

Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B₁ and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 μm in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 μm in diameter. The S-strain isolates generally produced high levels of aflatoxin B₁, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B₁ production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B₁ and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 μg of aflatoxin B₁ per ml. The percentages of isolates producing >10 μg of aflatoxin B₁ per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B₁ or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.     

The metabolism of biphenyl structures in lignin by the wood-rotting fungus Coriolus versicolor

Abstract In the degradation of lignin by the wood-rotting fungus Coriolus versicolor , understanding the metabolism of biphenyl structure is one of the most important steps in elucidation of the depolymerization process. This study gave the first evidence of aromatic ring fission of a biphenyl structure type by C. versicolor . The degradation of biphenyl structures of lignin by this fungus occurred to a slight extent as a result of a non-specific system. This phenomenon was observed only in a medium in which phenol oxidase was actively produced by this fungus.     

Reducing the cost of resistance; experimental evolution in the filamentous fungus Aspergillus nidulans

We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans.     

Spinal and sacroiliac assessment and treatment techniques used by osteopathic physicians in the United States

Background

Without systematic exposure to biomedical research concepts or applications, osteopathic medical students may be generally under-prepared to efficiently consume and effectively apply research and evidence-based medicine information in patient care. The academic literature suggests that although medical residents are increasingly expected to conduct research in their post graduate training specialties, they generally have limited understanding of research concepts. With grant support from the National Center for Complementary and Alternative Medicine, and a grant from the Osteopathic Heritage Foundation, the University of North Texas Health Science Center (UNTHSC) is incorporating research education in the osteopathic medical school curriculum. The first phase of this research education project involved a baseline assessment of students' understanding of targeted research concepts. This paper reports the results of that assessment and discusses implications for research education during medical school.

Methods

Using a novel set of research competencies supported by the literature as needed for understanding research information, we created a questionnaire to measure students' confidence and understanding of selected research concepts. Three matriculating medical school classes completed the on-line questionnaire. Data were analyzed for differences between groups using analysis of variance and t-tests. Correlation coefficients were computed for the confidence and applied understanding measures. We performed a principle component factor analysis of the confidence items, and used multiple regression analyses to explore how confidence might be related to the applied understanding.

Results

Of 496 total incoming, first, and second year medical students, 354 (71.4%) completed the questionnaire. Incoming students expressed significantly more confidence than first or second year students (F = 7.198, df = 2, 351, P = 0.001) in their ability to understand the research concepts. Factor analyses of the confidence items yielded conceptually coherent groupings. Regression analysis confirmed a relationship between confidence and applied understanding referred to as knowledge. Confidence scores were important in explaining variability in knowledge scores of the respondents.

Conclusion

Medical students with limited understanding of research concepts may struggle to understand the medical literature. Assessing medical students' confidence to understand and objectively measured ability to interpret basic research concepts can be used to incorporate competency based research material into the existing curriculum.     

Enhanced production of laccase in the fungus Trametes versicolor by the addition of xenobiotics

Agrochemicals, industrial compounds and their transformation products have been assayed for their ability to enhance laccase production in liquid cultures of Trametes versicolor, when added at 0.5 mM. After 3 days of treatment, enzymatic activity in the culture medium was increased 14-fold by 4-n-nonylphenol and 24-fold by aniline. Laccase activity was enhanced 10-fold by oxidised derivatives of the herbicide diquat, 17-fold by N,N-dimethyl-N-(5-chloro,4-hydroxyphenyl)urea and 22-fold by 9-fluorenone.     

Reevaluation of Energy Use in Wheat Production in the United States

Energy budgets for agricultural production can be used as building blocks for life-cycle assessments that include agricultural products, and can also serve as a first step toward identifying crop production processes that benefit most from increased efficiency. A general trend toward increased energy efficiency in U.S. agriculture has been reported. For wheat cultivation, in particular, this study updates cradle-to-gate process analyses produced in the seventies and eighties. Input quantities were obtained from official U.S. statistics and other sources and multiplied by calculated or recently published energy coefficients. The total energy input into the production of a kilogram of average U.S. wheat grain is estimated to range from 3.1 to 4.9 MJ/kg, with a best estimate at 3.9 MJ/kg. The dominant contribution is energy embodied in nitrogen fertilizer at 47% of the total energy input, followed by diesel fuel (25%), and smaller contributions such as energy embodied in seed grain, gasoline, electricity, and phosphorus fertilizer. This distribution is reflected in the energy carrier mix, with natural gas dominating (57%), followed by diesel fuel (30%). High variability in energy coefficients masks potential gains in total energy efficiency as compared to earlier, similar U.S. studies. Estimates from an input-output model for several input processes agree well with process analysis results, but the model's application can be limited by aggregation issues: Total energy inputs for generic food grain production were lower than wheat fertilizer inputs alone, possibly due to aggregation of diverse products into the food grain sector.     

Improvement of xylanase production in solid-state fermentation by alkali tolerant Aspergillus versicolor MKU3

AIMS: To optimize the media components for xylanase production by Aspergillus versicolor MKU3 in solid-state fermentation (SSF). METHODS AND RESULTS: Medium optimization was carried out using De Moe's fractional factorial design with seven components. Maximum production of xylanase 3249.9 U g(-1) was obtained in SSF with an optimized medium containing (g l(-1)): NaNO(3), 20; K(2)HPO(4), 20; MgSO(4), 10; FeSO(4), 0.001; KCl, 1; peptone, 10 and yeast extract, 10. Four components namely NaNO(3), MgSO(4), peptone and K(2)HPO(4) significantly increased the xylanase production by A. versicolor MKU3. CONCLUSIONS: Fractional factorial design was used to optimize the seven components in the fermentation medium for SSF. The optimized media increased xylanase production by 3.4-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus versicolor MKU3 produced maximum xylanase after two steps of media optimization under alkaline condition. This medium will be significant value for xylanase production in SSF.     

Suppressor mutations bypass the requirement of fluG for asexual sporulation and sterigmatocystin production in Aspergillus nidulans

Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.