Molecular characterization of ochratoxin A biosynthesis and producing fungi.
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Animals↗
Biomedical subjects
A D W Dobson
Publications and source records attributed to A D W Dobson.
Molecular Characterization of Ochratoxin A Biosynthesis and Producing Fungi.
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Journal Article↗
Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10.
A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.
Cloning, Molecular↗
Parameters affecting biological phosphate removal from wastewaters.
This paper reviews some of the key wastewater composition parameters, which influence the biological removal of phosphate from wastewaters, such as COD content, volatile fatty acid (VFA) content, cation concentration, phosphorus load, pH and food to microorganism ratio. The discussion also focuses on operational parameters affecting successful nutrient removal in wastewater treatment plants, such as temperature, sludge quality, sludge settlement, dissolved oxygen (DO) concentration, anaerobic P-release and secondary P-release. The aim of this review is to compile an updated document for researchers and operators of biological nutrient removal (BNR) systems. In addition, the article will provide a good foundation for readers with no prior knowledge of the process.
Fatty Acids, Volatile↗
The role of indigenous yeasts in traditional Irish cider fermentations.
AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.
Alcoholic Beverages↗
Real-time PCR analysis of carbon catabolite repression of cellobiose dehydrogenase gene transcription in Trametes versicolor.
Cellobiose dehydrogenase production in Trametes versicolor is repressed when additional carbon sources, such as glucose, maltose, galactose, arabinose, and xylose, are added to the fungal cultures growing on cellulose. Real-time quantitative reverse transcription-polymerase chain reaction has been used to demonstrate that the addition of galactose, arabinose, and xylose results in 19-, 92-, and 114-fold reductions, respectively, in cdh transcript levels 96 h post-addition. Glucose exhibits the greatest repressive effect, resulting in a 3400-fold decrease in cdh transcript levels.
Arabinose↗
Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.
Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.
Base Sequence↗
Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid.
AIMS: To develop a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect viable Candida krusei contaminations and examine the potential role of the citrate synthase (cs1) gene in adaptation to acetic acid. METHODS AND RESULTS: Fruit juice artificially contaminated with C. krusei cells was heat treated to inactivate the yeast cells, after which the improved ability of the RT-PCR over the PCR assay, through the amplification of the cs1 gene, to differentiate viable contaminations was shown. The sensitivity of the detection assay was 6 x 104 CFU ml-1. RT-PCR and densitometric analysis of the cs1 gene throughout the process of adaptation to acetic acid highlighted a potential role for the gene in the yeast's adaptive response. CONCLUSIONS: The RT-PCR assay through the targeting of the cs1 gene proved to be a specific, sensitive and direct method for the identification of a C. krusei contamination in a food environment. The cs1 gene was shown to play a potential role in the adaptation of the culture to the weak-acid preservative acetic acid. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: The development of a direct, sensitive and specific identification assay for C. krusei from a food environment and understanding the mechanism employed in adapting to a preservative challenge, represent important tools to the food industry in attempting to limit spoilage by this important food spoilage yeast.
Acetic Acid↗
The use of amplified flanking region-PCR in the isolation of laccase promoter sequences from the edible fungus Pleurotus sajor-caju.
AIMS: To determine the regulation of laccase isozyme gene transcription in Pleurotus sajor-caju in response to different aromatic inducers and physiological parameters. METHODS AND RESULTS: The promoter regions for each of four different laccase isozymes were cloned from P. sajor-caju, using amplified flanking region-PCR (AFR-PCR). Sequences stretching 724, 214, 840 and 1740 bp upstream from the predicted start codons for lac1, lac2, lac3 and lac4, respectively, were cloned in each case and analysed for the presence of putative transcriptional response elements. A number of putative response elements including metal response elements, xenobiotic response elements and antioxidant response elements appear to be present. In addition putative consensus sequences such as those for the binding of AP1, AP2, creA and NIT2 transcription factors, which are involved in nitrogen and carbon regulation in different fungi, are also present in the promoter regions of some of the isozymes. CONCLUSIONS: These elements may be involved in the transcriptional regulation of laccase gene expression in P. sajor-caju. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of a number of putative transcriptional response elements in the promoter regions of different isozyme genes indicates a potential role for these sites in regulating laccase gene transcription in P. sajor-caju. In addition this work demonstrates the potential usefulness of AFR-PCR as a technique to clone fungal DNA sequences located upstream from known sequences.
Base Sequence↗
A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus.
Ochratoxin A is an important nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus ochraceus as a polyketide-derived secondary metabolite. A portion of a putative polyketide synthase gene (pks) involved in the biosynthesis of this mycotoxin was cloned by using a suppression subtractive hybridization PCR-based approach. The predicted amino acid sequence of the 1.4 kb clone shared 28-35 % identity to acyl transferase regions from fungal polyketide synthases found in the databases. Based on reverse transcription PCR studies, the pks gene is expressed only under ochratoxin A permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted cannot synthesize ochratoxin A. This report is the first of the cloning and characterization of a gene involved in ochratoxin A biosynthesis.
Amino Acid Sequence↗
Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris.
A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 microM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation.
Alanine↗
Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host.
The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. The native Ple. sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic. pastoris. The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4.38. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3.3, 6, 6.5 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 degrees C and pH 3.5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.
Cloning, Molecular↗