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Hideo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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UV mutagenesis of Cupriavidus Necator for extracellular production of (R)-3-hydroxybutyric acid.
Journal of Applied Microbiology, 2008Co-Authors: Charles U. Ugwu, Hiroo Uchiyama, Yutaka Tokiwa, Hideki Aoyagi, Hideo TanakaAbstract:Aim: Ultraviolet (UV) mutagenesis was carried out to obtain mutant strains of Cupriavidus Necator that could produce (R)-3-hydroxybutyric acid [(R)-3-HB] in the culture supernatant. Methods and Results: C. Necator (formerly known as Ralstonia eutropha) was subjected to UV radiation to generate mutants that are capable of producing (R)-3-HB in the culture supernatant. Results indicated that UV mutagen disrupted the phbB (phbB knock-out) and thus, promoted production of (R)-3-HB in mutant strains. Inclusion of acetoacetate esters (carbonyl compounds) in the culture broth led to increased production of (R)-3-HB. Thus, acetoacetyl-CoA (an intermediate of the PHB synthetic pathway) might have been converted to acetoacetate, which in the presence of (R)-3-HB dehydrogenase and NADPH/NADP+, resulted in extracellular production of (R)-3-HB. Conclusions: UV mutagenesis proved to be a satisfactory method in generating interesting mutants for extracellular production of (R)-3-HB. Extracellular production of (R)-3-HB upon addition of acetoacetate esters would suggest a likely (R)-3-HB biosynthetic pathway in C. Necator. Significance and Impact of the Study: Mutants obtained in this study are very useful for production of (R)-3-HB. For the first time, the production of (R)-3-HB by C. Necator via acetoacetate is reported.
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UV mutagenesis of Cupriavidus Necator for extracellular production of (R)-3-hydroxybutyric acid.
Journal of applied microbiology, 2008Co-Authors: Charles U. Ugwu, Hiroo Uchiyama, Yutaka Tokiwa, Hideki Aoyagi, Hideo TanakaAbstract:Ultraviolet (UV) mutagenesis was carried out to obtain mutant strains of Cupriavidus Necator that could produce (R)-3-hydroxybutyric acid [(R)-3-HB] in the culture supernatant. C. Necator (formerly known as Ralstonia eutropha) was subjected to UV radiation to generate mutants that are capable of producing (R)-3-HB in the culture supernatant. Results indicated that UV mutagen disrupted the phbB (phbB knock-out) and thus, promoted production of (R)-3-HB in mutant strains. Inclusion of acetoacetate esters (carbonyl compounds) in the culture broth led to increased production of (R)-3-HB. Thus, acetoacetyl-CoA (an intermediate of the PHB synthetic pathway) might have been converted to acetoacetate, which in the presence of (R)-3-HB dehydrogenase and NADPH/NADP(+), resulted in extracellular production of (R)-3-HB. UV mutagenesis proved to be a satisfactory method in generating interesting mutants for extracellular production of (R)-3-HB. Extracellular production of (R)-3-HB upon addition of acetoacetate esters would suggest a likely (R)-3-HB biosynthetic pathway in C. Necator. Mutants obtained in this study are very useful for production of (R)-3-HB. For the first time, the production of (R)-3-HB by C. Necator via acetoacetate is reported.
Michael G. Milgroom - One of the best experts on this subject based on the ideXlab platform.
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Differential gene expression during conidiation in the grape powdery mildew pathogen, Erysiphe Necator.
Phytopathology, 2011Co-Authors: Laura Wakefield, David M. Gadoury, Michael G. Milgroom, Robert C. Seem, Qi Sun, Lance Cadle-davidsonAbstract:ABSTRACT Asexual sporulation (conidiation) is coordinately regulated in the grape powdery mildew pathogen Erysiphe Necator but nothing is known about its genetic regulation. We hypothesized that genes required for conidiation in other fungi would be upregulated at conidiophore initiation or full conidiation (relative to preconidiation vegetative growth and development of mature ascocarps), and that the obligate biotrophic lifestyle of E. Necator would necessitate some novel gene regulation. cDNA amplified fragment length polymorphism analysis with 45 selective primer combinations produced ≈1,600 transcript-derived fragments (TDFs), of which 620 (39%) showed differential expression. TDF sequences were annotated using BLAST analysis of GenBank and of a reference transcriptome for E. Necator developed by 454-FLX pyrosequencing of a normalized cDNA library. One-fourth of the differentially expressed, annotated sequences had similarity to fungal genes of unknown function. The remaining genes had annotated func...
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Phylogeography and population structure of the grape powdery mildew fungus, Erysiphe Necator, from diverse Vitis species
BMC evolutionary biology, 2010Co-Authors: Marin Talbot Brewer, Michael G. MilgroomAbstract:The grape powdery mildew fungus, Erysiphe Necator, was introduced into Europe more than 160 years ago and is now distributed everywhere that grapes are grown. To understand the invasion history of this pathogen we investigated the evolutionary relationships between introduced populations of Europe, Australia and the western United States (US) and populations in the eastern US, where E. Necator is thought to be native. Additionally, we tested the hypothesis that populations of E. Necator in the eastern US are structured based on geography and Vitis host species. We sequenced three nuclear gene regions covering 1803 nucleotides from 146 isolates of E. Necator collected from the eastern US, Europe, Australia, and the western US. Phylogeographic analyses show that the two genetic groups in Europe represent two separate introductions and that the genetic groups may be derived from eastern US ancestors. Populations from the western US and Europe share haplotypes, suggesting that the western US population was introduced from Europe. Populations in Australia are derived from European populations. Haplotype richness and nucleotide diversity were significantly greater in the eastern US populations than in the introduced populations. Populations within the eastern US are geographically differentiated; however, no structure was detected with respect to host habitat (i.e., wild or cultivated). Populations from muscadine grapes, V. rotundifolia, are genetically distinct from populations from other Vitis host species, yet no differentiation was detected among populations from other Vitis species. Multilocus sequencing analysis of the grape powdery mildew fungus is consistent with the hypothesis that populations in Europe, Australia and the western US are derived from two separate introductions and their ancestors were likely from native populations in the eastern US. The invasion history of E. Necator follows a pattern consistent with plant-mediated dispersal, however, more exhaustive sampling is required to make more precise conclusions as to origin. E. Necator shows no genetic structure across Vitis host species, except with respect to V. rotundifolia.
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Persistence and spatial autocorrelation of clones of Erysiphe Necator overwintering as mycelium in dormant buds in an isolated vineyard in northern Italy
Phytopathology, 2008Co-Authors: Paolo Cortesi, C. Pizzatti, D. Bertocchi, Michael G. MilgroomAbstract:The population structure of the grape powdery mildew fungus, Erysiphe Necator (formerly Uncinula Necator), has been hypothesized to vary from being clonal to highly diverse and recombining. We report here on the structure of an E. Necator population sampled during a 4-year period from an isolated vineyard in northern Italy (Voghera, Pavia Province). We obtained 54 isolates of E. Necator that overwintered asexually as mycelium in grapevine buds and caused severe symptoms on the emerging shoots, known as flag shoots. All isolates were genotyped for mating type, four multilocus polymerase chain reaction (PCR)-based markers (a total of 64 loci were scored), and two single-copy loci designed to identify genetic subgroups in E. Necator. All isolates had the same mating type and single-locus alleles that correlate to isolates from flag shoots in other areas. Only 2 of the 64 loci scored from multilocus markers were polymorphic; 46 of the 54 isolates had the same multilocus haplotype. Seven isolates had a second haplotype that was recovered over 3 years, and only a single isolate was found with a third haplotype. Both variant haplotypes differed from the main clonal haplotype by single loci. Spatial autocorrelation analyses showed that vines with flag shoots were not aggregated within years, but they were aggregated between consecutive years. These results demonstrate that this subpopulation of E. Necator on flag shoots is composed of a single clonal lineage that has persisted for at least 4 years. We speculate that the lack of diversity in the flag shoot subpopulation in this vineyard is the result of restricted immigration from surrounding areas and genetic drift operating through founder effects and periodic bottlenecks. We propose a model that integrates epidemiology and population genetics to explain the variation observed in genetic structure of E. Necator flag shoot subpopulations from different vineyards or viticultural regions.
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Spatial and Genetic Analysis of a Flag Shoot Subpopulation of Erysiphe Necator in Italy.
Phytopathology, 2004Co-Authors: Paolo Cortesi, Marie-paule Ottaviani, Michael G. MilgroomAbstract:ABSTRACT Erysiphe Necator overwinters as ascospores in cleistothecia and mycelium in dormant buds of grapevines. Shoots developing from infected buds early in the growing season are covered with dense mycelium and are known as "flag shoots". Combining epidemiological and genetic analyses, the objective of this study was to analyze the spatial and genetic structure of a flag shoot subpopulation of E. Necator as a way to assess the contribution of flag shoots as primary inoculum, and to determine if flag shoot subpopulations are clonal with only one mating type. One vineyard in Tuscany, Italy was surveyed intensively for flag shoots for 8 years; isolations of E. Necator were made from flag shoots for 5 years. We observed distinct disease foci developing around flag shoots early in epidemics, demonstrating a steep dispersal gradient of conidia and the importance of flag shoots as primary inoculum sources. Flag shoots were spatially aggregated within and between years, most likely as a result of short-distance dispersal of conidia from flags early in the season when dormant buds for the next year's shoots are formed and are susceptible to infection. The two mating types were found in 1:1 ratios in this flag shoot subpopulation. Genotypic diversity, based on inter-simple sequence repeat markers, was high in all years with only two haplotypes occurring twice, and subpopulations were genetically differentiated between years. Similarities between haplotypes were not spatially autocorrelated. One multilocus analysis of population structure is consistent with the hypothesis of random mating but another is not. These results are not consistent with expectations for a strictly clonal or strictly randomly mating flag shoot subpopulation. Instead, the hypothesis that the flag shoot subpopulation of E. Necator may reproduce clonally and sexually needs further testing.
David M. Gadoury - One of the best experts on this subject based on the ideXlab platform.
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Differential gene expression during conidiation in the grape powdery mildew pathogen, Erysiphe Necator.
Phytopathology, 2011Co-Authors: Laura Wakefield, David M. Gadoury, Michael G. Milgroom, Robert C. Seem, Qi Sun, Lance Cadle-davidsonAbstract:ABSTRACT Asexual sporulation (conidiation) is coordinately regulated in the grape powdery mildew pathogen Erysiphe Necator but nothing is known about its genetic regulation. We hypothesized that genes required for conidiation in other fungi would be upregulated at conidiophore initiation or full conidiation (relative to preconidiation vegetative growth and development of mature ascocarps), and that the obligate biotrophic lifestyle of E. Necator would necessitate some novel gene regulation. cDNA amplified fragment length polymorphism analysis with 45 selective primer combinations produced ≈1,600 transcript-derived fragments (TDFs), of which 620 (39%) showed differential expression. TDF sequences were annotated using BLAST analysis of GenBank and of a reference transcriptome for E. Necator developed by 454-FLX pyrosequencing of a normalized cDNA library. One-fourth of the differentially expressed, annotated sequences had similarity to fungal genes of unknown function. The remaining genes had annotated func...
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Specific Isolation of RNA from the Grape Powdery Mildew Pathogen Erysiphe Necator, an Epiphytic, Obligate Parasite
Journal of Phytopathology, 2010Co-Authors: Lance Cadle-davidson, Laura Wakefield, Robert C. Seem, David M. GadouryAbstract:RNA expression profiling of obligately parasitic plant microbes is hampered by the requisite interaction of host and parasite. This can be especially problematic in the case of powdery mildews, such as Erysiphe Necator (syn. Uncinula Necator), which grow superficially but tightly adhere to the plant epidermis. We developed and refined a simple and efficient technique in which nail polish was used to remove conidia, appressoria, hyphae, conidiophores, and developing ascocarps of E. Necator from grapevine (Vitis vinifera) leaves and showed that RNA isolated after removal was not contaminated with V. vinifera RNA. This approach can be applied to expression analyses throughout fungal development and could be extended to other epiphytic pathogens and saprophytes.
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effects of diffuse colonization of grape berries by uncinula Necator on bunch rots berry microflora and juice and wine quality
Phytopathology, 2007Co-Authors: David M. Gadoury, Robert C. Seem, Wayne F Wilcox, Thomas Henickkling, Lorenza Conterno, Andrea Day, Andrea FickeAbstract:ABSTRACT Production of grape (principally cultivars of Vitis vinifera) for high-quality wines requires a high level of suppression of powdery mildew (Uncinula Necator syn. Erysiphe Necator). Severe infection of either fruit or foliage has well-documented and deleterious effects upon crop and wine quality. We found that berries nearly immune to infection by U. Necator due to the development of ontogenic resistance may still support diffuse and inconspicuous mildew colonies when inoculated ≈3 weeks post-bloom. Fruit with diffuse mildew colonies appear to be healthy and free of powdery mildew in late-season vineyard assessments with the naked eye. Nonetheless, presence of these colonies on berries was associated with (i) elevated populations of spoilage microorganisms; (ii) increased evolution of volatile ethyl acetate, acetic acid, and ethanol; (iii) increased infestation by insects known to be attracted to the aforementioned volatiles; (iv) increased rotting by Botrytis cinerea; and (v) increased frequency...
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Heterothallism and pathogenic specialization in Uncinula Necator.
Phytopathology, 1991Co-Authors: David M. Gadoury, R. C. PearsonAbstract:A collection of 35 isolates of Uncinula Necator was established. Each isolate originated from a single chain of conidia. The collection included isolates from 10 species of Vitis and isolates from Vitis interspecific hybrids. Isolates were paired in all possible combinations on tissue culture plants of the Vitis interspecific hybrid cultivar Chancellor and were incubated for 60 days at 20 C. Initially, U. Necator appeared to be composed of two mating types that comprised a bipolar heterothallic system. Ascocarps were produced abundantly within 14 days in approximately one-half of the pairings (...)
Toshiaki Fukui - One of the best experts on this subject based on the ideXlab platform.
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identification of mutation points in cupriavidus Necator ncimb 11599 and genetic reconstitution of glucose utilization ability in wild strain h16 for polyhydroxyalkanoate production
Journal of Bioscience and Bioengineering, 2012Co-Authors: Izumi Orita, Satoshi Nakamura, Reiko Iwazawa, Toshiaki FukuiAbstract:Although the facultative chemolithoautotrophic Cupriavidus Necator (formerly Ralstonia eutropha) wild strain H16 is potentially useful as a host for metabolic engineering aimed at polyhydroxyalkanoate production, this organism is deficient in assimilating glucose, a major sugar in non-edible cellulosic resources. Growth properties of C. Necator H16 harboring heterologous glf (encoding glucose-facilitated diffusion transporter) and glk (encoding glucokinase) from Zymomonas mobilis strongly suggested that the lack of glucose-utilization ability of C. Necator H16 was caused by deficiency of both glucose-uptake and phosphorylation abilities. Next examination focused on previously unknown mutation points in a glucose-utilizing mutant of C. Necator NCIMB 11599. Direct sequencing of a region of genes for putative N-acetylglucosamine-specific phosphoenolpyruvate-dependent phosphotransferase system and its upstream region identified a missense mutation in nagE corresponding to Gly265Arg in the EIIC-EIIB component, and a nonsense mutation in nagR encoding a putative GntR-type transcriptional regulator. Further analyses demonstrated that the glucose-utilization ability of C. Necator NCIMB 11599 is attributed to extended sugar specificity of the mutated NagE and derepression of nagFE expression by inactivation of NagR. The mutation in nagE and disruption of nagR were then introduced onto chromosome 1 of wild strain H16 by homologous recombination. The resulting engineered strain C. Necator nagE_G265R∆nagR exhibited comparable growth and poly(3-hydroxybutyrate) accumulation on glucose to those of the wild strain on fructose, demonstrating successful reconstitution of functional glucose-uptake and phosphorylation system. This recombinant strain is expected to be useful in further engineering for efficient production of PHAs from inexpensive biomass resources.
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evaluation of promoters for gene expression in polyhydroxyalkanoate producing cupriavidus Necator h16
Applied Microbiology and Biotechnology, 2011Co-Authors: Toshiaki Fukui, Kei Ohsawa, Jun Mifune, Izumi Orita, Satoshi NakamuraAbstract:Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus Necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P lac , P tac , or P BAD derived from Escherichia coli, or promoter regions of phaC1 (P phaC ) or phaP1 (P phaP ) derived from C. Necator. Then, the gfp-expression profiles were determined in C. Necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P lac , P tac , P phaC , and P phaP mediated constitutive gene expression, among which P tac was the strongest promoter. lacI-P tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. Necator to uptake IPTG. Gene expression by araC-P BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. Necator.
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microbial synthesis of poly r 3 hydroxybutyrate co 3 hydroxypropionate from unrelated carbon sources by engineered cupriavidus Necator
Biomacromolecules, 2009Co-Authors: Toshiaki Fukui, Mamie Suzuki, Takeharu Tsuge, Satoshi NakamuraAbstract:Cupriavidus Necator was engineered aiming to synthesize poly[(R)-3-hydroxybutyrate-co-3-hydroxypropionate] copolyester, P(3HB-co-3HP), from structurally unrelated carbon sources without addition of any precursor compounds. We modified a metabolic pathway in C. Necator for generation of 3-hydroxypropionyl-CoA (3HP-CoA) by introducing malonyl-CoA reductase and the 3HP-CoA synthetase domain of trifunctional propionyl-CoA synthase; both members of the 3-hydroxypropionate cycle, a novel CO2-fixation pathway in the green nonsulfur bacterium Chloroflexus aurantiacus. In this recombinant strain, 3HP-CoA was expected to be provided from acetyl-CoA via malonyl-CoA, and then copolymerized by the function of polyhydroxyalkanoate synthase along with (R)-3-hydroxybutyryl-CoA synthesized from two acetyl-CoA molecules. C. Necator wild-type strains H16 and JMP134 harboring the two heterologous genes actually synthesized P(3HB-co-3HP) copolyester with 0.2−2.1 mol % of 3HP fraction from fructose or alkanoic acids of even ca...
Charles U. Ugwu - One of the best experts on this subject based on the ideXlab platform.
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UV mutagenesis of Cupriavidus Necator for extracellular production of (R)-3-hydroxybutyric acid.
Journal of Applied Microbiology, 2008Co-Authors: Charles U. Ugwu, Hiroo Uchiyama, Yutaka Tokiwa, Hideki Aoyagi, Hideo TanakaAbstract:Aim: Ultraviolet (UV) mutagenesis was carried out to obtain mutant strains of Cupriavidus Necator that could produce (R)-3-hydroxybutyric acid [(R)-3-HB] in the culture supernatant. Methods and Results: C. Necator (formerly known as Ralstonia eutropha) was subjected to UV radiation to generate mutants that are capable of producing (R)-3-HB in the culture supernatant. Results indicated that UV mutagen disrupted the phbB (phbB knock-out) and thus, promoted production of (R)-3-HB in mutant strains. Inclusion of acetoacetate esters (carbonyl compounds) in the culture broth led to increased production of (R)-3-HB. Thus, acetoacetyl-CoA (an intermediate of the PHB synthetic pathway) might have been converted to acetoacetate, which in the presence of (R)-3-HB dehydrogenase and NADPH/NADP+, resulted in extracellular production of (R)-3-HB. Conclusions: UV mutagenesis proved to be a satisfactory method in generating interesting mutants for extracellular production of (R)-3-HB. Extracellular production of (R)-3-HB upon addition of acetoacetate esters would suggest a likely (R)-3-HB biosynthetic pathway in C. Necator. Significance and Impact of the Study: Mutants obtained in this study are very useful for production of (R)-3-HB. For the first time, the production of (R)-3-HB by C. Necator via acetoacetate is reported.
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UV mutagenesis of Cupriavidus Necator for extracellular production of (R)-3-hydroxybutyric acid.
Journal of applied microbiology, 2008Co-Authors: Charles U. Ugwu, Hiroo Uchiyama, Yutaka Tokiwa, Hideki Aoyagi, Hideo TanakaAbstract:Ultraviolet (UV) mutagenesis was carried out to obtain mutant strains of Cupriavidus Necator that could produce (R)-3-hydroxybutyric acid [(R)-3-HB] in the culture supernatant. C. Necator (formerly known as Ralstonia eutropha) was subjected to UV radiation to generate mutants that are capable of producing (R)-3-HB in the culture supernatant. Results indicated that UV mutagen disrupted the phbB (phbB knock-out) and thus, promoted production of (R)-3-HB in mutant strains. Inclusion of acetoacetate esters (carbonyl compounds) in the culture broth led to increased production of (R)-3-HB. Thus, acetoacetyl-CoA (an intermediate of the PHB synthetic pathway) might have been converted to acetoacetate, which in the presence of (R)-3-HB dehydrogenase and NADPH/NADP(+), resulted in extracellular production of (R)-3-HB. UV mutagenesis proved to be a satisfactory method in generating interesting mutants for extracellular production of (R)-3-HB. Extracellular production of (R)-3-HB upon addition of acetoacetate esters would suggest a likely (R)-3-HB biosynthetic pathway in C. Necator. Mutants obtained in this study are very useful for production of (R)-3-HB. For the first time, the production of (R)-3-HB by C. Necator via acetoacetate is reported.
