The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completel... more The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE, cis -dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA , was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP) 2 . Direct N-terminal sequencing of...
The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially ... more The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.
Understanding the near-field fate of parent chemicals and their decay products in the atmosphere ... more Understanding the near-field fate of parent chemicals and their decay products in the atmosphere provides essential information for the development of remote chemical sensors. To elucidate the near-field fate of candidate chemical signatures, selected gas phase compounds were introduced into atmospheres of varying humidity, temperature and incident light flux. These atmospheres were maintained in an environmental wind tunnel for periods typical of near-field transport scenarios. The range of humidity and temperature into which the compounds were emitted encompassed arid, temperate, and tropical values. Simulated sunlight exposure was used to evaluate the impact of time of release on signature composition. The rates of compound decay and evolution of transformation products under the various environmental conditions were monitored in real time. A Fourier transform infrared spectrometer and a gas chromatograph/mass spectrometer were used to determine chemical concentration, evaluate detectability, and identify potential interferences to the detection capability. Specifically, this report describes the initial system function tests with pinacolyl alcohol and methyl iodide and subsequent atmospheric fate experiments with methylphosphonic dichloride and thionyl chloride. Test system function was evaluated using pinacolyl alcohol because as a relatively non-reactive compound, it served as a negative control for the system. Methyl iodide is a compound known to photodissociate in the atmosphere and therefore was used to evaluate the effectiveness of the test system to detect a known positive effect under specific conditions. Results from the function tests showed that sufficient vapor generation into the large volume of the wind tunnel could be accomplished within a reasonable time period and that the operating conditions of the wind tunnel did not appear to affect the decay rate of the two initial test chemicals. As expected, no near-field decay of pinacolyl alcohol was observed under a wide range of temperature and humidity conditions. Further, both analytical techniques could detect pinacolyl alcohol at and below field-relevant concentrations. No significant difference in the disappearance of pinacolyl alcohol from the wind tunnel atmosphere was observed when the contaminated atmosphere was illuminated with simulated sunlight. We also observed that plants exposed to pinacolyl alcohol absorbed it and then continued to outgas the absorbed compound for a period of hours after being removed from the contaminated atmosphere. Methyl iodide similarly proved unreactive as a function of humidity and temperature. It is known to photodissociate when exposed to ultraviolet light. Relative humidity was found to play an important role in the environmental fate of both compounds. DCMP exhibited an exponential decay rate whose lifetime was a function of the relative humidity. Compound dissociation was so rapid at high relative humidity that sufficient concentrations of the parent chemical could not be attained to permit reliable determination of the decay rate. These indicate an atmospheric lifetime of less than 10 minutes for typical conditions. This study looked at environmental conditions typical of midlatitude environmental conditions, particularly humidity. The results indicate that the near field fate of parent compounds must be considered when choosing chemical signatures for detection. Failure to do so may cause one to incorrectly identify pollution sources or inaccurately estimate their production.
The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the ru... more The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.
Page 1. WM2008 Conference, February 24 28, 2008, Phoenix, AZ Integration of 'omics tec... more Page 1. WM2008 Conference, February 24 28, 2008, Phoenix, AZ Integration of 'omics technologies for characterization of complex microbial ecosystems - 8119 AL Miracle, VL Bailey, SE Baker, AL Bunn, J. Magnuson, BJ ...
The present invention encompasses isolated gene regulatory elements and gene transcription termin... more The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant
High-throughput liquid chromatography mass spectrometry (LC-MS)-based proteomic analysis has emer... more High-throughput liquid chromatography mass spectrometry (LC-MS)-based proteomic analysis has emerged as a powerful tool for functional annotation of genome sequences. These analyses complement the bioinformatic and experimental tools used for deriving, verifying, and functionally annotating models of genes and their transcripts. Furthermore, proteomics extends verification and functional annotation to the level of the translation product of the gene model.
Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolym... more Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its ex...
Page 1. PNNL-18820 Prepared for the US Department of Energy under Contract DE-AC05-76RL01830 PNNL... more Page 1. PNNL-18820 Prepared for the US Department of Energy under Contract DE-AC05-76RL01830 PNNL Fungal Biotechnology Core DOE-OBP Project SE Baker JR Collett JK Magnuson KS Bruno DE Culley EA Panisko MG Butcher Z Dai November 2009 Page 2. ...
The low bioavailability of nutrients and oxygen in the soil environment has hampered successful e... more The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions
The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completel... more The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE, cis -dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA , was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP) 2 . Direct N-terminal sequencing of...
The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially ... more The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.
Understanding the near-field fate of parent chemicals and their decay products in the atmosphere ... more Understanding the near-field fate of parent chemicals and their decay products in the atmosphere provides essential information for the development of remote chemical sensors. To elucidate the near-field fate of candidate chemical signatures, selected gas phase compounds were introduced into atmospheres of varying humidity, temperature and incident light flux. These atmospheres were maintained in an environmental wind tunnel for periods typical of near-field transport scenarios. The range of humidity and temperature into which the compounds were emitted encompassed arid, temperate, and tropical values. Simulated sunlight exposure was used to evaluate the impact of time of release on signature composition. The rates of compound decay and evolution of transformation products under the various environmental conditions were monitored in real time. A Fourier transform infrared spectrometer and a gas chromatograph/mass spectrometer were used to determine chemical concentration, evaluate detectability, and identify potential interferences to the detection capability. Specifically, this report describes the initial system function tests with pinacolyl alcohol and methyl iodide and subsequent atmospheric fate experiments with methylphosphonic dichloride and thionyl chloride. Test system function was evaluated using pinacolyl alcohol because as a relatively non-reactive compound, it served as a negative control for the system. Methyl iodide is a compound known to photodissociate in the atmosphere and therefore was used to evaluate the effectiveness of the test system to detect a known positive effect under specific conditions. Results from the function tests showed that sufficient vapor generation into the large volume of the wind tunnel could be accomplished within a reasonable time period and that the operating conditions of the wind tunnel did not appear to affect the decay rate of the two initial test chemicals. As expected, no near-field decay of pinacolyl alcohol was observed under a wide range of temperature and humidity conditions. Further, both analytical techniques could detect pinacolyl alcohol at and below field-relevant concentrations. No significant difference in the disappearance of pinacolyl alcohol from the wind tunnel atmosphere was observed when the contaminated atmosphere was illuminated with simulated sunlight. We also observed that plants exposed to pinacolyl alcohol absorbed it and then continued to outgas the absorbed compound for a period of hours after being removed from the contaminated atmosphere. Methyl iodide similarly proved unreactive as a function of humidity and temperature. It is known to photodissociate when exposed to ultraviolet light. Relative humidity was found to play an important role in the environmental fate of both compounds. DCMP exhibited an exponential decay rate whose lifetime was a function of the relative humidity. Compound dissociation was so rapid at high relative humidity that sufficient concentrations of the parent chemical could not be attained to permit reliable determination of the decay rate. These indicate an atmospheric lifetime of less than 10 minutes for typical conditions. This study looked at environmental conditions typical of midlatitude environmental conditions, particularly humidity. The results indicate that the near field fate of parent compounds must be considered when choosing chemical signatures for detection. Failure to do so may cause one to incorrectly identify pollution sources or inaccurately estimate their production.
The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the ru... more The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.
Page 1. WM2008 Conference, February 24 28, 2008, Phoenix, AZ Integration of 'omics tec... more Page 1. WM2008 Conference, February 24 28, 2008, Phoenix, AZ Integration of 'omics technologies for characterization of complex microbial ecosystems - 8119 AL Miracle, VL Bailey, SE Baker, AL Bunn, J. Magnuson, BJ ...
The present invention encompasses isolated gene regulatory elements and gene transcription termin... more The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant
High-throughput liquid chromatography mass spectrometry (LC-MS)-based proteomic analysis has emer... more High-throughput liquid chromatography mass spectrometry (LC-MS)-based proteomic analysis has emerged as a powerful tool for functional annotation of genome sequences. These analyses complement the bioinformatic and experimental tools used for deriving, verifying, and functionally annotating models of genes and their transcripts. Furthermore, proteomics extends verification and functional annotation to the level of the translation product of the gene model.
Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolym... more Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its ex...
Page 1. PNNL-18820 Prepared for the US Department of Energy under Contract DE-AC05-76RL01830 PNNL... more Page 1. PNNL-18820 Prepared for the US Department of Energy under Contract DE-AC05-76RL01830 PNNL Fungal Biotechnology Core DOE-OBP Project SE Baker JR Collett JK Magnuson KS Bruno DE Culley EA Panisko MG Butcher Z Dai November 2009 Page 2. ...
The low bioavailability of nutrients and oxygen in the soil environment has hampered successful e... more The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions
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