Pat Unkefer - Academia.edu (original) (raw)
Papers by Pat Unkefer
Accurate measurement of carbon in soils remains one of the largest challenges in understanding so... more Accurate measurement of carbon in soils remains one of the largest challenges in understanding sources and sinks of carbon in terrestrial systems. Conventional methods of analyzing carbon require large initial volumes of soil, therefore obtaining samples for conventional analysis is time consuming and expensive. The application of laser-induced breakdown spectroscopy (LIBS) to carbon analysis has several advantages over conventional methods,
Canadian Journal of Microbiology, 1995
The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated ... more The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated alfalfa plants results in an increase in N/sub 2/-fixation potential and growth, but a 40-50% decrease in nodule glutamine synthetase (GS) activity, as compared to nodulated control plants. Tabtoxinine-..beta..-Lactam an exocellular toxin produced by Pseudomonas syringae pv tabaci irreversibly inhibits glutamine synthetase. Partial purification of nodule GS by DEAE-cellulose chromatography reveals two enzyme forms are present (GS/sub n1/ and GS/sub n2/). In vitro inactivation of the two glutamine synthetases associated with the nodule indicates a differential sensitivity to T-..beta..-L. The nodule specific GS/sub n1/ is much less sensitive to T-..beta..-L than the GS/sub n2/ enzyme, which was found to coelute with the root enzyme (GS/sub r/). However, both GS/sub n1/ and GS/sub n2/ are rapidly inactivated by methionine sulfoximine, another irreversible inhibitor of GS.
Extensive industrial and DOE use of chlorinated hydrocarbons has resulted in widespread soil and ... more Extensive industrial and DOE use of chlorinated hydrocarbons has resulted in widespread soil and groundwater contamination. Bioremediation is a potential remedy because various bacterial strains degrade chlorinated compounds, including trichloroethylene (TCE). Previous reports indicated that the propane monooxygenase (PMO) enzyme from Mycobacterium vaccae degraded TCE. These reports included additional substrates and resulting products that were inconsistent with PMO forming an epoxide intermediate; thus PMO appeared to be an attractive alternative to the epoxideforming methane monooxygenase (MMO) enzyme. PMO had not been isolated and was largely uncharacterized. This project characterized PMO and its mechanism. PMO had a multi-component quaternary structure that was remarkably similar to that of MMO. The products formed by PMO from two key substrates were not as previously reported and were precisely those predicted by an epoxide intermediate. The observed lack of unique character of PMO has caused us to forgo further study.
Environmental Injury to Plants, 1990
Pseudomonas syringae pv. tabaci, a pathogen of tobacco, is capable of colonizing the rhizosphere ... more Pseudomonas syringae pv. tabaci, a pathogen of tobacco, is capable of colonizing the rhizosphere of many plants. This pathogen excretes tabtoxinine-β-lactam (TβL), an active site directed, irreversible inhibitor of glutamine synthetase. TβL is produced in planta, in the rhizosphere, and under certain culture conditions. However, the factors which regulated TβL production in these environments are unknown. As a first step in characterizing TβL synthesis by P. syringae pv. tabaci, the authors have determined the effects of root exudates and various nutrients on production of TβL by P. syringae pv. tabaci PT113.
Applied Microbiology and Biotechnology, 1995
Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of pr... more Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT by M. vaccae. During the cometabolic process, there was transient production of a brown chromophore. This compound was identified as 4-amino-2,6-dinitrobenzoic acid. When M. vaccae was incubated with [14C]TNT and propane, 50% of the added radiolabel was incorporated into the cellular lipid fraction. These results suggest that ring cleavage occurred prior to the incorporation of radiolabeUed carbon into phosphatidyl-L-serine, phosphatidylethanolamine, cardiolipin, and other polar lipids.
Canadian Journal of Microbiology, 2000
Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro gro... more Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression o...
The DOE complex has generated vast quantities of complex heterogeneous mixed wastes. Paint stripp... more The DOE complex has generated vast quantities of complex heterogeneous mixed wastes. Paint stripper waste (PSW) is a complex waste that arose from decontamination and decommissioning activities. It contains paint stripper, cheesecloth, cellulose-based paints with Pb and Cr, and suspect Pu. Los Alamos National Laboratory has 150--200 barrels of PSW and other national laboratories such as Rocky Flats Plant have
We developed the necessary instrumentation using several Hamilton syringe pumps coupled to a L TQ... more We developed the necessary instrumentation using several Hamilton syringe pumps coupled to a L TQ-FT's ICR mass analyzer. The L TQ-FT's ICR mass analyzer allows detection of compounds with ppm mass accuracy, which allows assignment of molecular formula facilitating identification of potential ligands.
The main objective of NAABB was to combine science, technology, and engineering expertise from ac... more The main objective of NAABB was to combine science, technology, and engineering expertise from across the nation to break down critical technical barriers to commercialization of algae-based biofuels. The approach was to address technology development across the entire value chain of algal biofuels production, from selection of strains to cultivation, harvesting, extraction, fuel conversion, and agricultural coproduct production. Sustainable practices and financial feasibility assessments ununderscored the approach and drove the technology development
Improvement of biological nitrogen fixation represents a potential source of both increased food ... more Improvement of biological nitrogen fixation represents a potential source of both increased food production and decreased dependence on costly chemical fertilizer. They report the results of an investigation of the molecular basis of a unique, microbial-mediated mechanism for increased growth and nitrogen fixation rates in alfalfa. Inoculation of alfalfa plants with both Rhizobium meliloti and Pseudomonas syringae pv tabaci provides increased growth and N/sub 2/-fixation rates of alfalfa. Tabaci produces tabtoxinine-..beta..-lactam (T..beta..L), an exocellular product and glutamine synthetase (GS) inhibitor. The association of this pathogen with nodulating alfalfa plants appears to alter the normal regulation of nitrogen fixation such that nitrogenase activity is stimulated and GS activity is inhibited. Studies of the soluble amino acids in these nodules and the activities of the ammonia assimilatory enzymes indicate alternative pathways of ammonia assimilation are being employed.
Algal Research, 2017
In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium bega... more In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy's Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played by metabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels.
Accurate measurement of carbon in soils remains one of the largest challenges in understanding so... more Accurate measurement of carbon in soils remains one of the largest challenges in understanding sources and sinks of carbon in terrestrial systems. Conventional methods of analyzing carbon require large initial volumes of soil, therefore obtaining samples for conventional analysis is time consuming and expensive. The application of laser-induced breakdown spectroscopy (LIBS) to carbon analysis has several advantages over conventional methods,
Canadian Journal of Microbiology, 1995
The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated ... more The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated alfalfa plants results in an increase in N/sub 2/-fixation potential and growth, but a 40-50% decrease in nodule glutamine synthetase (GS) activity, as compared to nodulated control plants. Tabtoxinine-..beta..-Lactam an exocellular toxin produced by Pseudomonas syringae pv tabaci irreversibly inhibits glutamine synthetase. Partial purification of nodule GS by DEAE-cellulose chromatography reveals two enzyme forms are present (GS/sub n1/ and GS/sub n2/). In vitro inactivation of the two glutamine synthetases associated with the nodule indicates a differential sensitivity to T-..beta..-L. The nodule specific GS/sub n1/ is much less sensitive to T-..beta..-L than the GS/sub n2/ enzyme, which was found to coelute with the root enzyme (GS/sub r/). However, both GS/sub n1/ and GS/sub n2/ are rapidly inactivated by methionine sulfoximine, another irreversible inhibitor of GS.
Extensive industrial and DOE use of chlorinated hydrocarbons has resulted in widespread soil and ... more Extensive industrial and DOE use of chlorinated hydrocarbons has resulted in widespread soil and groundwater contamination. Bioremediation is a potential remedy because various bacterial strains degrade chlorinated compounds, including trichloroethylene (TCE). Previous reports indicated that the propane monooxygenase (PMO) enzyme from Mycobacterium vaccae degraded TCE. These reports included additional substrates and resulting products that were inconsistent with PMO forming an epoxide intermediate; thus PMO appeared to be an attractive alternative to the epoxideforming methane monooxygenase (MMO) enzyme. PMO had not been isolated and was largely uncharacterized. This project characterized PMO and its mechanism. PMO had a multi-component quaternary structure that was remarkably similar to that of MMO. The products formed by PMO from two key substrates were not as previously reported and were precisely those predicted by an epoxide intermediate. The observed lack of unique character of PMO has caused us to forgo further study.
Environmental Injury to Plants, 1990
Pseudomonas syringae pv. tabaci, a pathogen of tobacco, is capable of colonizing the rhizosphere ... more Pseudomonas syringae pv. tabaci, a pathogen of tobacco, is capable of colonizing the rhizosphere of many plants. This pathogen excretes tabtoxinine-β-lactam (TβL), an active site directed, irreversible inhibitor of glutamine synthetase. TβL is produced in planta, in the rhizosphere, and under certain culture conditions. However, the factors which regulated TβL production in these environments are unknown. As a first step in characterizing TβL synthesis by P. syringae pv. tabaci, the authors have determined the effects of root exudates and various nutrients on production of TβL by P. syringae pv. tabaci PT113.
Applied Microbiology and Biotechnology, 1995
Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of pr... more Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT by M. vaccae. During the cometabolic process, there was transient production of a brown chromophore. This compound was identified as 4-amino-2,6-dinitrobenzoic acid. When M. vaccae was incubated with [14C]TNT and propane, 50% of the added radiolabel was incorporated into the cellular lipid fraction. These results suggest that ring cleavage occurred prior to the incorporation of radiolabeUed carbon into phosphatidyl-L-serine, phosphatidylethanolamine, cardiolipin, and other polar lipids.
Canadian Journal of Microbiology, 2000
Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro gro... more Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression o...
The DOE complex has generated vast quantities of complex heterogeneous mixed wastes. Paint stripp... more The DOE complex has generated vast quantities of complex heterogeneous mixed wastes. Paint stripper waste (PSW) is a complex waste that arose from decontamination and decommissioning activities. It contains paint stripper, cheesecloth, cellulose-based paints with Pb and Cr, and suspect Pu. Los Alamos National Laboratory has 150--200 barrels of PSW and other national laboratories such as Rocky Flats Plant have
We developed the necessary instrumentation using several Hamilton syringe pumps coupled to a L TQ... more We developed the necessary instrumentation using several Hamilton syringe pumps coupled to a L TQ-FT's ICR mass analyzer. The L TQ-FT's ICR mass analyzer allows detection of compounds with ppm mass accuracy, which allows assignment of molecular formula facilitating identification of potential ligands.
The main objective of NAABB was to combine science, technology, and engineering expertise from ac... more The main objective of NAABB was to combine science, technology, and engineering expertise from across the nation to break down critical technical barriers to commercialization of algae-based biofuels. The approach was to address technology development across the entire value chain of algal biofuels production, from selection of strains to cultivation, harvesting, extraction, fuel conversion, and agricultural coproduct production. Sustainable practices and financial feasibility assessments ununderscored the approach and drove the technology development
Improvement of biological nitrogen fixation represents a potential source of both increased food ... more Improvement of biological nitrogen fixation represents a potential source of both increased food production and decreased dependence on costly chemical fertilizer. They report the results of an investigation of the molecular basis of a unique, microbial-mediated mechanism for increased growth and nitrogen fixation rates in alfalfa. Inoculation of alfalfa plants with both Rhizobium meliloti and Pseudomonas syringae pv tabaci provides increased growth and N/sub 2/-fixation rates of alfalfa. Tabaci produces tabtoxinine-..beta..-lactam (T..beta..L), an exocellular product and glutamine synthetase (GS) inhibitor. The association of this pathogen with nodulating alfalfa plants appears to alter the normal regulation of nitrogen fixation such that nitrogenase activity is stimulated and GS activity is inhibited. Studies of the soluble amino acids in these nodules and the activities of the ammonia assimilatory enzymes indicate alternative pathways of ammonia assimilation are being employed.
Algal Research, 2017
In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium bega... more In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy's Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played by metabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels.