The Experts below are selected from a list of 366 Experts worldwide ranked by ideXlab platform
Zhao-xun Liang - One of the best experts on this subject based on the ideXlab platform.
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A Flavin Cofactor-Binding PAS Domain Regulates c-di-GMP Synthesis in AxDGC2 from Acetobacter xylinum
Biochemistry, 2009Co-Authors: Feng Rao, Zhen Luo, Zhao-xun LiangAbstract:The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the Obligate Aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) domain and two putative catalytic domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential and unexpected modification of FAD in one of the mutants, none of the single mutations was able to completely disrupt the transmission of the signal to the GGDEF domain, indicating that the change in the FAD redox state can still trigger structural changes in the PAS domain probably by using substituted hydrogen-bonded water networks. Meanwhile, although the EAL domain of AxDGC2 was found to be catalytically inactive toward c-di-GMP, it was capable of hydrolyzing some phosphodiester bond-containing nonphysiological substrates. Together with the previously reported oxygen-dependent activity of the homologous AxPDEA1, the results provided new insight into relationships among oxygen level, c-di-GMP concentration, and cellulose synthesis in A. xylinum.
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a flavin cofactor binding pas domain regulates c di gmp synthesis in axdgc2 from acetobacter xylinum
Biochemistry, 2009Co-Authors: Yaning Qi, Zhao-xun LiangAbstract:The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the Obligate Aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) domain and two putative catalytic domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential...
Robert K Poole - One of the best experts on this subject based on the ideXlab platform.
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Biology and
2013Co-Authors: Susan Hill, Robert K Poole, Mark J S Kelly, Gary T Sawers, Biotechnology TheAbstract:The cydR gene product, required for regulation of cytochrome 6d expression in the Obligate Aerobe Azotobacter vinelandii, is an Fnr-like protei
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biosynthesis of poly β hydroxybutyrate phb is controlled by cydr fnr in the Obligate Aerobe azotobacter vinelandii
Fems Microbiology Letters, 2001Co-Authors: Arthur J G Moir, Susan Hill, Gary Sawers, Robert K PooleAbstract:CydR is an Fnr-like protein in the obligatory aerobic nitrogen-fixing bacterium Azotobacter vinelandii. The cydR mutant overproduces the cytochrome bd terminal oxidase. Using two-dimensional polyacrylamide gel electrophoresis, we showed that β-ketothiolase and acetoacetyl-CoA reductase were also overexpressed in the cydR mutant. Fumarase C and a coenzyme A transferase, possibly succinyl-SCoA transferase, were decreased in this mutant. Enzyme assays confirmed the elevated β-ketothiolase and acetoacetyl-CoA reductase activities in this mutant. The cydR mutant accumulated poly-β-hydroxybutyrate throughout the exponential growth phase, unlike the wild-type strain that only accumulated poly-β-hydroxybutyrate during stationary phase. The results demonstrate that CydR controls poly-β-hydroxybutyrate synthesis in A. vinelandii.
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regulation of cytochrome bd expression in the Obligate Aerobe azotobacter vinelandii by cydr fnr sensitivity to oxygen reactive oxygen species and nitric oxide
Journal of Biological Chemistry, 2000Co-Authors: Guanghui Wu, Hugo Cruzramos, Susan Hill, Jeffrey Green, Gary Sawers, Robert K PooleAbstract:Abstract Azotobacter vinelandii is an Obligately aerobic bacterium in which aerotolerant nitrogen fixation requires cytochrome bd. Regulation of cytochromebd expression is achieved by CydR (an Fnr homologue), which represses transcription of the oxidase genes cydAB. cydABmRNA was mapped by primer extension; the transcriptional start site was determined, and putative −10 and −35 regions were deduced. Two “CydR boxes,” one at the +1 site and one upstream of the −35 region, were identified. Transcriptionally inactive, purified CydR was converted, by adding NifS, cysteine, and Fe2+, into an active form possessing acid-labile sulfide and spectra suggesting a [4Fe-4S]2+ cluster. Reconstituted CydR specifically bound both CydR boxes cooperatively, with higher affinity for the nearer consensus +1 site. Low concentrations of O2 or NO ([O2]/[[CydR] or [NO]/[CydR] = 0.1–0.6) elicited loss of the 420 nm absorbance attributed to the [4Fe-4S]2+ cluster, formation of a 315 nm species, and loss of ability to retard DNA migration. Retardation by reconstituted CydR was enhanced by superoxide dismutase and/or catalase, suggesting a role for reactive oxygen species in CydR inactivation. The role of CydR in regulating cydAB expression in the supposedly anoxic cytoplasm of A. vinelandii and similarities to cydAB regulation by Fnr in Escherichia coli are discussed.
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the cydr gene product required for regulation of cytochrome bd expression in the Obligate Aerobe azotobacter vinelandii is an fnr like protein
Microbiology, 1997Co-Authors: Susan Hill, Gary Sawers, Mark J S Kelly, Robert K PooleAbstract:Summary: The cytochrome bd complex in the Obligately aerobic diazotroph Azotobacter vinelandii is an oxidase, which, in vivo, has a low affinity for oxygen and is required for respiratory protection of nitrogenase. Mutations caused by insertion of Tn5-B20 upstream of the structural genes (cydAB) for cytochrome bd result in over-expression of this oxidase and, for unexplained reasons, inability of the organism to grow microaerobically. Cloning and sequencing of this upstream region revealed a gene, cydR. The deduced amino acid sequence of CydR indicates that it is a new member of the Fnr class of regulators and that it represses cydAB expression. Refined mapping data for three insertions in cydR are presented. The cloned cydR gene complemented anaerobic growth of Escherichia coli fnr mutants and strongly enhanced expression of a narG-lacZ fusion in an E. coli fnr mutant.
Brett M Barney - One of the best experts on this subject based on the ideXlab platform.
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key factors affecting ammonium production by an azotobacter vinelandii strain deregulated for biological nitrogen fixation
Microbial Cell Factories, 2020Co-Authors: Mary H Plunkett, Carolann M Knutson, Brett M BarneyAbstract:The Obligate Aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.
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efforts toward optimization of aerobic biohydrogen reveal details of secondary regulation of biological nitrogen fixation by nitrogenous compounds in azotobacter vinelandii
Applied Microbiology and Biotechnology, 2018Co-Authors: Carolann M Knutson, Mary H Plunkett, Rachel A Liming, Brett M BarneyAbstract:Biological nitrogen fixation (BNF) through the enzyme nitrogenase is performed by a unique class of organisms known as diazotrophs. One interesting facet of BNF is that it produces molecular hydrogen (H2) as a requisite by-product. In the absence of N2 substrate, or under conditions that limit access of N2 to the enzyme through modifications of amino acids near the active site, nitrogenase activity can be redirected toward a role as a dedicated hydrogenase. In free-living diazotrophs, nitrogenases are tightly regulated to minimize BNF to meet only the growth requirements of the cell, and are often accompanied by uptake hydrogenases that oxidize the H2 by-product to recover the electrons from this product. The wild-type strain of Azotobacter vinelandii performs all of the tasks described above to minimize losses of H2 while also growing as an Obligate Aerobe. Individual alterations to A. vinelandii have been demonstrated that disrupt key aspects of the N2 reduction cycle, thereby diverting resources and energy toward the production of H2. In this work, we have combined three approaches to override the primary regulation of BNF and redirect metabolism to drive biological H2 production by nitrogenase in A. vinelandii. The resulting H2-producing strain was further utilized as a surrogate to study secondary, post-transcriptional regulation of BNF by several key nitrogen-containing metabolites. The improvement in yields of H2 that were achieved through various combinations of these three approaches was compared and is presented along with the insights into inhibition of BNF by several nitrogen compounds that are common in various waste streams. The findings indicate that both ammonium and nitrite hinder BNF through this secondary inhibition, but urea and nitrate do not. These results provide essential details to inform future biosynthetic approaches to yield nitrogen products that do not inadvertently inhibit BNF.
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gene deletions resulting in increased nitrogen release by azotobacter vinelandii application of a novel nitrogen biosensor
Applied and Environmental Microbiology, 2015Co-Authors: Brett M Barney, Carolann M Knutson, Lauren J Eberhart, Janet M Ohlert, Mary H PlunkettAbstract:ABSTRACT Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an Obligate Aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.
Mary H Plunkett - One of the best experts on this subject based on the ideXlab platform.
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key factors affecting ammonium production by an azotobacter vinelandii strain deregulated for biological nitrogen fixation
Microbial Cell Factories, 2020Co-Authors: Mary H Plunkett, Carolann M Knutson, Brett M BarneyAbstract:The Obligate Aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.
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efforts toward optimization of aerobic biohydrogen reveal details of secondary regulation of biological nitrogen fixation by nitrogenous compounds in azotobacter vinelandii
Applied Microbiology and Biotechnology, 2018Co-Authors: Carolann M Knutson, Mary H Plunkett, Rachel A Liming, Brett M BarneyAbstract:Biological nitrogen fixation (BNF) through the enzyme nitrogenase is performed by a unique class of organisms known as diazotrophs. One interesting facet of BNF is that it produces molecular hydrogen (H2) as a requisite by-product. In the absence of N2 substrate, or under conditions that limit access of N2 to the enzyme through modifications of amino acids near the active site, nitrogenase activity can be redirected toward a role as a dedicated hydrogenase. In free-living diazotrophs, nitrogenases are tightly regulated to minimize BNF to meet only the growth requirements of the cell, and are often accompanied by uptake hydrogenases that oxidize the H2 by-product to recover the electrons from this product. The wild-type strain of Azotobacter vinelandii performs all of the tasks described above to minimize losses of H2 while also growing as an Obligate Aerobe. Individual alterations to A. vinelandii have been demonstrated that disrupt key aspects of the N2 reduction cycle, thereby diverting resources and energy toward the production of H2. In this work, we have combined three approaches to override the primary regulation of BNF and redirect metabolism to drive biological H2 production by nitrogenase in A. vinelandii. The resulting H2-producing strain was further utilized as a surrogate to study secondary, post-transcriptional regulation of BNF by several key nitrogen-containing metabolites. The improvement in yields of H2 that were achieved through various combinations of these three approaches was compared and is presented along with the insights into inhibition of BNF by several nitrogen compounds that are common in various waste streams. The findings indicate that both ammonium and nitrite hinder BNF through this secondary inhibition, but urea and nitrate do not. These results provide essential details to inform future biosynthetic approaches to yield nitrogen products that do not inadvertently inhibit BNF.
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gene deletions resulting in increased nitrogen release by azotobacter vinelandii application of a novel nitrogen biosensor
Applied and Environmental Microbiology, 2015Co-Authors: Brett M Barney, Carolann M Knutson, Lauren J Eberhart, Janet M Ohlert, Mary H PlunkettAbstract:ABSTRACT Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an Obligate Aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.
Carolann M Knutson - One of the best experts on this subject based on the ideXlab platform.
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key factors affecting ammonium production by an azotobacter vinelandii strain deregulated for biological nitrogen fixation
Microbial Cell Factories, 2020Co-Authors: Mary H Plunkett, Carolann M Knutson, Brett M BarneyAbstract:The Obligate Aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.
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efforts toward optimization of aerobic biohydrogen reveal details of secondary regulation of biological nitrogen fixation by nitrogenous compounds in azotobacter vinelandii
Applied Microbiology and Biotechnology, 2018Co-Authors: Carolann M Knutson, Mary H Plunkett, Rachel A Liming, Brett M BarneyAbstract:Biological nitrogen fixation (BNF) through the enzyme nitrogenase is performed by a unique class of organisms known as diazotrophs. One interesting facet of BNF is that it produces molecular hydrogen (H2) as a requisite by-product. In the absence of N2 substrate, or under conditions that limit access of N2 to the enzyme through modifications of amino acids near the active site, nitrogenase activity can be redirected toward a role as a dedicated hydrogenase. In free-living diazotrophs, nitrogenases are tightly regulated to minimize BNF to meet only the growth requirements of the cell, and are often accompanied by uptake hydrogenases that oxidize the H2 by-product to recover the electrons from this product. The wild-type strain of Azotobacter vinelandii performs all of the tasks described above to minimize losses of H2 while also growing as an Obligate Aerobe. Individual alterations to A. vinelandii have been demonstrated that disrupt key aspects of the N2 reduction cycle, thereby diverting resources and energy toward the production of H2. In this work, we have combined three approaches to override the primary regulation of BNF and redirect metabolism to drive biological H2 production by nitrogenase in A. vinelandii. The resulting H2-producing strain was further utilized as a surrogate to study secondary, post-transcriptional regulation of BNF by several key nitrogen-containing metabolites. The improvement in yields of H2 that were achieved through various combinations of these three approaches was compared and is presented along with the insights into inhibition of BNF by several nitrogen compounds that are common in various waste streams. The findings indicate that both ammonium and nitrite hinder BNF through this secondary inhibition, but urea and nitrate do not. These results provide essential details to inform future biosynthetic approaches to yield nitrogen products that do not inadvertently inhibit BNF.
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gene deletions resulting in increased nitrogen release by azotobacter vinelandii application of a novel nitrogen biosensor
Applied and Environmental Microbiology, 2015Co-Authors: Brett M Barney, Carolann M Knutson, Lauren J Eberhart, Janet M Ohlert, Mary H PlunkettAbstract:ABSTRACT Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an Obligate Aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.
