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Herbert Böhme - One of the best experts on this subject based on the ideXlab platform.
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transcriptional and mutational analysis of the uptake hydrogenase of the filamentous cyanobacterium Anabaena variabilis atcc 29413
Journal of Bacteriology, 2000Co-Authors: Thomas Happe, Kathrin Schutz, Herbert BöhmeAbstract:A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase ( hupSL ) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis . The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N 2 -fixing conditions, the mutant strain exhibited significantly increased rates in H 2 accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H 2 developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.
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Identification of amino acids responsible for the oxygen sensitivity of ferredoxins from Anabaena variabilis using site-directed mutagenesis.
Biochimica et Biophysica Acta, 1999Co-Authors: Brij B Singh, I. Curdt, Prakash S. Bisen, Cornelis Jakobs, Dietmar Schomburg, Herbert BöhmeAbstract:Abstract The filamentous cyanobacterium Anabaena variabilis (ATCC 29413) possesses two molybdenum dependent nitrogenase systems, nif1 and nif2 . The nif1 system is regulated by a developmental program involving heterocyst differentiation; the nif2 system is expressed in all cells only under anaerobic conditions and the expression is controlled environmentally. The genes fdxH1 and fdxH2 , encoding two [2Fe–2S] ferredoxins, are part of the these two distinct and differently regulated nif gene clusters. The sensitivity of both ferredoxins to oxygen was different; the half-life of FdxH2 in air was only ≈1.5 h, while FdxH1 retained 80% of its nitrogenase activity after 24 h. We used site-directed mutagenesis to identify the role of individual amino acid residues responsible for oxygen sensitivity and found out that the FdxH2 double mutant I76A/V77L was much more resistant to oxygen than the wild-type ferredoxin (FdxH2) and similar to FdxH1. By modelling it was shown that the accessibility of the cavity around the iron–sulfur cluster was responsible for that.
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identification of amino acids responsible for the oxygen sensitivity of ferredoxins from Anabaena variabilis using site directed mutagenesis
Biochimica et Biophysica Acta, 1999Co-Authors: Brij B Singh, I. Curdt, Prakash S. Bisen, Cornelis Jakobs, Dietmar Schomburg, Herbert BöhmeAbstract:The filamentous cyanobacterium Anabaena variabilis (ATCC 29413) possesses two molybdenum dependent nitrogenase systems, nif1 and nif2. The nif1 system is regulated by a developmental program involving heterocyst differentiation; the nif2 system is expressed in all cells only under anaerobic conditions and the expression is controlled environmentally. The genes fdxH1 and fdxH2, encoding two [2Fe-2S] ferredoxins, are part of the these two distinct and differently regulated nif gene clusters. The sensitivity of both ferredoxins to oxygen was different; the half-life of FdxH2 in air was only approximately 1.5 h, while FdxH1 retained 80% of its nitrogenase activity after 24 h. We used site-directed mutagenesis to identify the role of individual amino acid residues responsible for oxygen sensitivity and found out that the FdxH2 double mutant I76A/V77L was much more resistant to oxygen than the wild-type ferredoxin (FdxH2) and similar to FdxH1. By modelling it was shown that the accessibility of the cavity around the iron-sulfur cluster was responsible for that.
Teresa Thiel - One of the best experts on this subject based on the ideXlab platform.
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Standards in Genomic Sciences (2014) 9:562-573 DOI:10.4056/sigs.3899418 The Genomic Standards Consortium Complete genome sequence of Anabaena variabilis ATCC
2014Co-Authors: Teresa Thiel, Brenda S Pratte, Jinshun Zhong, Lynne Goodwin, Susan Lucas, Cliff Han, Sam Pitluck, Alex Copel, Miriam L. L, Nikos C KyrpidesAbstract:Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic g rowth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Ana-baena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, g rowing well at approximately 40 ° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence
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complete genome sequence of Anabaena variabilis atcc 29413
Standards in Genomic Sciences, 2014Co-Authors: Teresa Thiel, Brenda S Pratte, Jinshun Zhong, Lynne Goodwin, Alex Copeland, Susan Lucas, Cliff Han, Sam PitluckAbstract:Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.
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Regulation of Fructose Transport and Its Effect on Fructose Toxicity
2008Co-Authors: Justin L. Ungerer, Brenda S Pratte, Teresa ThielAbstract:Anabaena variabilis grows heterotrophically using fructose, while the close relative Anabaena sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC transporter, herein named frtRABC, into Anabaena sp. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes are necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR, frt
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Transcription of hupSL in Anabaena variabilis ATCC 29413 Is Regulated by NtcA and Not by Hydrogen �
2007Co-Authors: Philip D. Weyman, Brenda Pratte, Teresa ThielAbstract:Nitrogen-fixing cyanobacteria such as Anabaena variabilis ATCC 29413 use an uptake hydrogenase, encoded by hupSL, to recycle hydrogen gas that is produced as an obligate by-product of nitrogen fixation. The regulation of hupSL in A. variabilis is likely to differ from that of the closely related Anabaena sp. strain PCC 7120 because A. variabilis lacks the excision element-mediated regulation that characterizes hupSL regulation in strain PCC 7120. An analysis of the hupSL transcript in a nitrogenase mutant of A. variabilis that does not produce any detectable hydrogen indicated that neither nitrogen fixation nor hydrogen gas was required for the induction of hupSL. Furthermore, exogenous addition of hydrogen gas did not stimulate hupSL transcription. Transcriptional reporter constructs indicated that the accumulation of hupSL transcript after nitrogen stepdown was restricted primarily to the microaerobic heterocysts. Anoxic conditions were not sufficient to induce hupSL transcription. The induction of hupSL after nitrogen step-down was reduced in a mutant in the global nitrogen regulator NtcA, but was not reduced in a mutant unable to form heterocysts. A consensus NtcAbinding site was identified upstream of hupSL, and NtcA was found to bind to this region. Thus, while neither hydrogen gas nor anoxia controlled the expression of hupSL, its expression was controlled by NtcA. Heterocyst differentiation was not required for hupSL induction in response to nitrogen step-down, but heterocystlocalized cues may add an additional level of regulation to hupSL
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High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413
2006Co-Authors: Brenda S Pratte, Teresa ThielAbstract:High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 � 10 �9 M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri. Vanadium, molybdenum, and tungsten cofactors are essential for a variety of enzymes. The three oxyanions are very similar in size and structure, with some ability to substitute fo
Peter Boger - One of the best experts on this subject based on the ideXlab platform.
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proteolytic degradation of dinitrogenase reductase from Anabaena variabilis atcc 29413 as a consequence of atp depletion and impact of oxygen
Journal of Bacteriology, 1996Co-Authors: Jorg Durner, Ines Bohm, Oliver C Knorzer, Peter BogerAbstract:Both components of nitrogenase, dinitrogenase and dinitrogenase reductase, are rapidly inactivated by oxygen. To investigate the proteolytic degradation of dinitrogenase reductase irreversibly destroyed by high oxygen concentrations, we carried out in vitro experiments with heterocyst extracts from Anabaena variabilis ATCC 29413. The results indicate a direct dependence of degradation on the applied oxygen concentration. Although the degrees of degradation were similar for both the modified and unmodified subunits of dinitrogenase reductase, there was a significant difference with respect to the cleavage products observed. The pattern of effective protease inhibitors suggests the involvement of serine proteases with chymotrypsin- and trypsin-like specificity. A protective effect was obtained by saturation of the nucleotide binding sites of dinitrogenase reductase with either ATP or ADP. As shown by gel filtration experiments, the adenylates prevented the nitrogenase subunits from extensive noncovalent aggregation, which is usually considered evidence for a denaturing process. The in vitro degradation of dinitrogenase reductase is discussed in connection with previous reports on degradation of nitrogenase in cyanobacteria under oxygen stress and/or starvation.
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in vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis atcc 29413
Journal of Bacteriology, 1992Co-Authors: Ines Bohm, Anneliese Ernst, Andrea Halbherr, S Smaglinski, Peter BogerAbstract:Abstract Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. Boger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) dinitrogenase reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.
Brenda S Pratte - One of the best experts on this subject based on the ideXlab platform.
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Standards in Genomic Sciences (2014) 9:562-573 DOI:10.4056/sigs.3899418 The Genomic Standards Consortium Complete genome sequence of Anabaena variabilis ATCC
2014Co-Authors: Teresa Thiel, Brenda S Pratte, Jinshun Zhong, Lynne Goodwin, Susan Lucas, Cliff Han, Sam Pitluck, Alex Copel, Miriam L. L, Nikos C KyrpidesAbstract:Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic g rowth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Ana-baena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, g rowing well at approximately 40 ° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence
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complete genome sequence of Anabaena variabilis atcc 29413
Standards in Genomic Sciences, 2014Co-Authors: Teresa Thiel, Brenda S Pratte, Jinshun Zhong, Lynne Goodwin, Alex Copeland, Susan Lucas, Cliff Han, Sam PitluckAbstract:Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.
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Regulation of Fructose Transport and Its Effect on Fructose Toxicity
2008Co-Authors: Justin L. Ungerer, Brenda S Pratte, Teresa ThielAbstract:Anabaena variabilis grows heterotrophically using fructose, while the close relative Anabaena sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC transporter, herein named frtRABC, into Anabaena sp. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes are necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR, frt
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High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413
2006Co-Authors: Brenda S Pratte, Teresa ThielAbstract:High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 � 10 �9 M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri. Vanadium, molybdenum, and tungsten cofactors are essential for a variety of enzymes. The three oxyanions are very similar in size and structure, with some ability to substitute fo
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effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis atcc 29413
Journal of Bacteriology, 2001Co-Authors: Teresa Thiel, Brenda S PratteAbstract:Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.
Hans Erik Molager Christensen - One of the best experts on this subject based on the ideXlab platform.
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characterization of conformational exchange of a histidine side chain protonation rotamerization and tautomerization of his61 in plastocyanin from Anabaena variabilis
Journal of the American Chemical Society, 2008Co-Authors: Mathias A S Hass, Flemming D Hansen, Hans Erik Molager ChristensenAbstract:A model describing conformational exchange of His 61 in plastocyanin from Anabaena variabilis is presented. A detailed picture of the exchange dynamics has been obtained using solution NMR relaxation measurements, chemical shift titrations, and structural information provided by a high-resolution crystal structure of the protein. A three-site model for chemical exchange that involves interconversion among the tautomeric and protonated forms of the histidine side chain with rates that are fast on the NMR chemical shift time scale can account for all of the data. In general, in the limit of fast exchange, it is not possible to obtain separate measures of chemical shift differences and populations of the participating states using NMR. However, we show here that when the data mentioned above are combined, it is possible to extract values of all of the parameters that characterize the exchange process, including rates, populations, and chemical shift changes, and to provide cross-validations that establish th...
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detection of short lived transient protein protein interactions by intermolecular nuclear paramagnetic relaxation plastocyanin from Anabaena variabilis
Journal of the American Chemical Society, 2003Co-Authors: Flemming D Hansen, Mathias A S Hass, Hans Erik Molager Christensen, Jens UlstrupAbstract:An NMR approach is presented that provides detailed information about short-lived, transient interactions between protein molecules in solution. The approach is based on the longitudinal paramagnetic relaxation rates of the protein nuclei and requires that at least one of the interacting molecules is paramagnetic. The specific interactions are monitored by the intermolecular paramagnetic contribution to the relaxation of protons at or close to the interaction surface. By applying the approach to plastocyanin from Anabaena variabilis, specific regions of interaction that may be involved in the electron self-exchange process of this plastocyanin were identified. This is in accord with recent 15N NMR relaxation studies of the backbone dynamics of Anabaena variabilis plastocyanin, with site-directed mutagenesis studies of the functional importance of the corresponding regions in Phormidium laminosum plastocyanin and with the crystal packing surface of P. laminosum plastocyanin.