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46 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ 54 -dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL–NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph,Azotobacter vinelandii.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in Escherichia coli

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe 8 S 7 ]) and M-cluster core (or L-cluster; [Fe 8 S 9 C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

The maturation and installation of the active site metal cluster (FeMo-co, Fe 7 S 9 CMo- R -homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe 8 S 9 C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDK RS contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK RS ). Apo-NifDK RS could be activated for N 2 reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK RS plus E. coli produced NifB RS and NifH RS were sufficient to yield active NifDK RS when incubated with the necessary substrates (homocitrate, molybdate, and S -adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifH RS and NifB RS proteins were independently shown to be functional. The reconstituted NifDK RS demonstrated reduction of N 2 , protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH RS , NifB RS , and apo-NifDK RS .

Payá-Tormo, Lucía↗
DOE OSTI2020

Switchgrass Rhizosphere Metabolite Chemistry Driven by Nitrogen Availability

Plants and soil microorganisms interact closely in the rhizosphere where plants may exchange carbon (C) for functional benefits from the microbial community. For example, the bioenergy crop, switchgrass (Panicum virgatum) is thought to exchange root-exuded C for nitrogen (N) fixed by diazotrophs (free-living N-fixers). However, this interaction is not well characterized and it is not known how or if switchgrass responds to diazotrophs or their activity. To explore this question, we assessed rhizosphere metabolite chemistry of switchgrass grown in a hydroponic system under two N levels and under inoculated or uninoculated conditions. Plants were grown with the inoculum Azotobacter vinelandii DJ for 3 days before harvest. We found switchgrass root exudate chemistry to be driven by N availability. Total metabolite concentrations were generally greater under high N versus low N and unaffected by inoculation. Examination of rhizosphere chemical fingerprints indicates metabolite chemistry was also driven strongly by N availability with a greater relative abundance of carbohydrates under high N and greater relative abundance of organic acids under low N. We also found evidence of changes in rhizosphere chemical fingerprints by inoculation treatment. However, we found little evidence of N treatment and inoculation interaction effects that suggests this response is not directly mediated by N availability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Data from: Switchgrass rhizosphere metabolite chemistry driven by nitrogen availability

Plants and soil microorganisms interact closely in the rhizosphere where plants may exchange carbon (C) for functional benefits from the microbial community. For example, the bioenergy crop, switchgrass (Panicum virgatum) is thought to exchange root-exuded C for nitrogen (N) fixed by diazotrophs (free-living N-fixers). However, this interaction is not well characterized and it is not known how or if switchgrass responds to diazotrophs or their activity. To explore this question, we assessed rhizosphere metabolite chemistry of switchgrass grown in a hydroponic system under two N levels and under inoculated or uninoculated conditions. Plants were grown with the inoculum Azotobacter vinelandii DJ for three days before harvest. We found switchgrass root exudate chemistry to be driven by N availability. Total metabolite concentrations were generally greater under high N versus low N and unaffected by inoculation. Examination of rhizosphere chemical fingerprints indicates metabolite chemistry was also driven strongly by N availability with a greater relative abundance of carbohydrates under high N and greater relative abundance of organic acids under low N. We also found evidence of changes in rhizosphere chemical fingerprints by inoculation treatment. However, we found little evidence of N treatment and inoculation interaction effects which suggests this response is not directly mediated by N availability.

09 BIOMASS FUELS↗
NASA NTRS1978

Assessment of free-living nitrogen fixing microorganisms for commercial nitrogen fixation

Ammonia production by Klebsiella pneumoniae is not economical with present strains and improving nitrogen fixation to its theoretical limits in this organism is not sufficient to achieve economic viability. Because the value of both the hydrogen produced by this organism and the methane value of the carbon source required greatly exceed the value of the ammonia formed, ammonia (fixed nitrogen) should be considered the by-product. The production of hydrogen by KLEBSIELLA or other anaerobic nitrogen fixers should receive additional study, because the activity of nitrogenase offers a significant improvement in hydrogen production. The production of fixed nitrogen in the form of cell mass by Azotobacter is also uneconomical and the methane value of the carbon substrate exceeds the value of the nitrogen fixed. Parametric studies indicate that as efficiencies approach the theoretical limits the economics may become competitive. The use of nif-derepressed microorganisms, particularly blue-green algae, may have significant potential for in situ fertilization in the environment.

Stokes, B. O.↗
NASA NTRS1985

New perspectives on bacterial ferredoxin evolution

Ferredoxins are low-molecular-weight, nonheme, iron proteins which function as electron carriers in a wide variety of electron transport chains. Howard et al. (1983) have suggested that the amino end of Azotobacter vinelandii ferredoxin shows a greater similarity to the carboxyl end of ferredoxin from Chromatium vinosum and that their half-chain sequences are homologous when the half-chains of either species are considered in inverse order. Examination of this proposition has made it necessary to reevaluate previous conclusions concerning the evolution of bacterial ferredoxin. Attention is given to the properties of the bacterial ferredoxin sequences, and the evolution of the bacterial ferredoxins.

George, D. G.↗
NASA NTRS1989

Biological nitrogen fixation under primordial Martian partial pressures of dinitrogen

One of the most striking differences between the conditions on early Mars and earth was a low (18 mb) partial pressure of N2 (pN2) on early Mars, as opposed to 780 mb N2 on earth. To investigate the possibility of biological nitrogen fixation under conditions of early Mars, experiments were carried out on the growth of Azotobacter vinelandii and Azomonas agilis in nitrogen-free synthetic medium under various partial pressures of N2 (ranging from 780 to 0 mb). It was found that, although the biomass, cell number, and growth rate of these bacteria decreased with decreasing pN2 values below pN2 of 400 mb, both microorganisms were capable of growing at pN2 as low as 5 mb (but not at of below 1 mb), indicating that biological fixation of nitrogen could have occurred on primordial Mars.

Klingler, J. M.↗
NASA NTRS1992

Survival of microorganisms in smectite clays - Implications for Martian exobiology

The survival of Baccillus subtilis, Azotobacter chroococcum, and the enteric bacteriophage MS2 has been examined in clays representing terrestrial (Wyoming type montmorillonite) and Martian (Fe3+ montmorillonite) soils exposed to terrestrial and Martian environmental conditions of temperature and atmospheric composition and pressure. An important finding is that MS2 survived simulated Mars conditions better than the terrestrial environment, probably owing to stabilization of the virus caused by the cold and dry conditions of the simulated Mars environment. This finding, the first published indication that viruses may be able to survive in Mars-type soils, may have important implications for future missions to Mars.

Moll, Deborah M.↗
NASA NTRS1994

Do prokaryotes contain microtubules?

In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins.

NASA Discipline Exobiology↗