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All sourcesNASA NTRSDOE OSTI
NASA NTRS2023

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

nitrogen fixation↗
NASA NTRS2022

CryoEM Structures of the Nitrogenase Complex During Catalytic Turnover

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multi-electron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here we report the cryogenic electron microscopic interrogation of the nitrogenase complex under enzymatic turnover conditions, which has enabled the structural characterization of the nitrogenase reaction intermediates at high resolution for the first time. Our structures show that asymmetry governs all aspects of nitrogenase mechanism including ATP hydrolysis, protein-protein interactions, and catalysis. Furthermore, they reveal several previously unobserved, mechanistically relevant conformational changes near the catalytic iron-molybdenum cofactor that are correlated with the nucleotide-hydrolysis state of the enzyme.

Hannah L. Rutledge↗
NASA NTRS2022

Structures of the Nitrogenase Complex Prepared Under Catalytic Turnover Conditions

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo–electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

Hannah L. Rutledge↗
NASA NTRS2003

Molecular Ecological and Stable Isotopic Studies of Nitrogen Fixation in Modern Microbial Mats

Nitrogen is usually the element limiting biological productivity in the marine environment. Microbial mats, laminated microbial communities analogous to some of the oldest forms of life on Earth, are often the sites of high rates of N fixation (the energetically expensive conversion of atmospheric dinitrogen into a biologically useful form). The N fixing enzyme nitrogenase is generally considered to be of ancient origin, and is widely distributed throughout the Bacterial and Archaeal domains of life, indicating an important role for this process over evolutionary time. The stable isotopic signature of N fixation is purportedly recognizable in organic matter (ancient kerogens as well as present-day microbial mats) as a delta (15)N(sub organic) near zero. We studied two microbial mats exhibiting different rates of N fixation in order to better understand the impact of N fixation on the delta (15)N (sub organic) of the mats, as well as what organisms are important in this process. Mats dominated by the cyanobacterium Microcoleus chthonoplastes grow in permanently submerged hypersaline salterns, and exhibit low rates of N fixation, whereas mats dominated by the cyanobacterium Lyngbya spp grow in an intertidal area, and exhibit rates of N fixation an order of magnitude higher. To examine successional stages in mat growth, both developing and established mats at each location were sampled. PCR and RT-PCR based approaches were used to identify, respectively, the organisms containing nifH (one of the genes that encode nitrogenase) as well as those expressing nifH in these mats. Both mats exhibited a distinct diel cycle of N fixation, with highest rates occurring at night. The delta (15)N(sub organic) of the subtidal Microcoleus mats is near zero whereas the delta (15)N(sub organic) is slightly more positive (+ 2-3%), in the intertidal Lyngbya mats, an interesting difference in view of the fact that overall rates of activity in the intertidal mats are much higher that those in the submerged hypersaline mats. Developing mats in both the subtidal and intertidal locations had delta (15)N(sub organic) values very near those of the established mats. Further work is necessary in order to determine the importance of other transformations of nitrogen on the delta (15)N(sub organic) signature of the mats.

Bebout, B. M.↗
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 NTRS1986

Biosynthetic Porphyrins and the Origin of Photosynthesis

Since the prebiotic atmosphere was anaerobic, if not reducing, a useful function of primordial photosynthesis would have been to photooxidize reduced substrates such as Fe(+2), S(-2) or reduced organic molecules and to emit hydrogen. Experiments have shown that the early biogenic pigments uroporphyrin and coproporphyrin do photooxidize organic compounds and emit hydrogen in the presence of a platinum catalyst. These experiments were carried out in dilute aqueous solution near neutral pH under anaerobic atmosphere, and quantum yields near 10-2 were obtained. Thus relevant prebiotic conditions were maintained. Rather then to further optimize conditions, attempts were made to replace the platinum catalyst by a more prebiotically suitable catalyst. Trials with an Fe4S4(SR)4 cluster, in analogy to the present hydrogenase and nitrogenase, were not successful. However, experiments using cobalt complexes to catalyze the formation of hydrogen are promising. In analogy with biological photosynthetic systems which group pigments, electron transfer molecules and enzymes in clusters for efficiency, it was found that binding the biogenic porphyrins to the polyvinyl alcohol used to support the platinum catalyst did increase the quantum yield of the reaction. It was also found that ultraviolet light can serve to photo-oxidize porphyrinogens to porphyrins under anaerobic conditions. Thus the formation of the colorless porphyriogens by the extraordinarily simple biosynthetic pathway would not be a problem because of the prevalence of UV light in the prebiotic, anoxic atmosphere.

David Mauzerall↗
NASA NTRS1997

Will Global Change Effect Primary Productivity in Coastal Ecosystems?

Algae are the base of coastal food webs because they provide the source of organic carbon for the remaining members of the community. Thus, the rate that they produce organic carbon to a large extent controls the productivity of the entire ecosystem. Factors that control algal productivity range from the physical (e.g., temperature, light), chemical (e.g., nutrient levels) to the biological (e.g., grazing). Currently, levels of atmospheric carbon dioxide surficial fluxes of ultraviolet radiation are rising. Both of these environmental variables can have a profound effect on algal productivity. Atmospheric carbon dioxide may increase surficial levels of dissolved inorganic carbon. Our laboratory and field studies of algal mats and phytoplankton cultures under ambient and elevated levels of pCO2 show that elevated levels of inorganic carbon can cause an increase in photosynthetic rates. In some cases, this increase will cause an increase in phytoplankton numbers. There may be an increase in the excretion of fixed carbon, which in turn may enhance bacterial productivity. Alternatively, in analogy with studies on the effect of elevated pCO2 on plants, the phytoplankton could change their carbon to nitrogen ratios, which will effect the feeding of the planktonic grazers. The seasonal depletion of stratospheric ozone has resulted in elevated fluxes of UVB radiation superimposed on the normal seasonal variation. Present surface UV fluxes have a significant impact on phytoplankton physiology, including the inhibition of the light and dark reactions of photosynthesis, inhibition of nitrogenase activity, inhibition of heterocyst formation, reduction in motility, increased synthesis of the UV-screening pigment scytonemin, and mutation. After reviewing these issues, recent work in our lab on measuring the effect of UV radiation on phytoplankton in the San Francisco Bay Estuary will be presented.

Rothschild, Lynn J.↗
NASA NTRS1992

Stimulation of nodulation in field peas (Pisum sativum) by low concentrations of ammonium in hydroponic culture

Although the inhibitory effects of high concentrations of mineral N (> 1.0 mM) on nodule development and function have often been studied, the effects of low, static concentrations of NH4+ (< 1.0 mM) on nodulation are unknown. In the present experiments we examine the effects of static concentrations of NH4+ at 0, 0.1 and 0.5 mM in flowing, hydroponic culture on nodule establishment and nitrogenase activity in field peas [Pisum sativum L. cv. Express (Svalof AB)] for the initial 28 days after planting (DAP). Peas grown in the presence of low concentrations of NH4+ had significantly greater nodule numbers (up to 4-fold) than plants grown without NH4+. Nodule dry weight per plant was significantly higher at 14, 21 and 28 DAP in plants grown in the presence of NH4+, but individual nodule mass was lower than in plants grown without NH4+. The nodulation pattern of the plants supplied with NH4+ was similar to that often reported for supernodulating mutants, however the plants did not express other growth habits associated with supernodulation. Estimates of N2 fixation indicate that the plus-NH4+ peas fixed as much or more N2 than the plants supplied with minus-NH4+ nutrient solution. There were no significant differences in nodule numbers, nodule mass or NH4+ uptake between the plants grown at the two concentrations of NH4+. Nodulation appeared to autoregulate by 14 DAP in the minus-NH4+ treatment. Plant growth and N accumulation in the minus-NH4+ plants lagged behind those of the plus-NH4+ treatments prior to N2 fixation becoming well established in the final week of the experiment. The plus-NH4+ treatments appeared not to elicit autoregulation and plants continued to initiate nodules throughout the experiment.

NASA Program CELSS↗
NASA NTRS1997

Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

NASA Discipline Cell Biology↗
NASA NTRS1994

Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142

It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.

Non-NASA Center↗