SEARCH · Engineering Papers
Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products.
Frankiaceae are bacterial endosymbionts that are also found free-living in soil. Here, we present the genome sequences of two novel bacterial members of the order Frankiales , class Actinobacteria , isolated from temperate terrestrial forest soils. The genomes for MT45 and GAS493 indicate a genetic capacity for carbohydrate degradation but not nitrogen fixation.
Forty-two bacterial strains were isolated from root samples of Sorghum bicolor. The strains spanned 17 genera, including Dechloromonas, Duganella, Dyella, Flavobacterium, Herbaspirillum, Lutibacter, Mucilaginibacter, Novosphingobium, Paraburkholderia, Pedobacter, Pleomorphomonas, Rhizobacter, Rhizobium, Rhizomicrobium, Rugamonas, Variovorax, and Xanthobacter. Their whole-genome sequences revealed diverse metabolic processes, including biological nitrogen fixation, in sorghum root microbiota.
We report the genomic features of Bradyrhizobium sp. strain SRS-191, which was isolated from a former nuclear legacy site in Aiken, South Carolina, USA. With a genome size of 7,621,400 bp, the strain harbored genes not only for environmentally beneficial traits (e.g., heavy metal resistance, nitrogen fixation, and aromatic biodegradation) but also for antimicrobial resistance.
A large portion of activity in soil microbial communities occurs in short time frames in response to an increase in C availability, affecting the biogeochemical cycling of nitrogen. These changes are of particular importance as nitrogen represents both a limiting nutrient for terrestrial plants as well as a potential pollutant.
Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood. We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways. In addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products.
Cyanobacteria are photoautotrophic prokaryotes that exhibit robust growth under diverse environmental conditions with minimal nutritional requirements. They can use solar energy to convert CO 2 and other reduced carbon sources into biofuels and chemical products. The genus Cyanothece includes unicellular nitrogen-fixing cyanobacteria that have been shown to offer high levels of hydrogen production and nitrogen fixation. The reconstruction of quality genome-scale metabolic models for organisms with limited annotation resources remains a challenging task. Here we reconstruct and subsequently analyze and compare the metabolism of five Cyanothece strains, namely Cyanothece sp. PCC 7424, 7425, 7822, 8801 and 8802, as the genome-scale metabolic reconstructions iCyc792, iCyn731, iCyj826, iCyp752, and iCyh755 respectively. We compare these phylogenetically related Cyanothece strains to assess their bio-production potential. A systematic workflow is introduced for integrating and prioritizing annotation information from the Universal Protein Resource (Uniprot), NCBI Protein Clusters, and the Rapid Annotations using Subsystems Technology (RAST) method. The genome-scale metabolic models include fully traced photosynthesis reactions and respiratory chains, as well as balanced reactions and GPR associations. Metabolic differences between the organisms are highlighted such as the non-fermentative pathway for alcohol production found in only Cyanothece 7424, 8801, and 8802. Our development workflow provides a path for constructing models using information from curated models of related organisms and reviewed gene annotations. This effort lays the foundation for the expedient construction of curated metabolic models for organisms that, while not being the target of comprehensive research, have a sequenced genome and are related to an organism with a curated metabolic model. Organism-specific models, such as the five presented in this paper, can be used to identify optimal genetic manipulations for targeted metabolite overproduction as well as to investigate the biology of diverse organisms.
The type strain of the prospective Bradyrhizobium shewense sp. nov. ERR11 T , was isolated from a nodule of the leguminous tree Erythrina brucei native to Ethiopia. The type strain Bradyrhizobium yuanmingense CCBAU 10071 T , was isolated from the nodules of Lespedeza cuneata in Beijing, China. The genomes of ERR11 T and CCBAU 10071 T were sequenced by DOE–JGI and deposited at the DOE–JGI genome portal as well as at the European Nucleotide Archive. The genome of ERR11 T is 9,163,226 bp in length and has 102 scaffolds, containing 8548 protein–coding and 86 RNA genes. The CCBAU 10071 T genome is arranged in 108 scaffolds and consists of 8,201,522 bp long and 7776 protein–coding and 85 RNA genes. Both genomes contain symbiotic genes, which are homologous to the genes found in the complete genome sequence of Bradyrhizobium diazoefficiens USDA110 T . The genes encoding for nodulation and nitrogen fixation in ERR11 T showed high sequence similarity with homologous genes found in the draft genome of peanut–nodulating Bradyrhizobium arachidis LMG 26795 T . The nodulation genes nolYA-nodD2D1YABCSUIJ-nolO-nodZ of ERR11 T and CCBAU 10071 T are organized in a similar way to the homologous genes identified in the genomes of USDA110 T , Bradyrhizobium ottawaense USDA 4 and Bradyrhizobium liaoningense CCBAU 05525. The genomes harbor hupSLCFHK and hypBFDE genes that code the expression of hydrogenase, an enzyme that helps rhizobia to uptake hydrogen released by the N 2 -fixation process and genes encoding denitrification functions napEDABC and norCBQD for nitrate and nitric oxide reduction, respectively. The genome of ERR11 T also contains nosRZDFYLX genes encoding nitrous oxide reductase. Based on multilocus sequence analysis of housekeeping genes, the novel species, which contains eight strains formed a unique group close to the B. ottawaense branch. Genome Average Nucleotide Identity (ANI) calculated between the genome sequences of ERR11 T and closely related sequences revealed that strains belonging to B. ottawaense branch (USDA4 and CCBAU15615), were the closest strains to the strain ERR11 T with 95.2% ANI. Type strain ERR11 T showed the highest DDH predicted value with CCBAU15615 (58.5%), followed by USDA 4 (53.1%). Nevertheless, the ANI and DDH values obtained between ERR11 T and CCBAU 15615 or USDA 4 were below the cutoff values (ANI ≥ 96.5%; DDH ≥ 70%) for strains belonging to the same species, suggesting that ERR11 T is a new species. Therefore, based on the phylogenetic analysis, ANI and DDH values, we formally propose the creation of B. shewense sp. nov. with strain ERR11 T (HAMBI 3532 T =LMG 30162 T ) as the type strain.
Sorghum ( Sorghum bicolor ) is an important food, feed, and fodder crop worldwide and is gaining popularity as an energy crop due to its high potential for biomass production. Some sorghum accessions develop many aerial roots and produce an abundant carbohydrate-rich mucilage after rain. This aerial root mucilage is similar to that observed in landraces of maize ( Zea mays ) from southern Mexico, which have been previously shown to host diazotrophs. In this study, we characterized the aerial root development of several sorghum accessions and the impact of humidity on this trait. We conducted a microbiome study of the aerial root mucilage of maize and sorghum and isolated numerous diazotrophs from field sorghum mucilage. We observed that the prevailing phyla in the mucilage were Pseudomonadota, Bacteroidota, and Bacillota. However, bacterial abundances varied based on the genotype and the location. Using acetylene reduction, 15 N 2 gas feeding, and 15 N isotope dilution assays, we confirmed that these sorghum accessions can acquire about 40% of their nitrogen from the atmosphere through these associations on aerial roots. Nitrogen fixation in sorghum aerial root mucilage offers a promising avenue to reduce reliance on synthetic fertilizers and promote sustainable agricultural practices for food, feed, fodder, and bioenergy production.
Arsenic (As) contamination in soil and groundwater has become a serious problem to public health. To examine how microbial communities and functional genes respond to long-term arsenic contamination in vertical soil profile, soil samples were collected from the surface to the depth of 4 m (with an interval of 1 m) after 16-year arsenic downward infiltration. Integrating BioLog and functional gene microarray (GeoChip 3.0) technologies, we showed that microbial metabolic potential and diversity substantially decreased, and community structure was markedly distinct along the depth. Variations in microbial community functional genes, including genes responsible for As resistance, carbon and nitrogen cycling, phosphorus utilization and cytochrome c oxidases were detected. In particular, changes in community structures and activities were correlated with the biogeochemical features along the vertical soil profile when using the rbcL and nifH genes as biomarkers, evident for a gradual transition from aerobic to anaerobic lifestyles. The C/N showed marginally significant correlations with arsenic resistance (p = 0.069) and carbon cycling genes (p = 0.073), and significant correlation with nitrogen fixation genes (p = 0.024). The combination of C/N, NO 3 - and P showed the highest correlation (r = 0.779, p = 0.062) with the microbial community structure. Contradict to our hypotheses, a long-term arsenic downward infiltration was not the primary factor, while the spatial isolation and nutrient availability were the key forces in shaping the community structure. This study provides new insights about the heterogeneity of microbial community metabolic potential and future biodiversity preservation for arsenic bioremediation management.
In this study, the Principal Investigator, F.R. Tabita has teamed up with J. C. Liao from UCLA. This project's main goal is to manipulate regulatory networks in phototrophic bacteria to affect and maximize the production of large amounts of hydrogen gas under conditions where wild-type organisms are constrained by inherent regulatory mechanisms from allowing this to occur. Unrestrained production of hydrogen has been achieved and this will allow for the potential utilization of waste materials as a feed stock to support hydrogen production. By further understanding the means by which regulatory networks interact, this study will seek to maximize the ability of currently available “unrestrained” organisms to produce hydrogen. The organisms to be utilized in this study, phototrophic microorganisms, in particular nonsulfur purple (NSP) bacteria, catalyze many significant processes including the assimilation of carbon dioxide into organic carbon, nitrogen fixation, sulfur oxidation, aromatic acid degradation, and hydrogen oxidation/evolution. Moreover, due to their great metabolic versatility, such organisms highly regulate these processes in the cell and since virtually all such capabilities are dispensable, excellent experimental systems to study aspects of molecular control and biochemistry/physiology are available.
Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.
Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel and other high-value chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory recently elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction. This remarkable transformation, which involves a 6-electron reduction, the formation of a γ-lactam ring, and the generation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the adenosine triphosphate (ATP)-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, specific aims were pursued for the 1) identification of physiological electron donors and in vivo coenzyme F430 synthesis, 2) analysis of the iron sulfur centers, structure, and oligomerization state changes, and 3) characterization of transient intermediates and the intercomponent electron transfer.
Every year, Haber-Bosch nitrogen fixation to form ammonia releases immense volumes of CO2. At the same time, nitrate contamination from untreated wastewater threatens human health across the US. Development of circular processes to efficiently upgrade waste nitrate for reuse is critical to sustainably address this growing environmental hazard. Electrochemical reduction of nitrate operates at ambient temperatures and pressures, and can leverage distributed renewable energy sources and water as a hydrogen source for ammonia production. However, current catalysts lack electron efficiency in reducing nitrate versus water, lack selectivity in forming ammonia as a product, or rely on expensive rare metals, making widespread implementation unfeasible. To overcome these limitations, this proposal seeks a mechanistic understanding of nitrate electrochemical reduction on earth abundant metals and their alloys, with the goal of identifying active-site properties that improve both Faradaic efficiency and product selectivity. This insight will extend to other electrocatalytic reductive process that compete with water reduction.
Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel or other high-value commodity chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction that converts Ni-sirohydrochlorin a , c -diamide to 15,17 3 -seco-F430-17 3 -acid. This remarkable transformation, which involves a 6-electron reduction of the isobacteriochlorin ring system, cyclization of the c -acetamide side chain to form a γ-lactam ring, and the formation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the ATP-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes that catalyze multi-electron redox reactions. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, the following Specific Aims will be pursued to determine the: 1) Identity of the CfbCD reaction product. The exact reaction catalyzed by CfbCD, including the number of electrons transferred and whether it involves enzymatic cyclization, is unclear. Several approaches, including reaction stoichiometry measurements, spectroelectrochemistry, and magnetic resonance spectroscopy will be applied to elucidate the structure of the reaction product and establish whether CfbCD is a reductase or reductive cyclase. 2) Structure, conformational dynamics, and oligomerization state changes of CfbCD. Significant insight into the mechanism and allosteric regulation of CfbCD can be obtained by assessing changes in the structure and dynamics of the complex during the catalytic cycle. To accomplish this, a combination of size-exclusion chromatography, hydrogen-deuterium exchange mass spectrometry, molecular dynamics simulations, and high-resolution structural methods will be employed. 3) Source, order, and stereochemistry of proton additions during CfbCD catalysis. Details regarding the order and stereochemistry of proton additions during the CfbCD reaction will be uncovered using a combined spectroscopic and computational approach. Complementary mechanistic studies employing site-directed mutagenesis and substrate analogs will establish the identity of active site acid residues and the possible involvement of substrate-assisted catalysis during the CfbCD reaction.
Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico$-$ permanently submerged Microcoleus microbial mat (GN-S), and intertidal Lyngbya microbial mat (GN-I)$-$were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H 2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H 2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO 2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of DsrA phylotypes were responsible for H 2 consumption. Incubation with 13 C-acetate and NanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.
Microbes play crucial roles in various biogeochemical processes in the ocean, including carbon (C), nitrogen (N), and phosphorus (P) cycling. Functional gene diversity and the structure of the microbial community determines its metabolic potential and therefore its ecological function in the marine ecosystem. However, little is known about the functional gene composition and metabolic potential of bacterioplankton in estuary areas. The East China Sea (ECS) is a dynamic marginal ecosystem in the western Pacific Ocean that is mainly affected by input from the Changjiang River and the Kuroshio Current. Here, using a high-throughput functional gene microarray (GeoChip), we analyzed the functional gene diversity, composition, structure, and metabolic potential of microbial assemblages in different ECS water masses. Four water masses determined by temperature and salinity relationship showed different patterns of functional gene diversity and composition. Generally, functional gene diversity [Shannon-Weaner's H and reciprocal of Simpson's 1/(1-D)] in the surface water masses was higher than that in the bottom water masses. The different presence and proportion of functional genes involved in C, N, and P cycling among the bacteria of the different water masses showed different metabolic preferences of the microbial populations in the ECS. Genes involved in starch metabolism ( amyA and nplT ) showed higher proportion in microbial communities of the surface water masses than of the bottom water masses. In contrast, a higher proportion of genes involved in chitin degradation was observed in microorganisms of the bottom water masses. Moreover, we found a higher proportion of nitrogen fixation ( nifH ), transformation of hydroxylamine to nitrite ( hao ) and ammonification ( gdh ) genes in the microbial communities of the bottom water masses compared with those of the surface water masses. The spatial variation of microbial functional genes was significantly correlated with salinity, temperature, and chlorophyll based on canonical correspondence analysis, suggesting a significant influence of hydrologic conditions on water microbial communities. Our data provide new insights into better understanding of the functional potential of microbial communities in the complex estuarine-coastal environmental gradient of the ECS.
Integrated virus genomes (prophages) are commonly found in sequenced bacterial genomes but have rarely been described in detail for rhizobial genomes. Cupriavidus taiwanensis STM 6018 is a rhizobial Betaproteobacteria strain that was isolated in 2006 from a root nodule of a Mimosa pudica host in French Guiana, South America. Here we describe features of the genome of STM 6018, focusing on the characterization of two different types of prophages that have been identified in its genome. The draft genome of STM 6018 is 6,553,639 bp, and consists of 80 scaffolds, containing 5,864 protein-coding genes and 61 RNA genes. STM 6018 contains all the nodulation and nitrogen fixation gene clusters common to symbiotic Cupriavidus species; sharing >99.97% bp identity homology to the nod / nif / noeM gene clusters from C. taiwanensis LMG19424 T and “ Cupriavidus neocalidonicus” STM 6070. The STM 6018 genome contains the genomes of two prophages: one complete Mu-like capsular phage and one filamentous phage, which integrates into a putative dif site. This is the first characterization of a filamentous phage found within the genome of a rhizobial strain. Further examination of sequenced rhizobial genomes identified filamentous prophage sequences in several Beta-rhizobial strains but not in any Alphaproteobacterial rhizobia.