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1. Characterizing the interplay of rubisco 1 and nitrogenase enzymes in anaerobic-photoheterotrophically grown Rhodopseudomonas palustris CGA009 through a genome-scale metabolic and expression model

   https://doi.org/10.1101/2022.03.03.482919  

   Chowdhury, Niaz B.; Alsiyabi, A.; Saha, R. (March 2022, bioRxiv)

   Rhodopseudomonas palustris CGA009 (R. palustris) is a gram negative purple non-sulfur bacteria that grows phototrophically or chemotrophically by fixing or catabolizing a wide array of substrates including lignin breakdown products (e.g., p-coumarate) for its carbon and nitrogen requirements. It can grow aerobically or anaerobically and can use light, inorganic, and organic compounds for energy production. Due to its ability to convert different carbon sources into useful products in anaerobic mode, this study, for the first time, reconstructed a metabolic and expression (ME-) model of R. palustris to investigate its anaerobic-photoheterotrophic growth. Unlike metabolic (M-) models, ME-models include transcription and translation reactions along with macromolecules synthesis and couple these reactions with growth rate. This unique feature of the ME-model led to nonlinear growth curve predictions which matched closely with experimental growth rate data. At the theoretical maximum growth rate, the ME-model suggested a diminishing rate of carbon fixation and predicted malate dehydrogenase and glycerol-3 phosphate dehydrogenase as alternate electron sinks. Moreover, the ME-model also identified ferredoxin as a key regulator in distributing electrons between major redox balancing pathways. Since ME-models include turnover rate for each metabolic reaction, it was used to successfully capture experimentally observed temperature regulation of different nitrogenases. Overall, these unique features of the ME-model demonstrated the influence of nitrogenases and rubiscos on R. palustris growth and predicted a key regulator in distributing electrons between major redox balancing pathways, thus establishing a platform for in silico investigation of R. palustris metabolism from a multi-omics perspective. 

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2. Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

   https://doi.org/10.1111/1462-2920.14955  

   Luxem, Katja_E; Kraepiel, Anne_M_L; Zhang, Lichun; Waldbauer, Jacob_R; Zhang, Xinning (February 2020, Environmental Microbiology)

   Summary Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)‐only nitrogenase metalloenzymes. Studies with purified enzymes have found that the ‘alternative’ V‐ and Fe‐nitrogenases generally reduce N₂more slowly and produce more byproduct H₂than the Mo‐nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotrophRhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V‐nitrogenase supports growth as fast as the Mo‐nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V‐nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V‐nitrogenase on acetate, the wild‐type strain uses exclusively the Mo‐nitrogenase on both carbon substrates. Notably, the differences in H₂:N₂stoichiometry by alternative nitrogenases (~1.5 for V‐nitrogenase, ~4–7 for Fe‐nitrogenase) and Mo‐nitrogenase (~1) measured here are lower than priorin vitroestimates. These results indicate that the metabolic costs of V‐based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment. 

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3. Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins

   https://doi.org/10.1093/gbe/evac031  

   Garcia, Amanda K; Kolaczkowski, Bryan; Kaçar, Betül (March 2022, Genome Biology and Evolution)

   Archibald, John (Ed.)

   Abstract The evolution of biological nitrogen fixation, uniquely catalyzed by nitrogenase enzymes, has been one of the most consequential biogeochemical innovations over life’s history. Though understanding the early evolution of nitrogen fixation has been a longstanding goal from molecular, biogeochemical, and planetary perspectives, its origins remain enigmatic. In this study, we reconstructed the evolutionary histories of nitrogenases, as well as homologous maturase proteins that participate in the assembly of the nitrogenase active-site cofactor but are not able to fix nitrogen. We combined phylogenetic and ancestral sequence inference with an analysis of predicted functionally divergent sites between nitrogenases and maturases to infer the nitrogen-fixing capabilities of their shared ancestors. Our results provide phylogenetic constraints to the emergence of nitrogen fixation and are consistent with a model wherein nitrogenases emerged from maturase-like predecessors. Though the precise functional role of such a predecessor protein remains speculative, our results highlight evolutionary contingency as a significant factor shaping the evolution of a biogeochemically essential enzyme. 

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4. Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

   https://doi.org/10.1128/AEM.00849-20  

   Luxem, Katja E.; Leavitt, William D.; Zhang, Xinning; Atomi, Haruyuki (September 2020, Applied and Environmental Microbiology)

   ABSTRACT Biological nitrogen fixation is catalyzed by the enzyme nitrogenase. Two forms of this metalloenzyme, the vanadium (V)- and iron (Fe)-only nitrogenases, were recently found to reduce small amounts of carbon dioxide (CO 2 ) into the potent greenhouse gas methane (CH 4 ). Here, we report carbon ( 13 C/ 12 C) and hydrogen ( 2 H/ 1 H) stable isotopic compositions and fractionations of methane generated by V- and Fe-only nitrogenases in the metabolically versatile nitrogen fixer Rhodopseudomonas palustris . The stable carbon isotope fractionation imparted by both forms of alternative nitrogenase are within the range observed for hydrogenotrophic methanogenesis ( 13 α CO2/CH4 = 1.051 ± 0.002 for V-nitrogenase and 1.055 ± 0.001 for Fe-only nitrogenase; values are means ± standard errors). In contrast, the hydrogen isotope fractionations ( 2 α H2O/CH4 = 2.071 ± 0.014 for V-nitrogenase and 2.078 ± 0.018 for Fe-only nitrogenase) are the largest of any known biogenic or geogenic pathway. The large 2 α H2O/CH4 shows that the reaction pathway nitrogenases use to form methane strongly discriminates against 2 H, and that 2 α H2O/CH4 distinguishes nitrogenase-derived methane from all other known biotic and abiotic sources. These findings on nitrogenase-derived methane will help constrain carbon and nitrogen flows in microbial communities and the role of the alternative nitrogenases in global biogeochemical cycles. IMPORTANCE All forms of life require nitrogen for growth. Many different kinds of microbes living in diverse environments make inert nitrogen gas from the atmosphere bioavailable using a special enzyme, nitrogenase. Nitrogenase has a wide substrate range, and, in addition to producing bioavailable nitrogen, some forms of nitrogenase also produce small amounts of the greenhouse gas methane. This is different from other microbes that produce methane to generate energy. Until now, there was no good way to determine when microbes with nitrogenases are making methane in nature. Here, we present an isotopic fingerprint that allows scientists to distinguish methane from microbes making it for energy versus those making it as a by-product of nitrogen acquisition. With this new fingerprint, it will be possible to improve our understanding of the relationship between methane production and nitrogen acquisition in nature. 

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5. Phototrophic Lactate Utilization by Rhodopseudomonas palustris Is Stimulated by Coutilization with Additional Substrates

   https://doi.org/10.1128/AEM.00048-19  

   Govindaraju, Alekhya; McKinlay, James B.; LaSarre, Breah; Parales, Rebecca E. (June 2019, Applied and Environmental Microbiology)

   ABSTRACT The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris is known for its metabolic versatility and is of interest for various industrial and environmental applications. Despite decades of research on R. palustris growth under diverse conditions, patterns of R. palustris growth and carbon utilization with mixtures of carbon substrates remain largely unknown. R. palustris readily utilizes most short-chain organic acids but cannot readily use lactate as a sole carbon source. Here we investigated the influence of mixed-substrate utilization on phototrophic lactate consumption by R. palustris . We found that lactate was simultaneously utilized with a variety of other organic acids and glycerol in time frames that were insufficient for R. palustris growth on lactate alone. Thus, lactate utilization by R. palustris was expedited by its coutilization with additional substrates. Separately, experiments using carbon pairs that did not contain lactate revealed acetate-mediated inhibition of glycerol utilization in R. palustris . This inhibition was specific to the acetate-glycerol pair, as R. palustris simultaneously utilized acetate or glycerol when either was paired with succinate or lactate. Overall, our results demonstrate that (i) R. palustris commonly employs simultaneous mixed-substrate utilization, (ii) mixed-substrate utilization expands the spectrum of readily utilized organic acids in this species, and (iii) R. palustris has the capacity to exert carbon catabolite control in a substrate-specific manner. IMPORTANCE Bacterial carbon source utilization is frequently assessed using cultures provided single carbon sources. However, the utilization of carbon mixtures by bacteria (i.e., mixed-substrate utilization) is of both fundamental and practical importance; it is central to bacterial physiology and ecology, and it influences the utility of bacteria as biotechnology. Here we investigated mixed-substrate utilization by the model organism Rhodopseudomonas palustris . Using mixtures of organic acids and glycerol, we show that R. palustris exhibits an expanded range of usable carbon substrates when provided substrates in mixtures. Specifically, coutilization enabled the prompt consumption of lactate, a substrate that is otherwise not readily used by R. palustris . Additionally, we found that R. palustris utilizes acetate and glycerol sequentially, revealing that this species has the capacity to use some substrates in a preferential order. These results provide insights into R. palustris physiology that will aid the use of R. palustris for industrial and commercial applications. 

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