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The splitting of dinitrogen (N₂) and reduction to ammonia (NH₃) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N₂ reduction is accomplished at high temperature and pressure, whereas N₂ fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N₂ into NH₃. The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N₂ reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N₂ reduction to NH₃.

The world’s dependence on industrially produced nitrogenous fertilizers has created a dichotomy of issues. First, parts of the globe lack access to fertilizers, leading to poor crop yields that significantly limit nutrition while contributing to disease and starvation. There is considerable interest in promoting biological nitrogen fixation (BNF) as a mechanism to reduce the inputs of nitrogenous fertilizers in agriculture, but considerable fundamental knowledge gaps still need to be addressed. BNF is catalyzed by nitrogenase, which requires a large input of energy in the form of ATP and low potential electrons. Diazotrophs that respire aerobically have an advantage in meeting the ATP demands of BNF but face challenges in protecting nitrogenase from inactivation by oxygen. Here, we constructed a genome-scale metabolic model of the nitrogen-fixing bacterium Azotobacter vinelandii , which uses a complex respiratory protection mechanism to consume oxygen at a high rate to keep intracellular conditions microaerobic. Our model accurately predicts growth rate under high oxygen and substrate concentrations, consistent with a large electron flux directed to the respiratory protection mechanism. While a partially decoupled electron transport chain compensates for some of the energy imbalance under high-oxygen conditions, it does not account for all substrate intake, leading to increased maintenance rates. Interestingly, the respiratory protection mechanism is required for accurate predictions even when ammonia is supplemented during growth, suggesting that the respiratory protection mechanism might be a core principle of metabolism and not just used for nitrogenase protection. We have also shown that rearrangement of flux through the electron transport system allows A. vinelandii to adapt to different oxygen concentrations, metal availability, and genetic disruption, which cause an ammonia excretion phenotype. Accurately determining the energy balance in an aerobic nitrogen-fixing metabolic model is required for future engineering approaches. IMPORTANCE The world’s dependence on industrially produced nitrogenous fertilizers has created a dichotomy of issues. First, parts of the globe lack access to fertilizers, leading to poor crop yields that significantly limit nutrition while contributing to disease and starvation. In contrast, abundant nitrogenous fertilizers and associated overuse in large agricultural systems result in compromised soil quality and downstream environmental issues. Thus, there is considerable interest in expanding the impacts of BNF to promote improved crop yields in places struggling with access to industrial fertilizers while reducing fertilizer input in areas where overuse results in the degradation of soil health. A more robust and fundamental understanding of BNF biochemistry and microbial physiology will enable strategies to promote new and more robust associations between nitrogen-fixing microorganisms and crop plants.

Geologic, chemical and isotopic evidence indicate that Earth has experienced numerous intervals of widespread glaciation throughout its history, with roughly 11% of present day Earth’s land surface covered in ice. Despite the pervasive nature of glacial ice both today and in Earth’s past and the potential contribution of these systems to global biogeochemical cycles, the composition and phylogenetic structure of an active microbial community in subglacial systems has yet to be described. Here, using RNA-based approaches, we demonstrate the presence of active and endogenous archaeal, bacterial and eukaryal assemblages in cold (0–1 °C) subglacial sediments sampled from Robertson Glacier, Alberta, Canada. Patterns in the phylogenetic structure and composition of subglacial sediment small subunit (SSU) ribosomal RNA (rRNA) assemblages indicate greater diversity and evenness than in glacial surface environments, possibly due to facilitative or competitive interactions among populations in the subglacial environment. The combination of phylogenetically more even and more diverse assemblages in the subglacial environment suggests minimal niche overlap and optimization to capture a wider spectrum of the limited nutrients and chemical energy made available from weathering of bedrock minerals. The prevalence of SSU rRNA affiliated with lithoautotrophic bacteria, autotrophic methane producing archaea and heterotrophic eukarya in the subglacial environment is consistent with this hypothesis and suggests an active contribution to the global carbon cycle. Collectively, our findings demonstrate that subglacial environments harbor endogenous active ecosystems that have the potential to impact global biogeochemical cycles over extended periods of time.

The availability of fixed nitrogen limits overall agricultural crop production worldwide. The so-called modern “green revolution” catalyzed by the widespread application of nitrogenous fertilizer has propelled global population growth. It has led to imbalances in global biogeochemical nitrogen cycling, resulting in a “nitrogen problem” that is growing at a similar trajectory to the “carbon problem”. As a result of the increasing imbalances in nitrogen cycling and additional environmental problems such as soil acidification, there is renewed and increasing interest in increasing the contributions of biological nitrogen fixation to reduce the inputs of nitrogenous fertilizers in agriculture. Interestingly, biological nitrogen fixation, or life’s ability to convert atmospheric dinitrogen to ammonia, is restricted to microbial life and not associated with any known eukaryotes. It is not clear why plants never evolved the ability to fix nitrogen and rather form associations with nitrogen-fixing microorganisms. Perhaps it is because of the large energy demand of the process, the oxygen sensitivity of the enzymatic apparatus, or simply failure to encounter the appropriate selective pressure. Whatever the reason, it is clear that this ability of crop plants, especially cereals, would transform modern agriculture once again. Successfully engineering plants will require creating an oxygen-free niche that can supply ample energy in a tightly regulated manner to minimize energy waste and ensure the ammonia produced is assimilated. Nitrogen-fixing aerobic bacteria can perhaps provide a blueprint for engineering nitrogen-fixing plants. This short review discusses the key features of robust nitrogen fixation in the model nitrogen-fixing aerobe, gamma proteobacteria Azotobacter vinelandii, in the context of the basic requirements for engineering nitrogen-fixing plants.

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188 Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: ( i ) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; ( ii ) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188 Ser ; ( iii ) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188 Ser ) are important for communication between the two halves.

Histone deacetylases (HDACs) are a family of enzymes responsible for regulating DNA transcription by modulating its binding to histone proteins. HDACs are overexpressed in several types of cancers and are recognised as drug targets. Vorinostat, or suberanilohydroxamic acid (SAHA), is an histone deacetylase (HDAC) inhibitor with a hydroxamic acid as a zinc-binding group (ZBG), and it has been FDA approved for the treatment of T-cell lymphoma. In this work, phosphorus-based SAHA analogues were synthesised to assess their zinc-binding effectiveness compared to the hydroxamic acid of SAHA. Specifically, we examined phosphate, phosphoramidate and phosphorothiolate groups as isosteres of the canonical hydroxamic acid motif of conventional HDAC inhibitors. The compounds were screened for binding to HDAC enzymes from HeLa cell lysate. The most potent derivatives were then screened against HDAC3 and HDAC8 isoforms. HDAC inhibition assays demonstrated that these phosphorus-based SAHA analogs exhibited slow binding to HDACs but with greater potency than phosphonate SAHA analogs examined previously. All compounds inhibited HDACs, the most potent having an IC of 50 µM.

The ability of an arthropod to harbor and transmit pathogens is termed “vector competency.” Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro , in vivo , and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed “vector competency.” Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease ( Borrelia burgdorferi ) and anaplasmosis ( Anaplasma phagocytophilum ). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.

The Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydFEG), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydFEG. The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.