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1. The Importance of Protein Phosphorylation for Signaling and Metabolism in Response to Diel Light Cycling and Nutrient Availability in a Marine Diatom

   https://doi.org/10.3390/biology9070155  

   Tan, Maxine H.; Smith, Sarah R.; Hixson, Kim K.; Tan, Justin; McCarthy, James K.; Kustka, Adam B.; Allen, Andrew E. (July 2020, Biology)

   null (Ed.)

   Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms. 

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2. Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

   https://doi.org/10.1128/spectrum.02815-23  

   Rivier, Alex J.; Myers, Kevin S.; Garcia, Amanda K.; Sobol, Morgan S.; Kaçar, Betül (October 2023, Microbiology Spectrum)

   Youssef, Noha H. (Ed.)

   ABSTRACT 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 ofAzotobacter vinelandiiengineered 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. IMPORTANCEAzotobacter vinelandiiis a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineeredA. vinelandiistrains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts. 

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3. Bacterial Approaches for Assembling Iron-Sulfur Proteins

   https://doi.org/10.1128/mBio.02425-21  

   Esquilin-Lebron, Karla; Dubrac, Sarah; Barras, Frédéric; Boyd, Jeffrey M. (December 2021, mBio)

   Yount, Jacob (Ed.)

   ABSTRACT Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target. 

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4. Warming Iron-Limited Oceans Enhance Nitrogen Fixation and Drive Biogeographic Specialization of the Globally Important Cyanobacterium Crocosphaera

   https://doi.org/10.3389/fmars.2021.628363  

   Yang, Nina; Merkel, Carlin A.; Lin, Yu-An; Levine, Naomi M.; Hawco, Nicholas J.; Jiang, Hai-Bo; Qu, Ping-Ping; DeMers, Michelle A.; Webb, Eric A.; Fu, Fei-Xue; et al (February 2021, Frontiers in Marine Science)

   null (Ed.)

   Primary productivity in the nutrient-poor subtropical ocean gyres depends on new nitrogen inputs from nitrogen fixers that convert inert dinitrogen gas into bioavailable forms. Temperature and iron (Fe) availability constrain marine nitrogen fixation, and both are changing due to anthropogenic ocean warming. We examined the physiological responses of the globally important marine nitrogen fixer, Crocosphaera watsonii across its full thermal range as a function of iron availability. At the lower end of its thermal range, from 22 to 27°C, Crocosphaera growth, nitrogen fixation, and Nitrogen-specific Iron Use Efficiencies (N-IUEs, mol N fixed hour –1 mol Fe –1 ) increased with temperature. At an optimal growth temperature of 27°C, N-IUEs were 66% higher under iron-limited conditions than iron-replete conditions, indicating that low-iron availability increases metabolic efficiency. However, Crocosphaera growth and function decrease from 27 to 32°C, temperatures that are predicted for an increasing fraction of tropical oceans in the future. Altogether, this suggests that Crocosphaera are well adapted to iron-limited, warm waters, within prescribed limits. A model incorporating these results under the IPCC RCP 8.5 warming scenario predicts that Crocosphaera N-IUEs could increase by a net 47% by 2100, particularly in higher-latitude waters. These results contrast with published responses of another dominant nitrogen fixer ( Trichodesmium ), with predicted N-IUEs that increase most in low-latitude, tropical waters. These models project that differing responses of Crocosphaera and Trichodesmium N-IUEs to future warming of iron-limited oceans could enhance their current contributions to global marine nitrogen fixation with rates increasing by ∼91 and ∼22%, respectively, thereby shifting their relative importance to marine new production and also intensifying their regional divergence. Thus, interactive temperature and iron effects may profoundly transform existing paradigms of nitrogen biogeochemistry and primary productivity in open ocean regimes. 

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5. Characterizing the Interplay of Rubisco and Nitrogenase Enzymes in Anaerobic-Photoheterotrophically Grown Rhodopseudomonas palustris CGA009 through a Genome-Scale Metabolic and Expression Model

   https://doi.org/10.1128/spectrum.01463-22  

   Chowdhury, Niaz Bahar; Alsiyabi, Adil; Saha, Rajib (June 2022, Microbiology Spectrum)

   Gralnick, Jeffrey A. (Ed.)

   ABSTRACT Rhodopseudomonas palustris CGA009 is a Gram-negative purple nonsulfur bacterium that grows phototrophically by fixing carbon dioxide and nitrogen or chemotrophically by fixing or catabolizing a wide array of substrates, including lignin breakdown products for its carbon and fixing nitrogen for its 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 during anaerobic growth, this study 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. Because ME models include the 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 multiomics perspective. IMPORTANCE In this work, we reconstructed the first ME model for a purple nonsulfur bacterium (PNSB). Using the ME model, different aspects of R. palustris metabolism were examined. First, the ME model was used to analyze how reducing power entering the R. palustris cell through organic carbon sources gets partitioned into biomass, carbon dioxide fixation, and nitrogen fixation. Furthermore, the ME model predicted electron flux through ferredoxin as a major bottleneck in distributing electrons to nitrogenase enzymes. Next, the ME model characterized different nitrogenase enzymes and successfully recapitulated experimentally observed temperature regulations of those enzymes. Identifying the bottleneck responsible for transferring an electron to nitrogenase enzymes and recapitulating the temperature regulation of different nitrogenase enzymes can have profound implications in metabolic engineering, such as hydrogen production from R. palustris . Another interesting application of this ME model can be to take advantage of its redox balancing strategy to gain an understanding of the regulatory mechanism of biodegradable plastic production precursors, such as polyhydroxybutyrate (PHB). 

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