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1. Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and ¹³ C-Tracer Analysis

   https://doi.org/10.1128/mSystems.00173-21  

   Lawson, Christopher E. ; Mundinger, Aniela B. ; Koch, Hanna ; Jacobson, Tyler B. ; Weathersby, Coty A. ; Jetten, Mike S. ; Pabst, Martin ; Amador-Noguez, Daniel ; Noguera, Daniel R. ; McMahon, Katherine ; et al ( August 2021 , mSystems)

   Hallam, Steven J. (Ed.)

   ABSTRACT Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis ( i Nmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [ 13 C]bicarbonate and [ 13 C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO 2 fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO 2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira ’s in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting theirmore »metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13 C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria.« less
2. Vitamin B ₁₂ -dependent biosynthesis ties amplified 2-methylhopanoid production during oceanic anoxic events to nitrification

   https://doi.org/10.1073/pnas.2012357117  

   Elling, Felix J. ; Hemingway, Jordon D. ; Evans, Thomas W. ; Kharbush, Jenan J. ; Spieck, Eva ; Summons, Roger E. ; Pearson, Ann ( December 2020 , Proceedings of the National Academy of Sciences)

   Bacterial hopanoid lipids are ubiquitous in the geologic record and serve as biomarkers for reconstructing Earth’s climatic and biogeochemical evolution. Specifically, the abundance of 2-methylhopanoids deposited during Mesozoic ocean anoxic events (OAEs) and other intervals has been interpreted to reflect proliferation of nitrogen-fixing marine cyanobacteria. However, there currently is no conclusive evidence for 2-methylhopanoid production by extant marine cyanobacteria. As an alternative explanation, here we report 2-methylhopanoid production by bacteria of the genusNitrobacter, cosmopolitan nitrite oxidizers that inhabit nutrient-rich freshwater, brackish, and marine environments. The model organismNitrobacter vulgarisproduced only trace amounts of 2-methylhopanoids when grown in minimal medium or with added methionine, the presumed biosynthetic methyl donor. Supplementation of cultures with cobalamin (vitamin B₁₂) increased nitrite oxidation rates and stimulated a 33-fold increase of 2-methylhopanoid abundance, indicating that the biosynthetic reaction mechanism is cobalamin dependent. BecauseNitrobacterspp. cannot synthesize cobalamin, we postulate that they acquire it from organisms inhabiting a shared ecological niche—for example, ammonia-oxidizing archaea. We propose that during nutrient-rich conditions, cobalamin-based mutualism intensifies upper water column nitrification, thus promoting 2-methylhopanoid deposition. In contrast, anoxia underlying oligotrophic surface ocean conditions in restricted basins would prompt shoaling of anaerobic ammonium oxidation, leading to low observed 2-methylhopanoid abundances. The first scenario ismore »consistent with hypotheses of enhanced nutrient loading during OAEs, while the second is consistent with the sedimentary record of Pliocene–Pleistocene Mediterranean sapropel events. We thus hypothesize that nitrogen cycling in the Pliocene–Pleistocene Mediterranean resembled modern, highly stratified basins, whereas no modern analog exists for OAEs.

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3. Metabolic Profiling Reveals Biochemical Pathways Responsible for Eelgrass Response to Elevated CO2 and Temperature

   https://doi.org/10.1038/s41598-020-61684-x  

   Zayas-Santiago, Carmen C. ; Rivas-Ubach, Albert ; Kuo, Li-Jung ; Ward, Nicholas D. ; Zimmerman, Richard C. ( March 2020 , Scientific Reports)

   As CO₂levels in Earth’s atmosphere and oceans steadily rise, varying organismal responses may produce ecological losers and winners. Increased ocean CO₂can enhance seagrass productivity and thermal tolerance, providing some compensation for climate warming. However, the metabolic shifts driving the positive response to elevated CO₂by these important ecosystem engineers remain unknown. We analyzed whole-plant performance and metabolic profiles of two geographically distinct eelgrass (Zostera marinaL.) populations in response to CO₂enrichment. In addition to enhancing overall plant size, growth and survival, CO₂enrichment increased the abundance of Calvin Cycle and nitrogen assimilation metabolites while suppressing the abundance of stress-related metabolites. Overall metabolome differences between populations suggest that some eelgrass phenotypes may be better suited than others to cope with an increasingly hot and sour sea. Our results suggest that seagrass populations will respond variably, but overall positively, to increasing CO₂concentrations, generating negative feedbacks to climate change.
4. Genome Streamlining, Proteorhodopsin, and Organic Nitrogen Metabolism in Freshwater Nitrifiers

   https://doi.org/10.1128/mbio.02379-21  

   Podowski, Justin C. ; Paver, Sara F. ; Newton, Ryan J. ; Coleman, Maureen L. ( June 2022 , mBio)

   Giovannoni, Stephen J. (Ed.)

   ABSTRACT Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earth’s largest lakes. Surprisingly, ammonia-oxidizing bacteria (AOB) related to Nitrosospira dominated over ammonia-oxidizing archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival under conditions where ammonia oxidation is inhibited or substrate limited. Nitrite-oxidizing bacteria (NOB) “ Candidatus Nitrotoga” and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less-productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a criticalmore »freshwater resource experiencing rapid environmental change. IMPORTANCE Microorganisms play critical roles in Earth’s nitrogen cycle. In lakes, microorganisms called nitrifiers derive energy from reduced nitrogen compounds. In doing so, they transform nitrogen into a form that can ultimately be lost to the atmosphere by a process called denitrification, which helps mitigate nitrogen pollution from fertilizer runoff and sewage. Despite their importance, freshwater nitrifiers are virtually unexplored. To understand their diversity and function, we reconstructed genomes of freshwater nitrifiers across some of Earth’s largest freshwater lakes, the Laurentian Great Lakes. We discovered several new species of nitrifiers specialized for clear low-nutrient waters and distinct species in comparatively turbid Lake Erie. Surprisingly, one species may be able to harness light energy by using a protein called proteorhodopsin, despite the fact that nitrifiers typically live in deep dark water. Our work reveals the unique biodiversity of the Great Lakes and fills key gaps in our knowledge of an important microbial group, the nitrifiers.« less
5. Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

   https://doi.org/10.1038/s41396-020-00805-w  

   Lawson, Christopher E. ; Nuijten, Guylaine H. ; de Graaf, Rob M. ; Jacobson, Tyler B. ; Pabst, Martin ; Stevenson, David. M. ; Jetten, Mike S. ; Noguera, Daniel R. ; McMahon, Katherine D. ; Amador-Noguez, Daniel ; et al ( October 2020 , The ISME Journal)

   Abstract Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13 C and 2 H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO 2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis , and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO 2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and thatmore »acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.« less