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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 their 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. 

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2. “ Candidatus Subterrananammoxibiaceae,” a New Anammox Bacterial Family in Globally Distributed Marine and Terrestrial Subsurfaces

   https://doi.org/10.1128/aem.00800-23  

   Zhao, Rui; Le Moine Bauer, Sven; Babbin, Andrew R. (January 2023, Applied and Environmental Microbiology)

   Spear, John R. (Ed.)

   ABSTRACT Bacteria specialized in anaerobic ammonium oxidation (anammox) are widespread in many anoxic habitats and form an important functional guild in the global nitrogen cycle by consuming bio-available nitrogen for energy rather than biomass production. Due to their slow growth rates, cultivation-independent approaches have been used to decipher their diversity across environments. However, their full diversity has not been well recognized. Here, we report a new family of putative anammox bacteria, “ Candidatus Subterrananammoxibiaceae,” existing in the globally distributed terrestrial and marine subsurface (groundwater and sediments of estuary, deep-sea, and hadal trenches). We recovered a high-quality metagenome-assembled genome of this family, tentatively named “ Candidatus Subterrananammoxibius californiae,” from a California groundwater site. The “ Ca. Subterrananammoxibius californiae” genome not only contains genes for all essential components of anammox metabolism (e.g., hydrazine synthase, hydrazine oxidoreductase, nitrite reductase, and nitrite oxidoreductase) but also has the capacity for urea hydrolysis. In an Arctic ridge sediment core where redox zonation is well resolved, “ Ca. Subterrananammoxibiaceae” is confined within the nitrate-ammonium transition zone where the anammox rate maximum occurs, providing environmental proof of the anammox activity of this new family. Phylogenetic analysis of nitrite oxidoreductase suggests that a horizontal transfer facilitated the spreading of the nitrite oxidation capacity between anammox bacteria (in the Planctomycetota phylum) and nitrite-oxidizing bacteria from Nitrospirota and Nitrospinota . By recognizing this new anammox family, we propose that all lineages within the “ Ca. Brocadiales” order have anammox capacity. IMPORTANCE Microorganisms called anammox bacteria are efficient in removing bioavailable nitrogen from many natural and human-made environments. They exist in almost every anoxic habitat where both ammonium and nitrate/nitrite are present. However, only a few anammox bacteria have been cultured in laboratory settings, and their full phylogenetic diversity has not been recognized. Here, we present a new bacterial family whose members are present across both the terrestrial and marine subsurface. By reconstructing a high-quality genome from the groundwater environment, we demonstrate that this family has all critical enzymes of anammox metabolism and, notably, also urea utilization. This bacterium family in marine sediments is also preferably present in the niche where the anammox process occurs. These findings suggest that this novel family, named “ Candidatus Subterrananammoxibiaceae,” is an overlooked group of anammox bacteria, which should have impacts on nitrogen cycling in a range of environments. 

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3. Metabolic Remodeling during Biofilm Development of Bacillus subtilis

   https://doi.org/10.1128/mBio.00623-19  

   Pisithkul, Tippapha; Schroeder, Jeremy W.; Trujillo, Edna A.; Yeesin, Ponlkrit; Stevenson, David M.; Chaiamarit, Tai; Coon, Joshua J.; Wang, Jue D.; Amador-Noguez, Daniel; Ghigo, Jean-Marc; et al (June 2019, mBio)

   ABSTRACT Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis , very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism. 

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4. Hierarchical routing in carbon metabolism favors iron-scavenging strategy in iron-deficient soil Pseudomonas species

   https://doi.org/10.1073/pnas.2016380117  

   Mendonca, Caroll M.; Yoshitake, Sho; Wei, Hua; Werner, Anne; Sasnow, Samantha S.; Thannhauser, Theodore W.; Aristilde, Ludmilla (December 2020, Proceedings of the National Academy of Sciences)

   null (Ed.)

   High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple 13 C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil Pseudomonas species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated ( Pseudomonas putida KT2440, Pseudomonas protegens Pf-5, and Pseudomonas putida S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis–anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency. 

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5. Marine and terrestrial nitrifying bacteria are sources of diverse bacteriohopanepolyols

   https://doi.org/10.1111/gbi.12484  

   Elling, Felix J.; Evans, Thomas W.; Nathan, Vinitra; Hemingway, Jordon D.; Kharbush, Jenan J.; Bayer, Barbara; Spieck, Eva; Husain, Fatima; Summons, Roger E.; Pearson, Ann (January 2022, Geobiology)

   Abstract Hopanoid lipids, bacteriohopanols and bacteriohopanepolyols, are membrane components exclusive to bacteria. Together with their diagenetic derivatives, they are commonly used as biomarkers for specific bacterial groups or biogeochemical processes in the geologic record. However, the sources of hopanoids to marine and freshwater environments remain inadequately constrained. Recent marker gene studies suggest a widespread potential for hopanoid biosynthesis in marine bacterioplankton, including nitrifying (i.e., ammonia‐ and nitrite‐oxidizing) bacteria. To explore their hopanoid biosynthetic capacities, we studied the distribution of hopanoid biosynthetic genes in the genomes of cultivated and uncultivated ammonia‐oxidizing (AOB), nitrite‐oxidizing (NOB), and complete ammonia‐oxidizing (comammox) bacteria, finding that biosynthesis of diverse hopanoids is common among seven of the nine presently cultivated clades of nitrifying bacteria. Hopanoid biosynthesis genes are also conserved among the diverse lineages of bacterial nitrifiers detected in environmental metagenomes. We selected seven representative NOB isolated from marine, freshwater, and engineered environments for phenotypic characterization. All tested NOB produced diverse types of hopanoids, with some NOB producing primarily diploptene and others producing primarily bacteriohopanepolyols. Relative and absolute abundances of hopanoids were distinct among the cultures and dependent on growth conditions, such as oxygen and nitrite limitation. Several novel nitrogen‐containing bacteriohopanepolyols were tentatively identified, of which the so called BHP‐743.6 was present in all NOB. Distinct carbon isotopic signatures of biomass, hopanoids, and fatty acids in four tested NOB suggest operation of the reverse tricarboxylic acid cycle inNitrospiraspp. andNitrospina gracilisand of the Calvin–Benson–Bassham cycle for carbon fixation inNitrobacter vulgarisandNitrococcus mobilis. We suggest that the contribution of hopanoids by NOB to environmental samples could be estimated by their carbon isotopic compositions. The ubiquity of nitrifying bacteria in the ocean today and the antiquity of this metabolic process suggest the potential for significant contributions to the geologic record of hopanoids. 

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