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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. 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 critical 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. 

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3. Methylphosphonate Oxidation in Prochlorococcus Strain MIT9301 Supports Phosphate Acquisition, Formate Excretion, and Carbon Assimilation into Purines

   https://doi.org/10.1128/AEM.00289-19  

   Sosa, Oscar A. ; Casey, John R. ; Karl, David M. ; Liu, Shuang-Jiang ( July 2019 , Applied and Environmental Microbiology)

   ABSTRACT The marine unicellular cyanobacterium Prochlorococcus is an abundant primary producer and widespread inhabitant of the photic layer in tropical and subtropical marine ecosystems, where the inorganic nutrients required for growth are limiting. In this study, we demonstrate that Prochlorococcus high-light strain MIT9301, an isolate from the phosphate-depleted subtropical North Atlantic Ocean, can oxidize methylphosphonate (MPn) and hydroxymethylphosphonate (HMPn), two phosphonate compounds present in marine dissolved organic matter, to obtain phosphorus. The oxidation of these phosphonates releases the methyl group as formate, which is both excreted and assimilated into purines in RNA and DNA. Genes encoding the predicted phosphonate oxidative pathway of MIT9301 were predominantly present in Prochlorococcus genomes from parts of the North Atlantic Ocean where phosphate availability is typically low, suggesting that phosphonate oxidation is an ecosystem-specific adaptation of some Prochlorococcus populations to cope with phosphate scarcity. IMPORTANCE Until recently, MPn was only known to be degraded in the environment by the bacterial carbon-phosphorus (CP) lyase pathway, a reaction that releases the greenhouse gas methane. The identification of a formate-yielding MPn oxidative pathway in the marine planctomycete Gimesia maris (S. R. Gama, M. Vogt, T. Kalina, K. Hupp, et al., ACS Chem Biol 14:735–741, 2019, https://doi.org/10.1021/acschembio.9b00024 ) and the presence of this pathway in Prochlorococcus indicate that this compound can follow an alternative fate in the environment while providing a valuable source of P to organisms. In the ocean, where MPn is a major component of dissolved organic matter, the oxidation of MPn to formate by Prochlorococcus may direct the flow of this one-carbon compound to carbon dioxide or assimilation into biomass, thus limiting the production of methane. 

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4. Microbial Nitrogen Metabolism in Chloraminated Drinking Water Reservoirs

   https://doi.org/10.1128/mSphere.00274-20  

   Potgieter, Sarah C. ; Dai, Zihan ; Venter, Stephanus N. ; Sigudu, Makhosazana ; Pinto, Ameet J. ; McMahon, Katherine ( April 2020 , mSphere)

   ABSTRACT Ammonia availability due to chloramination can promote the growth of nitrifying organisms, which can deplete chloramine residuals and result in operational problems for drinking water utilities. In this study, we used a metagenomic approach to determine the identity and functional potential of microorganisms involved in nitrogen biotransformation within chloraminated drinking water reservoirs. Spatial changes in the nitrogen species included an increase in nitrate concentrations accompanied by a decrease in ammonium concentrations with increasing distance from the site of chloramination. This nitrifying activity was likely driven by canonical ammonia-oxidizing bacteria (i.e., Nitrosomonas ) and nitrite-oxidizing bacteria (i.e., Nitrospira ) as well as by complete-ammonia-oxidizing (i.e., comammox) Nitrospira -like bacteria. Functional annotation was used to evaluate genes associated with nitrogen metabolism, and the community gene catalogue contained mostly genes involved in nitrification, nitrate and nitrite reduction, and nitric oxide reduction. Furthermore, we assembled 47 high-quality metagenome-assembled genomes (MAGs) representing a highly diverse assemblage of bacteria. Of these, five MAGs showed high coverage across all samples, which included two Nitrosomonas, Nitrospira, Sphingomonas , and Rhizobiales -like MAGs. Systematic genome-level analyses of these MAGs in relation to nitrogen metabolism suggest that under ammonia-limited conditions, nitrate may be also reduced back to ammonia for assimilation. Alternatively, nitrate may be reduced to nitric oxide and may potentially play a role in regulating biofilm formation. Overall, this study provides insight into the microbial communities and their nitrogen metabolism and, together with the water chemistry data, improves our understanding of nitrogen biotransformation in chloraminated drinking water distribution systems. IMPORTANCE Chloramines are often used as a secondary disinfectant when free chlorine residuals are difficult to maintain. However, chloramination is often associated with the undesirable effect of nitrification, which results in operational problems for many drinking water utilities. The introduction of ammonia during chloramination provides a potential source of nitrogen either through the addition of excess ammonia or through chloramine decay. This promotes the growth of nitrifying microorganisms and provides a nitrogen source (i.e., nitrate) for the growth for other organisms. While the roles of canonical ammonia-oxidizing and nitrite-oxidizing bacteria in chloraminated drinking water systems have been extensively investigated, those studies have largely adopted a targeted gene-centered approach. Further, little is known about the potential long-term cooccurrence of complete-ammonia-oxidizing (i.e., comammox) bacteria and the potential metabolic synergies of nitrifying organisms with their heterotrophic counterparts that are capable of denitrification and nitrogen assimilation. This study leveraged data obtained for genome-resolved metagenomics over a time series to show that while nitrifying bacteria are dominant and likely to play a major role in nitrification, their cooccurrence with heterotrophic organisms suggests that nitric oxide production and nitrate reduction to ammonia may also occur in chloraminated drinking water systems. 

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5. Comammox Nitrospira bacteria outnumber canonical nitrifiers irrespective of electron donor mode and availability in biofiltration systems

   https://doi.org/10.1093/femsec/fiac032  

   Vilardi, Katherine J ; Cotto, Irmarie ; Sevillano, Maria ; Dai, Zihan ; Anderson, Christopher L ; Pinto, Ameet ( April 2022 , FEMS Microbiology Ecology)

   Abstract Complete ammonia oxidizing bacteria coexist with canonical ammonia and nitrite oxidizing bacteria in a wide range of environments. Whether this is due to competitive or cooperative interactions, or a result of niche separation is not yet clear. Understanding the factors driving coexistence of nitrifiers is critical to manage nitrification processes occurring in engineered and natural ecosystems. In this study, microcosm-based experiments were used to investigate the impact of nitrogen source and loading on the population dynamics of nitrifiers in drinking water biofilter media. Shotgun sequencing of DNA followed by co-assembly and reconstruction of metagenome assembled genomes revealed clade A2 comammox bacteria were likely the primary nitrifiers within microcosms and increased in abundance over Nitrosomonas-like ammonia and Nitrospira-like nitrite oxidizing bacteria irrespective of nitrogen source type or loading. Changes in comammox bacterial abundance did not correlate with either ammonia or nitrite oxidizing bacterial abundance in urea-amended systems, where metabolic reconstruction indicated potential for cross-feeding between strict ammonia and nitrite oxidizers. In contrast, comammox bacterial abundance demonstrated a negative correlation with nitrite oxidizers in ammonia-amended systems. This suggests potentially weaker synergistic relationships between strict ammonia and nitrite oxidizers might enable comammox bacteria to displace strict nitrite oxidizers from complex nitrifying communities. 

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