Metabolic potential of Nitrososphaera-associated clades.

Soil ammonia-oxidizing archaea (AOA) play a crucial role in converting ammonia to nitrite, thereby mobilizing reactive nitrogen species into their soluble form, with a significant impact on nitrogen losses from terrestrial soils. Yet, our knowledge regarding their diversity and functions remains limited. In this study, we reconstructed 97 high-quality AOA metagenome-assembled genomes (MAGs) from 180 soil samples collected in Central Germany during 2014-2019 summers. These MAGs were affiliated with the order Nitrososphaerales and clustered into four family-level clades (NS-α/γ/δ/ε). Among these MAGs, 75 belonged to the most abundant but least understood δ-clade. Within the δ-clade, the amoA genes in three MAGs from neutral soils showed a 99.5% similarity to the fosmid clone 54d9, which has served as representative of the δ-clade for the past two decades since even today no cultivated representatives are available. Seventy-two MAGs constituted a distinct δ sub-clade, and their abundance and expression activity were more than twice that of other MAGs in slightly acidic soils. Unlike the less abundant clades (α, γ, and ε), the δ-MAGs possessed multiple highly expressed intracellular and extracellular carbohydrate-active enzymes responsible for carbohydrate binding (CBM32) and degradation (GH5), along with highly expressed genes involved in ammonia oxidation. Together, these results suggest metabolic versatility of uncultured soil AOA and a potential mixotrophic or chemolithoheterotrophic lifestyle among 54d9-like AOA.

Analysis of biomass productivity and physiology of Nitrososphaera viennensis grown in continuous culture.

Microbial ammonia oxidation is the first and usually rate limiting step in nitrification and is therefore an important step in the global nitrogen cycle. Ammonia-oxidizing archaea (AOA) play an important role in nitrification. Here, we report a comprehensive analysis of biomass productivity and the physiological response of Nitrososphaera viennensis to different ammonium and carbon dioxide (CO2) concentrations aiming to understand the interplay between ammonia oxidation and CO2 fixation of N. viennensis. The experiments were performed in closed batch in serum bottles as well as in batch, fed-batch, and continuous culture in bioreactors. A reduced specific growth rate (µ) of N. viennensis was observed in batch systems in bioreactors. By increasing CO2 gassing µ could be increased to rates comparable to that of closed batch systems. Furthermore, at a high dilution rate (D) in continuous culture (≥ 0.7 of µmax) the biomass to ammonium yield (Y(X/NH3)) increased up to 81.7% compared to batch cultures. In continuous culture, biofilm formation at higher D prevented the determination of D crit. Due to changes in Y(X/NH3) and due to biofilm, nitrite concentration becomes an unreliable proxy for the cell number in continuous cultures at D towards µmax. Furthermore, the obscure nature of the archaeal ammonia oxidation prevents an interpretation in the context of Monod kinetics and thus the determination of K S. Our findings indicate that the physiological response of N. viennensis might be regulated with different enzymatic make-ups, according to the ammonium catalysis rate. We reveal novel insights into the physiology of N. viennensis that are important for biomass production and the biomass yield of AOA. Moreover, our study has implications to the field of archaea biology and microbial ecology by showing that bioprocess technology and quantitative analysis can be applied to decipher environmental factors affecting the physiology and productivity of AOA.

Unexpected complexity of the ammonia monooxygenase in archaea.

Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this ecologically important nitrification complex.

Genome wide transcriptomic analysis of the soil ammonia oxidizing archaeon Nitrososphaera viennensis upon exposure to copper limitation.

Ammonia-oxidizing archaea (AOA) are widespread in nature and are involved in nitrification, an essential process in the global nitrogen cycle. The enzymes for ammonia oxidation and electron transport rely heavily on copper (Cu), which can be limited in nature. In this study the model soil archaeon Nitrososphaera viennensis was investigated via transcriptomic analysis to gain insight regarding possible Cu uptake mechanisms and compensation strategies when Cu becomes limiting. Upon Cu limitation, N. viennensis exhibited impaired nitrite production and thus growth, which was paralleled by downregulation of ammonia oxidation, electron transport, carbon fixation, nucleotide, and lipid biosynthesis pathway genes. Under Cu-limitation, 1547 out of 3180 detected genes were differentially expressed, with 784 genes upregulated and 763 downregulated. The most highly upregulated genes encoded proteins with a possible role in Cu binding and uptake, such as the Cu chelator and transporter CopC/D, disulfide bond oxidoreductase D (dsbD), and multicopper oxidases. While this response differs from the marine strain Nitrosopumilus maritimus, conserved sequence motifs in some of the Cu-responsive genes suggest conserved transcriptional regulation in terrestrial AOA. This study provides possible gene regulation and energy conservation mechanisms linked to Cu bioavailability and presents the first model for Cu uptake by a soil AOA.

Copper limiting threshold in the terrestrial ammonia oxidizing archaeon Nitrososphaera viennensis.

Ammonia oxidizing archaea (AOA) inhabiting soils have a central role in the global nitrogen cycle. Copper (Cu) is central to many enzymes in AOA including ammonia monooxygenase (AMO), the enzyme involved in the first step of ammonia oxidation. This study explored the physiological response of the AOA soil isolate, Nitrososphaera viennensis (EN76T) to Cu-limiting conditions in order to approach its limiting threshold under laboratory conditions. The chelator TETA (1,4,8,11-tetraazacyclotetradecane N, N', N″, N‴-tetraacetic acid hydrochloride hydrate) with selective affinity for Cu2+ was used to lower bioavailable Cu2+ in culture experiments as predicted by thermodynamic speciation calculations. Results show that N. viennensis is Cu-limited at concentrations ≤10-15 mol L-1 free Cu2+ compared to standard conditions (10-12 mol L-1). This Cu2+ limiting threshold is similar to pure cultures of denitrifying bacteria and other AOA and AOB inhabiting soils, freshwaters and sewage (<10-16 mol L-1), and lower than pure cultures of the marine AOA Nitrosopumilus maritimus (<10-12.7 mol L-1), which also possesses a high amount of Cu-dependent enzymes.