Metabolic potential of Nitrososphaera-associated clades

Abstract

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.

Keywords: 54d9; Nitrososphaerales; ammonia-oxidizing archaea; metagenomics.

Figure 1

Layout of the GCEF research station and the plots from which the high-quality NS MAGs were retrieved; for land-use types and soil pH values, see Supplementary Fig. S1 and Supplementary Table S2; photo: UFZ.

Figure 2

Phylogenomic and comparative analysis of recovered AOA MAGs; (A) maximum-likelihood phylogenomic tree of recovered NS MAGs and reference genomes; the tree was inferred from concatenated phylogenetic markers and rooted with the Ca. Nitrosocaldales strains 3F and SCU2; the black dots represent bootstrap values > 90%; (B) alignment of the 54d9 clone and three δ2-MAGs in this study; (C) pairwise ANI and amoA genes of recovered δ-MAGs and reference genomes; (D) global distribution of the NS-δ1 MAGs based on the BLASTN of amoA genes; for amoA gene sequences of our MAGs, see Supplementary Files; WWTP, wastewater treatment plant.

Figure 3

Metagenomic and metatranscriptomic abundance of recovered AOA MAGs; (A) metagenomic abundance of recovered MAGs across 180 soil samples during the 2014–2019 summers; lines indicate the Pearson correlation between soil pH and MAG relative abundance in metagenomic datasets; (B) metatranscriptomic abundance of recovered MAGs across 24 soil samples in May and July 2022; boxplots show median, upper and lower quartile, and minimum and maximum values; L and H denote groups of plots with mean soil pH values of 6.4 and 7.2, respectively.

Figure 4

Metabolic potential of AOA genomes; the presence and absence of selected genes are indicated by a filled or empty circle, respectively; Amo, ammonia monooxygenase; MoCo, molybdenum cofactor; Msm, multiple sugar metabolism (ATP-binding protein); Amt, ammonium transporter; UT/SSS, urea transporters; YrbG, Ca²⁺/Na⁺ antiporter; CPA, cation/proton antiporter; Cha, Na⁺(Ca²⁺)/H⁺ antiporter; NahD, Na⁺/H⁺ antiporter; CLC, Cl^- channels; CorA/MgtC, mg²⁺ transport system; Trk/Kch, K⁺ transport system; KdpABC, K⁺ transport system; CBM, carbohydrate-binding module; GH, glycoside hydrolase; CE, carbohydrate esterase.

Figure 5

Expression levels of selected genes in recovered AOA MAGs; mean values were reported only if they were observed in at least two out of six replicates; dashes represent the absence of the selected gene in MAGs; a grey background indicates genes in MAGs that may lack sufficient coverage from metatranscriptome sequencing; L and H denote groups of plots with mean soil pH values of 6.4 and 7.2, respectively; GDH, glutamate dehydrogenase; Acc/Pcc, acetyl-CoA/propionyl-CoA carboxylase; FBP A/P, fructose 1,6-bisphosphate aldolase/phosphatase; TKT, transketolase; OFORs, 2-oxoacid: ferredoxin oxidoreductases; ACS, acetyl-CoA synthetase.

Figure 6

Phylogeny and predicted protein structure of CAZymes in recovered AOA MAGs; (A) protein structure of the 3CBM32 gene in NS-δ1 MAGs as predicted by AlphaFold2; (B) a maximum-likelihood tree of GH5 genes encoded by AOA genomes/MAGs; four GH5 gene clusters derived from δ-MAGs were colored; bootstrap values higher than 90% are indicated; (C) AlphaFold protein structures of the five GH5 gene copies in NS-δ1 MAGs; AP, acid phosphatase; SP, signal peptide; for GH5 and 3CBM32 gene sequences of the NS-δ1 MAGs, see Supplementary Files.

Figure 7

Metabolic reconstruction and gene expression of major pathways in NS-delta AOA MAGs; mean values were reported only if they were observed in at least two out of six replicates; PGM, phosphoglucomutase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; PK, pyruvate kinase; PckA, phosphoenolpyruvate carboxykinase; TAL, transaldolase; GDH, glutamate dehydrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; NirK, nitrite reductase; SOD, superoxide dismutase; Trx, thioredoxin; Hsp, heat shock protein; MPGS, mannosyl-3-phosphoglycerate synthase; GrpE, molecular chaperone.