Comparative Genomics of Ecologically Differentiated Nitrososphaera-Related Ammonia-Oxidizing Archaea

Soil acidification strongly influences the ecology of terrestrial ammonia-oxidizing archaea (AOA). Within this group, the Nitrososphaera-related alpha (NS-α) and delta (NS-δ) lineages exhibit pronounced pH-associated ecological differentiation. In the preceding microcosm experiment, NS-α populations showed greater persistence than NS-δ during ammonium-driven soil acidification, yet the genomic basis underlying this ecological differentiation remains poorly understood. In this study, metagenomic sequencing of one near-neutral baseline microcosm (pH 7.35) and one acidified microcosm (pH 4.73) was used to reconstruct 12 Nitrososphaera-affiliated metagenome-assembled genomes (MAGs), including 2 NS-α and 10 NS-δ MAGs. These genomes were compared using genome-based phylogenetic analysis, amoA phylogeny, average amino acid identity (AAI), average nucleotide identity (ANI), and KEGG functional annotation to investigate lineage-level genomic differentiation and candidate functional genomic features. Genome-based phylogenetic and whole-genome similarity analyses supported clear differentiation between NS-α and NS-δ, while also revealing substantial genomic heterogeneity within NS-δ. Functions related to nitrogen acquisition and regulation, respiratory energy metabolism, autotrophic carbon fixation, and oxidative-stress defense were broadly distributed across both lineages and showed no clear NS-α-specific pattern. In contrast, Mre11, HerA, NurA, and RadA were detected in both reconstructed NS-α MAGs. Comparison with representative NS-α reference genomes, including the neutrophilic Nitrososphaera viennensis EN76, further showed that this DNA-repair-related gene repertoire is not restricted to acidic environments and is more consistent with a broader NS-α-associated genomic feature. Na⁺/H⁺ antiporters and Trk/Ktr-type K⁺ transport systems were not detected within either reconstructed NS-α MAG, although MAG-level non-detection was not interpreted as definitive lineage-level absence. Overall, the results identify genomic differences between NS-α and NS-δ but do not establish a functional advantage under acidification. The potential physiological significance of the DNA-repair-related pattern therefore remains to be experimentally tested.