Genomics and physiological characterizations of an acidotolerant nitrite-oxidizing <i>Nitrospira</i> enriched from freshwater pond

Nitrite-oxidizing bacteria (NOB) play a crucial role in global nitrogen cycling, yet their presence and adaptations in acidic environments remain poorly understood. This study unveils the cultivation and characterization of a novel acid-tolerant NOB, NS4 culture, affiliated with lineage II (<i>Nitrospira</i>_<i>D</i>) within the genus <i>Nitrospira</i>. Enriched and isolated from a freshwater pond sediment, NS4 culture exhibits remarkable physiological and genomic traits that shed light on NOB survival strategies in low pH conditions. NS4 culture demonstrates the optimal growth at pH 6 and 0.5 mM nitrite concentration, with a maximum growth rate of 0.62 day⁻¹. Kinetic analyses reveal a high affinity for nitrite (<i>K_m</i>_(app) = 4.02 µM), suggesting adaptation to oligotrophic environments. Phylogenomic and genomic-relatedness analyses position NS4 culture as a novel member within the genus <i>Nitrospira</i>, for which we propose as "<i>Candidatus</i> Nitrospira acidotolerans." Genomic investigations indicate the presence of a complete reductive tricarboxylic acid cycle and genes for nitrite oxidation, confirming its chemolithoautotrophic lifestyle. Intriguingly, NS4 genome lacks complete pathways for cobalamin biosynthesis, implying a potential dependence on symbiotic partners for this essential cofactor. The NS4 genome harbors genes associated with acid resistance, including chaperones, transporters, and amino acid metabolism, suggesting a genetic potential for adaptation or resistance to low pH conditions. This discovery expands our understanding of NOB diversity and adaptability, offering insights into nitrogen cycling in acid-impacted ecosystems. The physiological and genomic traits of this acid-tolerant NOB open new insights for exploring the ecological significance of NOB in previously overlooked acidic habitats.IMPORTANCENitrite-oxidizing bacteria (NOB) are integral to the global nitrogen cycle, yet their adaptations to acidic environments remain poorly understood. This study introduces <i>Candidatus</i> Nitrospira acidotolerans, an acid-tolerant NOB highly enriched from freshwater pond sediment. By combining physiological and genomic analyses, this work reveals unique adaptations that enable survival and nitrite oxidation under low pH conditions. Notably, the NS4 culture demonstrates high nitrite affinity and resistance to acidic stress, suggesting its ecological significance in acid-impacted ecosystems. Additionally, NS4 genomic traits reveal genetic potential of metabolic dependencies, including reliance on symbiotic partners for cobalamin synthesis. These findings expand our understanding of NOB diversity and their role in nitrogen cycling under extreme conditions, offering novel insights into microbial ecology and potential applications in managing nitrogen processes in acidic environments.