Acid tolerance and metabolic potential of comammox and nitrite-oxidizing <i>Nitrospira</i> enriched from soil

Nitrification is the two-step microbial oxidation of ammonia to nitrate via nitrite, and it can contribute to environmental problems in soils. Some nitrifiers have been cultivated from acidic soils at pH <5.5, allowing their metabolic potential and phylogeny to be investigated through genomic analyses. However, the genomic features of the genus <i>Nitrospira</i> remain poorly understood in the context of acid tolerance, despite its wide distribution in acidic environments. This study aimed to characterize the physiology and genomics of acid-tolerant <i>Nitrospira</i> enriched from an acidic soil. Using a metagenomic approach, two closed genomes of <i>Nitrospira</i> were reconstructed: a complete ammonia-oxidizing (comammox) bacterium and a nitrite-oxidizing bacterium (NOB). Both enriched <i>Nitrospira</i> survived at pH <5.5 in physiological tests, and the enriched comammox <i>Nitrospira</i> was phylogenetically close to clones derived from acidic soils. The active-site residues of hydroxylamine oxidase, a key nitrification enzyme, were conserved between the comammox <i>Nitrospira</i> characterized in this study and the previously reported betaproteobacterial ammonia oxidizers. This conservation suggests that existing nitrification inhibitors targeting this enzyme may also inhibit ammonia oxidation by comammox <i>Nitrospira</i> in acidic soils. Although the comammox and NOB <i>Nitrospira</i> in this study shared nearly all key metabolic pathways with <i>Nitrospira</i> species identified from neutral pH environments, both possessed passive urea transporters homologous to those found in acid-tolerant bacteria. These results revealed the acid tolerance of the enriched <i>Nitrospira</i> at pH <5.5, as well as their genomic features shared with acid-tolerant bacteria, rather than with previously reported <i>Nitrospira</i> species.