Characterization of the First “ Candidatus Nitrotoga” Isolate Reveals Metabolic Versatility and Separate Evolution of Widespread Nitrite-Oxidizing Bacteria

Abstract

Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “Candidatus Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “Ca. Nitrotoga” culture was available. Here we obtained the first “Ca. Nitrotoga” isolate from activated sludge. This organism, “Candidatus Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “Ca. Nitrotoga” enrichments, which were described as cold-adapted NOB. “Ca. Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO2−) and nitrate (up to 25 mM NO3−). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent Km (Km(app)) of ~89 µM nitrite and a Vmax of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “Ca. Nitrotoga fabula” were reconstructed from the closed genome. “Ca. Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “Ca. Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “Ca. Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “Ca. Nitrotoga fabula” to survive nitrite depletion and colonize new niches.

Low Temperature and Neutral pH Define “ Candidatus Nitrotoga sp.” as a Competitive Nitrite Oxidizer in Coculture with Nitrospira defluvii

“…The presence of such an enzyme in Nitrotoga sp. BS still awaits investigation, but reported Nitrotoga genomes contain genes for assimilatory nitrite reduction to ammonia (17,34).…”

“…Accordingly, cultivated members of the genus Nitrotoga show optimal growth at between 10°C and 22°C (9,11,16). However, the paradigm of strict cold adaptation of the genus Nitrotoga has been questioned just recently by the isolation of the mesophilic Nitrotoga fabula from a WWTP (17). Previously, it was shown by Kruse and colleagues (18) that the Nitrotoga-typical fatty acid 16:1 cis9 was labeled by [ 13 C]bicarbonate using 3 mM and 30 mM nitrite, indicating metabolically active cells in activated sludge at 28°C.…”

Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth

“…In oxic environments, complete nitrification is accomplished through the complementary metabolisms of ammonia-oxidizing bacteria (AOB)/archaea (AOA) and nitrite-oxidizing bacteria (NOB) or by comammox bacteria (2,3). The existence of nitrite-oxidizing archaea (NOA) has been proposed but not yet confirmed (4). Although an essential process during wastewater and drinking water treatment, nitrification is also a major cause of nitrogen (N) loss from N-amended soils.…”

Discovery of New Nitrite-Oxidizing Bacteria Increases Phylogenetic and Metabolic Diversity within This Niche

“…Following in the footsteps of Winogradsky, Kitzinger et al (7) now report the first isolation and characterization of a nitrite-oxidizing “ Ca . Nitrotoga” obtained from a wastewater treatment plant.…”