Aalborg Universitet Characterization of the first “ Candidatus nitrotoga ” isolate reveals metabolic versatility and separate evolution of widespread nitrite-oxidizing bacteria

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 coldadapted 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 M nitrite and a Vmax of ~28 mol 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. IMPORTANCE Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus “Candidatus Nitrotoga” and performed a detailed physiological and genomic characterization of this organism (“Candidatus Nitrotoga fabula”). Differences between key phenotypic properties of “Ca. Nitrotoga fabula” and those of previously enriched “Ca. Nitrotoga” members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that “Ca. Nitrotoga” are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in “Ca. Nitrotoga fabula” Received 29 May 2018 Accepted 14 June 2018 Published 10 July 2018 Citation Kitzinger K, Koch H, Lücker S, Sedlacek CJ, Herbold C, Schwarz J, Daebeler A, Mueller AJ, Lukumbuzya M, Romano S, Leisch N, Karst SM, Kirkegaard R, Albertsen M, Nielsen PH, Wagner M, Daims H. 2018. Characterization of the first “Candidatus Nitrotoga” isolate reveals metabolic versatility and separate evolution of widespread nitrite-oxidizing bacteria. mBio 9:e01186-18. https://doi.org/10.1128/mBio . 01186-18. Editor Douglas G. Capone, University of Southern California Copyright © 2018 Kitzinger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Holger Daims, daims@microbial-ecology.net. * Present address: Hanna Koch, Department of Microbiology, Radboud University, Nijmegen, The Netherlands. This article is a direct contribution from a Fellow of the American Academy of Microbiology. Solicited external reviewers: Willm Martens-Habbena, University of Florida; Daniel Noguera, University of WisconsinMadison. RESEARCH ARTICLE