SummaryNitrification is a two-step process where ammonia is considered to first be oxidized to nitrite by ammonia-oxidizing bacteria (AOB) and/or archaea (AOA), and subsequently to nitrate by nitrite-oxidizing bacteria (NOB). Described by Winogradsky already in 18901, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle2. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible and it was postulated that this process could occur under conditions selecting for species with lower growth-rates but higher growth-yields than canonical ammonia-oxidizing microorganisms3. Still, organisms catalysing this process have not yet been discovered. Here, we report the enrichment and initial characterization of two Nitrospira species that encode all enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding on the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of "Candidatus Nitrospira defluvii" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.

Nitrogen fixation in the surface ocean impacts global marine nitrogen bioavailability and thus microbial primary productivity. Until now, cyanobacterial populations have been viewed as the main suppliers of bioavailable nitrogen in this habitat. Although PCR amplicon surveys targeting the nitrogenase reductase gene have revealed the existence of diverse non-cyanobacterial diazotrophic populations, subsequent quantitative PCR surveys suggest that they generally occur in low abundance. Here, we use state-of-the-art metagenomic assembly and binning strategies to recover nearly one thousand non-redundant microbial population genomes from the TARA Oceans metagenomes. Among these, we provide the first genomic evidence for non-cyanobacterial diazotrophs inhabiting surface waters of the open ocean, which correspond to lineages within the Proteobacteria and, most strikingly, the Planctomycetes. Members of the latter phylum are prevalent in aquatic systems, but have never been linked to nitrogen fixation previously. Moreover, using genome-wide quantitative read recruitment, we demonstrate that the discovered diazotrophs were not only widespread but also remarkably abundant (up to 0.3% of metagenomic reads for a single population) in both the Pacific Ocean and the Atlantic Ocean northwest. Our results extend decades of PCR-based gene surveys, and substantiate the importance of heterotrophic bacteria in the fixation of nitrogen in the surface ocean.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, nitrite oxidation to nitrate, which is an important process of the biogeochemical nitrogen cycle. NOB were traditionally perceived as physiologically restricted organisms and were less intensively studied than other nitrogen-cycling microorganisms. This picture is in contrast to new discoveries of an unexpected high diversity of mostly uncultured NOB and a great physiological versatility, which includes complex microbe-microbe interactions and lifestyles outside the nitrogen cycle. Most surprisingly, close relatives to NOB perform complete nitrification (ammonia oxidation to nitrate) and this finding will have far-reaching implications for nitrification research. We review recent work that has changed our perspective on NOB and provides a new basis for future studies on these enigmatic organisms.

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.