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.

The recent discovery of ammonia-oxidizing archaea (AOA) dramatically changed our perception of the diversity and evolutionary history of microbes involved in nitrification. In this study, a moderately thermophilic (46°C) ammonia-oxidizing enrichment culture, which had been seeded with biomass from a hot spring, was screened for ammonia oxidizers. Although gene sequences for crenarchaeotal 16S rRNA and two subunits of the ammonia monooxygenase (amoA and amoB) were detected via PCR, no hints for known ammonia-oxidizing bacteria were obtained. Comparative sequence analyses of these gene fragments demonstrated the presence of a single operational taxonomic unit and thus enabled the assignment of the amoA and amoB sequences to the respective 16S rRNA phylotype, which belongs to the widely distributed group I.1b (soil group) of the Crenarchaeota. Catalyzed reporter deposition (CARD)-FISH combined with microautoradiography (MAR) demonstrated metabolic activity of this archaeon in the presence of ammonium. This finding was corroborated by the detection of amoA gene transcripts in the enrichment. CARD-FISH/ MAR showed that the moderately thermophilic AOA is highly active at 0.14 and 0.79 mM ammonium and is partially inhibited by a concentration of 3.08 mM. The enriched AOA, which is provisionally classified as ''Candidatus Nitrososphaera gargensis,'' is the first described thermophilic ammonia oxidizer and the first member of the crenarchaeotal group I.1b for which ammonium oxidation has been verified on a cellular level. Its preference for thermophilic conditions reinvigorates the debate on the thermophilic ancestry of AOA.ammonia oxidation ͉ archaea ͉ nitrification ͉ thermophile ͉ amoA N itrification, the successive microbial oxidation of ammonia via nitrite to nitrate, is a crucial step in the biogeochemical nitrogen cycle, and ammonia-oxidizing microorganisms catalyze the first, rate-limiting step of this process. Until recently, the microbiology of ammonia oxidation was thought to be well understood. Aerobic, chemolithoautotrophic bacteria within the Beta-and Gammaproteobacteria were the only known ammoniaoxidizing microorganisms (1, 2). However, in the last few years, this understanding has been radically changed, first, by the discovery that ammonium can also be oxidized anaerobically by a clade of deep branching planctomycetes (3, 4), and later by the equally surprising cultivation of ammonia-oxidizing archaea (AOA) belonging to the Crenarchaeota (5). Since then, AOA were found to outnumber ammonia-oxidizing bacteria (AOB) in several terrestrial and marine systems, including different soils (6), the North Sea and Atlantic Ocean (7), the Pacific Ocean (8), and the Black Sea (9). Furthermore, molecular analyses demonstrated that AOA also occur in association with marine sponges (10-13), and amoA sequences related to recognized AOA were retrieved in numerous studies from a wide variety of other habitats (7-9, 14-18), including two moderately thermophilic sites with a temperature below 50°C (19,20). The latter findin...

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.

In marine systems, nitrate is the major reservoir of inorganic fixed nitrogen. The only known biological nitrate-forming reaction is nitrite oxidation, but despite its importance, our knowledge of the organisms catalyzing this key process in the marine N-cycle is very limited. The most frequently encountered marine NOB are related to Nitrospina gracilis, an aerobic chemolithoautotrophic bacterium isolated from ocean surface waters. To date, limited physiological and genomic data for this organism were available and its phylogenetic affiliation was uncertain. In this study, the draft genome sequence of N. gracilis strain 3/211 was obtained. Unexpectedly for an aerobic organism, N. gracilis lacks classical reactive oxygen defense mechanisms and uses the reductive tricarboxylic acid cycle for carbon fixation. These features indicate microaerophilic ancestry and are consistent with the presence of Nitrospina in marine oxygen minimum zones. Fixed carbon is stored intracellularly as glycogen, but genes for utilizing external organic carbon sources were not identified. N. gracilis also contains a full gene set for oxidative phosphorylation with oxygen as terminal electron acceptor and for reverse electron transport from nitrite to NADH. A novel variation of complex I may catalyze the required reverse electron flow to low-potential ferredoxin. Interestingly, comparative genomics indicated a strong evolutionary link between Nitrospina, the nitrite-oxidizing genus Nitrospira, and anaerobic ammonium oxidizers, apparently including the horizontal transfer of a periplasmically oriented nitrite oxidoreductase and other key genes for nitrite oxidation at an early evolutionary stage. Further, detailed phylogenetic analyses using concatenated marker genes provided evidence that Nitrospina forms a novel bacterial phylum, for which we propose the name Nitrospinae.

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