Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N(2)) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N(2)H(4)). Here we show that N(2)H(4) is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N(2)H(4). We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N(2)H(4) synthesis and its oxidation to N(2). These results present a new biochemical reaction forging an N-N bond and fill a lacuna in our understanding of the biochemical synthesis of the N(2) in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the ‘Candidatus Scalindua’ species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by ‘Candidatus Scalindua profunda’ to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater ‘Candidatus Kuenenia stuttgartiensis’. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K. stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm.

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.

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Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N‐[1′‐(2″‐amino‐4″‐hydroxy‐7″‐methyl‐6″‐pteridinyl)ethyl]‐4‐[2′,3′,4′,5′‐tetrahydroxypent‐1′‐yl(5′‐1″)O‐α‐ribofuranosyl‐5″‐phosphoric acid] aniline in which the phosphate group is esterified with α‐hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5′→ 1″ glycosidic bond and yielded a ‘hydrolytic product’ which was identified as N‐[1′‐(2″‐amino‐4″‐hydroxy‐7″‐methyl‐6″‐pteridinyl)ethyl]‐4‐[2′, 3′,4′, 5′‐tetrahydroxypent‐1′‐yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7‐methylpterin‐6‐carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6‐ethyl‐7‐methyl‐7,8‐dihydropterin, 6‐ethyl‐7‐methylpterin and a third compound, named methaniline which was identified as 4‐[2′, 3′,4′,5′‐tetrahydroxypent‐1′‐yl(5′→ 1″)O‐α‐ribofuranosyl‐5″‐phosphoric acid]aniline, in which the phosphate group is esterified with α‐hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a l‐glutamyl derivative of methanopterin, where the glutamate moiety is attached to the α‐carboxylic acid group of the α‐hydroxyglutaric acid moiety of methanopterin via an amide linkage.