Overlooked Genetic Diversity of Ammonia Oxidizing Archaea Lineages in the Global Oceans

Nitrification, the microbial oxidation of ammonia to nitrite and nitrate, occurs in a wide variety of environments and plays a central role in the global nitrogen cycle. Catalyzed by the enzyme ammonia monooxygenase, the ability to oxidize ammonia was previously thought to be restricted to a few groups within the beta- and gamma-Proteobacteria. However, recent metagenomic studies have revealed the existence of unique ammonia monooxygenase alpha-subunit (amoA) genes derived from uncultivated, nonextremophilic Crenarchaeota. Here, we report molecular evidence for the widespread presence of ammonia-oxidizing archaea (AOA) in marine water columns and sediments. Using PCR primers designed to specifically target archaeal amoA, we find AOA to be pervasive in areas of the ocean that are critical for the global nitrogen cycle, including the base of the euphotic zone, suboxic water columns, and estuarine and coastal sediments. Diverse and distinct AOA communities are associated with each of these habitats, with little overlap between water columns and sediments. Within marine sediments, most AOA sequences are unique to individual sampling locations, whereas a small number of sequences are evidently cosmopolitan in distribution. Considering the abundance of nonextremophilic archaea in the ocean, our results suggest that AOA may play a significant, but previously unrecognized, role in the global nitrogen cycle.

Marine Crenarchaeota are the most abundant single group of prokaryotes in the ocean, but their physiology and role in marine biogeochemical cycles are unknown. Recently, a member of this clade was isolated from a sea aquarium and shown to be capable of nitrification, tentatively suggesting that Crenarchaeota may play a role in the oceanic nitrogen cycle. We enriched a crenarchaeote from North Sea water and showed that its abundance, and not that of bacteria, correlates with ammonium oxidation to nitrite. A time series study in the North Sea revealed that the abundance of the gene encoding for the archaeal ammonia monooxygenase alfa subunit (amoA) is correlated with a decline in ammonium concentrations and with the abundance of Crenarchaeota. Remarkably, the archaeal amoA abundance was 1-2 orders of magnitude higher than those of bacterial nitrifiers, which are commonly thought to mediate the oxidation of ammonium to nitrite in marine environments. Analysis of Atlantic waters of the upper 1,000 m, where most of the ammonium regeneration and oxidation takes place, showed that crenarchaeotal amoA copy numbers are also 1-3 orders of magnitude higher than those of bacterial amoA. Our data thus suggest a major role for Archaea in oceanic nitrification.

Mesophilic crenarchaeota are frequently found in terrestrial and marine habitats worldwide, but despite their considerable abundance the physiology of these as yet uncultivated archaea has remained unknown. From a 1.2 Gb large-insert environmental fosmid library of a calcareous grassland soil, a 43 kb genomic fragment was isolated with a ribosomal RNA that shows its affiliation to group 1.1b of crenarchaeota repeatedly found in soils. The insert encoded a homologue of a copper-containing nitrite reductase with an unusual C-terminus that encoded a potential amicyanin-like electron transfer domain as well as two proteins related to subunits of ammonia monooxygenases or particulate methane monooxygenases (AmoAB/PmoAB) respectively. Expression of nirK and the amoA-like gene was shown by reverse transcription polymerase chain reaction (PCR) analyses in soil samples, the latter being found at higher levels when the soil was incubated with ammonia (measured by quantitative PCR). Further variants of both genes were amplified from soil samples and were found in the environmental database from the Sargasso Sea plankton. Taken together, our findings suggest that mesophilic terrestrial and marine crenarchaeota might be capable of ammonia oxidation under aerobic and potentially also under anaerobic conditions.