Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus “ Nitrosopumilus maritimus ” strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Significance: Ammonia-oxidizing archaea (AOA) influence the form and availability of nitrogen in marine environments and are a major contributor to N ₂ O release and plausible indirect source of methane in the upper ocean. Thus, their sensitivity to ocean acidification and other physicochemical changes associated with climate change has global significance. Here, we report on the physiological response of marine AOA isolates to key environmental variables. Although reported as highly sensitive to reduction in ocean pH, we now show that some coastal marine AOA can remain active with increasing acidification of the oceans. All AOA isolates assimilate fixed carbon and two are obligate mixotrophs, suggesting this globally significant assemblage serves a significant function in coupling chemolithotrophy with organic matter assimilation in marine food webs.

Four mesophilic, neutrophilic, and aerobic marine ammonia-oxidizing archaea, designated strains SCM1T, HCA1T, HCE1T and PS0T, were isolated from a tropical marine fish tank, dimly lit deep coastal waters, the lower euphotic zone of coastal waters, and near-surface sediment in the Puget Sound estuary, respectively. Cells are straight or slightly curved small rods, 0.15–0.26 µm in diameter and 0.50–1.59 µm in length. Motility was not observed, although strain PS0T possesses genes associated with archaeal flagella and chemotaxis, suggesting it may be motile under some conditions. Cell membranes consist of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, with crenarchaeol as the major component. Strain SCM1T displays a single surface layer (S-layer) with p6 symmetry, distinct from the p3-S-layer reported for the soil ammonia-oxidizing archaeon Nitrososphaera viennensis EN76T. Respiratory quinones consist of fully saturated and monounsaturated menaquinones with 6 isoprenoid units in the side chain. Cells obtain energy from ammonia oxidation and use carbon dioxide as carbon source; addition of an α-keto acid (α-ketoglutaric acid) was necessary to sustain growth of strains HCA1T, HCE1T, and PS0T. Strain PS0T uses urea as a source of ammonia for energy production and growth. All strains synthesize vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), and B12 (cobalamin). Optimal growth occurs between 25 and 32 °C, between pH 6.8 and 7.3, and between 25 and 37 ‰ salinity. All strains have a low mol% G+C content of 33.0–34.2. Strains are related by 98 % or greater 16S rRNA gene sequence identity, sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. All four isolates are well separated by phenotypic and genotypic characteristics and are here assigned to distinct species within the genus Nitrosopumilus gen. nov. Isolates SCM1T (=ATCC TSD-97T =NCIMB 15022T), HCA1T (=ATCC TSD-96T), HCE1T (=ATCC TSD-98T), and PS0T (=ATCC TSD-99T) are type strains of the species Nitrosopumilus maritimus sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., respectively. In addition, we propose the family Nitrosopumilaceae fam. nov. and the order Nitrosopumilales ord. nov. within the class Nitrososphaeria .

Summary: Nitrification is a critical process for the balance of reduced and oxidized nitrogen pools in nature, linking mineralization to the nitrogen loss processes of denitrification and anammox. Recent studies indicate a significant contribution of ammonia‐oxidizing archaea ( AOA ) to nitrification. However, quantification of the relative contributions of AOA and ammonia‐oxidizing bacteria ( AOB ) to in situ ammonia oxidation remains challenging. We show here the production of nitric oxide ( NO ) by N itrosopumilus maritimus SCM 1. Activity of SCM 1 was always associated with the release of NO with quasi‐steady state concentrations between 0.05 and 0.08 μM. NO production and metabolic activity were inhibited by the nitrogen free radical scavenger 2‐phenyl‐4,4,5,5,‐tetramethylimidazoline‐1‐oxyl‐3‐oxide ( PTIO ). Comparison of marine and terrestrial AOB strains with SCM 1 and the recently isolated marine AOA strain HCA 1 demonstrated a differential sensitivity of AOB and AOA to PTIO and allylthiourea ( ATU ). Similar to the investigated AOA strains, bulk water column nitrification at coastal and open ocean sites with sub‐micromolar ammonia/ammonium concentrations was inhibited by PTIO and insensitive to ATU . These experiments support predictions from kinetic, molecular and biogeochemical studies, indicating that marine nitrification at low ammonia/ammonium concentrations is largely driven by archaea and suggest an important role of NO in the archaeal metabolism.