Ammonia-oxidizing archaea (AOA), that is, members of the Thaumarchaeota phylum, occur ubiquitously in the environment and are of major significance for global nitrogen cycling. However, controls on cell growth and organic carbon assimilation by AOA are poorly understood. We isolated an ammonia-oxidizing archaeon (designated strain DDS1) from seawater and used this organism to study the physiology of ammonia oxidation. These findings were confirmed using four additional Thaumarchaeota strains from both marine and terrestrial habitats. Ammonia oxidation by strain DDS1 was enhanced in coculture with other bacteria, as well as in artificial seawater media supplemented with α-keto acids (e.g., pyruvate, oxaloacetate). α-Keto acid-enhanced activity of AOA has previously been interpreted as evidence of mixotrophy. However, assays for heterotrophic growth indicated that incorporation of pyruvate into archaeal membrane lipids was negligible. Lipid carbon atoms were, instead, derived from dissolved inorganic carbon, indicating strict autotrophic growth. α-Keto acids spontaneously detoxify H 2 O 2 via a nonenzymatic decarboxylation reaction, suggesting a role of α-keto acids as H 2 O 2 scavengers. Indeed, agents that also scavenge H 2 O 2 , such as dimethylthiourea and catalase, replaced the α-keto acid requirement, enhancing growth of strain DDS1. In fact, in the absence of α-keto acids, strain DDS1 and other AOA isolates were shown to endogenously produce H 2 O 2 (up to ∼4.5 μM), which was inhibitory to growth. Genomic analyses indicated catalase genes are largely absent in the AOA. Our results indicate that AOA broadly feature strict autotrophic nutrition and implicate H 2 O 2 as an important factor determining the activity, evolution, and community ecology of AOA ecotypes.

Nitrification of excess ammonia in soil causes eutrophication of water resources and emission of atmospheric N(2) O gas. The first step of nitrification, ammonia oxidation, is mediated by Archaea as well as Bacteria. The physiological reactions mediated by ammonia-oxidizing archaea (AOA) and their contribution to soil nitrification are still unclear. Results of non-culture-based studies have shown the thaumarchaeotal group I.1b lineage of AOA to be dominant over both AOA of group I.1a and ammonia-oxidizing bacteria in various soils. We obtained from an agricultural soil a highly enriched ammonia-oxidizing culture dominated by a single archaeal population [c. 90% of total cells, as determined microscopically (by fluorescence in situ hybridization) and by quantitative PCR of its 16S rRNA gene]. The archaeon (termed 'strain JG1') fell within thaumarchaeotal group I.1b and was related to the moderately thermophilic archaeon, Candidatus Nitrososphaera gargensis, and the mesophilic archaeon, Ca. Nitrososphaera viennensis with 97.0% and 99.1% 16S rRNA gene sequence similarity respectively. Strain JG1 was neutrophilic (growth range pH 6.0-8.0) and mesophilic (growth range temperature 25-40°C). The optimum temperature of strain JG1 (35-40°C) is > 10°C higher than that of ammonia-oxidizing bacteria (AOB). Membrane analysis showed that strain JG1 contained a glycerol dialkyl glycerol tetraether, GDGT-4, and its regioisomer as major core lipids; this crenarchaeol regioisomer was previously detected in similar abundance in the thermophile, Ca. N. gargensis and has been frequently observed in tropical soils. Substrate uptake assays showed that the affinity of strain JG1 for ammonia and oxygen was much higher than those of AOB. These traits may give a competitive advantage to AOA related to strain JG1 in oligotrophic environments. (13) C-bicarbonate incorporation into archaeal lipids of strain JG1 established its ability to grow autotrophically. Strain JG1 produced a significant amount of N(2) O gas - implicating AOA as a possible source of N(2) O emission from soils. Sequences of archaeal amoA and 16S rRNA genes closely related to those of strain JG1 have been retrieved from various terrestrial environments in which lineage of strain JG1 is likely engaged in autotrophic nitrification.

ABSTRACTEcological studies of thaumarchaeota often apply glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids. However, these components have only been characterized for thaumarchaeota from aquatic environments. Thaumarchaeota have been shown to play an important role in the nitrogen cycle in soil as ammonium oxidizers, and GDGTs are common lipids encountered in soil. We report the core and intact polar lipid (IPL) GDGTs produced by three newly available thaumarchaeota isolated from grassland soil in Austria (“Nitrososphaera viennensis,” group I.1b) and enriched from agricultural soils in South Korea (“CandidatusNitrosoarchaeum koreensis” MY1, group I.1a; and “CandidatusNitrososphaera” strain JG1, group I.1b). The soil thaumarchaeota all synthesize crenarchaeol as their major core GDGT, in agreement with the fact that crenarchaeol has also been detected in thaumarchaeota from aquatic environments. The crenarchaeol regioisomer apparently is produced in significant quantities only by soil thaumarchaeota of the I.1b subgroup. In addition, GDGTs with 0 to 4 cyclopentane moieties and GDGTs containing an additional hydroxyl group were detected. The IPL head groups of their membrane lipids comprised mainly monohexose, dihexose, trihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose head group bound to crenarchaeol occurred in all soil thaumarchaeota, and this IPL is at present the only lipid that is detected in all thaumarchaeota analyzed so far. This specificity and its lability indicate that it is the most suitable biomarker lipid to trace living thaumarchaeota. This study, in combination with previous studies, also suggests that hydroxylated GDGTs occur in the I.1a, but not in the I.1b, subgroup of the thaumarchaeota.

N 2 O gas is involved in global warming and ozone depletion. The major sources of N 2 O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N 2 O produced by soil AOA and associated N 2 O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N 2 O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), d 15 N bulk and d 18 O -N 2 O of soil AOA strains were 13-30%, À 13 to À 35% and 22-36%, respectively, and strains MY1-3 and other soil AOA strains had distinct isotopic signatures. A 15 N-NH 4 þ -labeling experiment indicated that N 2 O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N 2 O from AOA may be attributable to the relative contributions of these two processes. The highest N 2 O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N 2 O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N 2 O emissions from soil nitrification may be attributable to AOA.

A wide diversity of ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota exists and plays a key role in the N cycle in a variety of habitats. In this study, we isolated and characterized an ammonia-oxidizing archaeon, strain MY3, from a coal tar-contaminated sediment. Phylogenetically, strain MY3 falls in clade 'Nitrosocosmicus' of the thaumarchaeotal group I.1b. The cells of strain MY3 are large 'walnut-like' cocci, divide by binary fission along a central cingulum, and form aggregates. Strain MY3 is mesophilic and neutrophilic. An assay of C-bicarbonate incorporation into archaeal membrane lipids indicated that strain MY3 is capable of autotrophy. In contrast to some other AOA, TCA cycle intermediates, i.e. pruvate, oxaloacetate and α-ketoglutarate, did not affect the growth rates and yields of strain MY3. The attachment of cells of strain MY3 to XAD-7 hydrophobic beads and to the adsorbent vermiculite demonstrated the potential of strain MY3 to form biofilms. The cell surface was confirmed to be hydrophobic by the extraction of strain MY3 from an aqueous medium with p-xylene. Our finding of a strong potential for surface attachment by strain MY3 may reflect an adaptation to the selective pressures in hydrophobic terrestrial environments.

Polynyas, areas of open water surrounded by sea ice, are sites of intense primary production and ecological hotspots in the Antarctic Ocean. This study determined the spatial variation in communities of prokaryotes in a polynya in the Amundsen Sea using 454 pyrosequencing technology, and the results were compared with biotic and abiotic environmental factors. The bacterial abundance was correlated with that of phytoplankton, Phaeocystis spp. and diatoms. A cluster analysis indicated that the bacterial communities in the surface waters of the polynya were distinct from those under the sea ice. Overall, two bacterial clades, Polaribacter (20-64%) and uncultivated Oceanospirillaceae (7-34%), dominated the surface water in the polynya while the Pelagibacter clade was abundant at all depths (7-42%). The archaeal communities were not as diverse as the bacterial communities in the polynya, and marine group I was dominant (> 80%). Canonical correspondence analysis indicated that the oceanographic properties facilitated the development of distinct prokaryotic assemblages in the polynya. This analysis of the diversity and composition of the psychrophilic prokaryotes associated with high phytoplankton production provides new insights into the roles of prokaryotes in biogeochemical cycles in high-latitude polynyas.

Ammonia-oxidizing archaea (AOA) from the phylum Thaumarchaeota are ubiquitous in marine ecosystems and play a prominent role in carbon and nitrogen cycling. Previous studies have suggested that, like all microbes, thaumarchaea are infected by viruses and that viral predation has a profound impact on thaumarchaeal functioning and mortality, thereby regulating global biogeochemical cycles. However, not a single virus capable of infecting thaumarchaea has been reported thus far. Here we describe the isolation and characterization of three Nitrosopumilus spindle-shaped viruses (NSVs) that infect AOA and are distinct from other known marine viruses. Although NSVs have a narrow host range, they efficiently infect autochthonous Nitrosopumilus strains and display high rates of adsorption to their host cells. The NSVs have linear double-stranded DNA genomes of ∼28 kb that do not display appreciable sequence similarity to genomes of other known archaeal or bacterial viruses and could be considered as representatives of a new virus family, the “Thaspiviridae.” Upon infection, NSV replication leads to inhibition of AOA growth, accompanied by severe reduction in the rate of ammonia oxidation and nitrite reduction. Nevertheless, unlike in the case of lytic bacteriophages, NSV propagation is not associated with detectable degradation of the host chromosome or a decrease in cell counts. The broad distribution of NSVs in AOA-dominated marine environments suggests that NSV predation might regulate the diversity and dynamics of AOA communities. Collectively, our results shed light on the diversity, evolution, and potential impact of the virosphere associated with ecologically important mesophilic archaea.

dSoil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia-oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautotrophic ammonia oxidizer that belongs to a distinct clade of nonmarine thaumarchaeal group I.1a, which is widespread in terrestrial environments. The archaeal strain MY2 was cultivated from a deep oligotrophic soil horizon. The similarity of the 16S rRNA gene sequence of strain MY2 to those of other cultivated group I.1a thaumarchaeota members, i.e., Nitrosopumilus maritimus and "Candidatus Nitrosoarchaeum koreensis," is 92.9% for both species. Extensive growth assays showed that strain MY2 is chemolithoautotrophic, mesophilic (optimum temperature, 30°C), and neutrophilic (optimum pH, 7 to 7.5). The accumulation of nitrite above 1 mM inhibited ammonia oxidation, while ammonia oxidation itself was not inhibited in the presence of up to 5 mM ammonia. The genome size of strain MY2 was 1.76 Mb, similar to those of N. maritimus and "Ca. Nitrosoarchaeum koreensis," and the repertoire of genes required for ammonia oxidation and carbon fixation in thaumarchaeal group I.1a was conserved. A high level of representation of conserved orthologous genes for signal transduction and motility in the noncore genome might be implicated in niche adaptation by strain MY2. On the basis of phenotypic, phylogenetic, and genomic characteristics, we propose the name "Candidatus Nitrosotenuis chungbukensis" for the ammonia-oxidizing archaeal strain MY2.