Physical mixing in coastal waters controls and decouples nitrification via biomass dilution.

Nitrification is a central process of the aquatic nitrogen cycle that controls the supply of nitrate used in other key processes, such as phytoplankton growth and denitrification. Through time series observation and modeling of a seasonally stratified, eutrophic coastal basin, we demonstrate that physical dilution of nitrifying microorganisms by water column mixing can delay and decouple nitrification. The findings are based on a 4-y, weekly time series in the subsurface water of Bedford Basin, Nova Scotia, Canada, that included measurement of functional (amoA) and phylogenetic (16S rRNA) marker genes. In years with colder winters, more intense winter mixing resulted in strong dilution of resident nitrifiers in subsurface water, delaying nitrification for weeks to months despite availability of ammonium and oxygen. Delayed regrowth of nitrifiers also led to transient accumulation of nitrite (3 to 8 µmol · kgsw-1) due to decoupling of ammonia and nitrite oxidation. Nitrite accumulation was enhanced by ammonia-oxidizing bacteria (Nitrosomonadaceae) with fast enzyme kinetics, which temporarily outcompeted the ammonia-oxidizing archaea (Nitrosopumilus) that dominated under more stable conditions. The study reveals how physical mixing can drive seasonal and interannual variations in nitrification through control of microbial biomass and diversity. Variable, mixing-induced effects on functionally specialized microbial communities are likely relevant to biogeochemical transformation rates in other seasonally stratified water columns. The detailed study reveals a complex mechanism through which weather and climate variability impacts nitrogen speciation, with implications for coastal ecosystem productivity. It also emphasizes the value of high-frequency, multiparameter time series for identifying complex controls of biogeochemical processes in aquatic systems.

Conexivisphaera calida gen. nov., sp. nov., a thermophilic sulfur- and iron-reducing archaeon, and proposal of Conexivisphaeraceae fam. nov., Conexivisphaerales ord. nov., and Conexivisphaeria class. nov. in the phylum Thaumarchaeota.

A thermoacidophilic, anaerobic, and iron- and sulfur-reducing archaeon, strain NAS-02T, was isolated from a terrestrial hot spring in Japan, as previously reported. This organism is the first non-ammonia-oxidizing isolate in the phylum Thaumarchaeota. Here, we propose Conexivisphaera calida gen. nov., sp. nov. to accommodate this strain. The type strain of the type species is NAS-02T (=JCM 31663T=DSM 105898T). The values of 16S rRNA gene similarity and average amino acid identity between NAS-02T and its closest relatives are <86 and <42 %, respectively. Based on the phylogeny and physiology, we propose the family Conexivisphaeraceae fam. nov., the order Conexivisphaerales ord. nov. and the class Conexivisphaeria class. nov. to accommodate the novel genus.

Nitrosopumilus zosterae sp. nov., an autotrophic ammonia-oxidizing archaeon of phylum Thaumarchaeota isolated from coastal eelgrass sediments of Japan.

A novel mesophilic and aerobic ammonia-oxidizing archaeon of the phylum Thaumarchaeota, strain NM25T, was isolated from coastal eelgrass zone sediment sampled in Shimoda (Japan). The cells were rod-shaped with an S-layer cell wall. The temperature range for growth was 20-37 °C, with an optimum at 30 °C. The pH range for growth was pH 6.1-7.7, with an optimum at pH 7.1. The salinity range for growth was 5-40 %, with an optimum range of 15-32 %. Cells obtained energy from ammonia oxidation and used bicarbonate as a carbon source. Utilization of urea was not observed for energy generation and growth. Strain NM25T required a hydrogen peroxide scavenger, such as α-ketoglutarate, pyruvate or catalase, for sustained growth on ammonia. Growth of strain NM25T was inhibited by addition of low concentrations of some organic compounds and organic mixtures, including complete inhibition by glycerol, peptone and yeast extract. Phylogenetic analysis of four concatenated housekeeping genes (16S rRNA, rpoB, rpsI and atpD) and concatenated AmoA, AmoB, AmoC amino acid sequences indicated that the isolate is similar to members of the genus Nitrosopumilus. The closest relative is Nitrosopumilus ureiphilus PS0T with sequence similarities of 99.5 % for the 16S rRNA gene and 97.2 % for the amoA gene. Genome relatedness between strain NM25T and N. ureiphilus PS0T was assessed by average nucleotide identity and digital DNA-DNA hybridization, giving results of 85.4 and 40.2 %, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strain NM25T represents a novel species of the genus Nitrosopumilus, for which the name sp. nov, is proposed. The type strain is NM25T (=NBRC 111181T=ATCC TSD-147T).

Genome-resolved metagenomics analysis provides insights into the ecological role of Thaumarchaeota in the Amazon River and its plume.

BACKGROUND: Thaumarchaeota are abundant in the Amazon River, where they are the only ammonia-oxidizing archaea. Despite the importance of Thaumarchaeota, little is known about their physiology, mainly because few isolates are available for study. Therefore, information about Thaumarchaeota was obtained primarily from genomic studies. The aim of this study was to investigate the ecological roles of Thaumarchaeota in the Amazon River and the Amazon River plume. RESULTS: The archaeal community of the shallow in Amazon River and its plume is dominated by Thaumarchaeota lineages from group 1.1a, which are mainly affiliated to Candidatus Nitrosotenuis uzonensis, members of order Nitrosopumilales, Candidatus Nitrosoarchaeum, and Candidatus Nitrosopelagicus sp. While Thaumarchaeota sequences have decreased their relative abundance in the plume, Candidatus Nitrosopelagicus has increased. One genome was recovered from metagenomic data of the Amazon River (ThauR71 [1.05 Mpb]), and two from metagenomic data of the Amazon River plume (ThauP25 [0.94 Mpb] and ThauP41 [1.26 Mpb]). Phylogenetic analysis placed all three Amazon genome bins in Thaumarchaeota Group 1.1a. The annotation revealed that most genes are assigned to the COG subcategory coenzyme transport and metabolism. All three genomes contain genes involved in the hydroxypropionate/hydroxybutyrate cycle, glycolysis, tricarboxylic acid cycle, oxidative phosphorylation. However, ammonia-monooxygenase genes were detected only in ThauP41 and ThauR71. Glycoside hydrolases and auxiliary activities genes were detected only in ThauP25. CONCLUSIONS: Our data indicate that Amazon River is a source of Thaumarchaeota, where these organisms are important for primary production, vitamin production, and nitrification.

Diverse Thaumarchaeota Dominate Subsurface Ammonia-oxidizing Communities in Semi-arid Floodplains in the Western United States.

Subsurface microbial communities mediate biogeochemical transformations that drive both local and ecosystem-level cycling of essential elements, including nitrogen. However, their study has been largely limited to the deep ocean, terrestrial mines, caves, and topsoils (< 30 cm). Here, we present regional insights into the microbial ecology of aerobic ammonia oxidation within the terrestrial subsurface of five semi-arid riparian sites spanning a 900-km N-S transect. We sampled sediments, profiled communities to depths of ≤ 10 m, and compared them to reveal trends regionally within and surrounding the Upper Colorado River Basin (CRB). The diversity and abundance of ammonia-oxidizing microbial communities were evaluated in the context of subsurface geochemistry by applying a combination of amoA (encoding ammonia monooxygenase subunit A) gene sequencing, quantitative PCR, and geochemical techniques. Analysis of 898 amoA sequences from ammonia-oxidizing archaea (AOA) and bacteria (AOB) revealed extensive ecosystem-scale diversity, including archaeal amoA sequences from four of the five major AOA lineages currently found worldwide as well as distinct AOA ecotypes associated with naturally reduced zones (NRZs) and hydrogeochemical zones (unsaturated, capillary fringe, and saturated). Overall, AOA outnumber AOB by 2- to 5000-fold over this regional scale, suggesting that AOA may play a prominent biogeochemical role in nitrification within terrestrial subsurface sediments.

Two distinct pools of B12 analogs reveal community interdependencies in the ocean.

Organisms within all domains of life require the cofactor cobalamin (vitamin B12), which is produced only by a subset of bacteria and archaea. On the basis of genomic analyses, cobalamin biosynthesis in marine systems has been inferred in three main groups: select heterotrophic Proteobacteria, chemoautotrophic Thaumarchaeota, and photoautotrophic Cyanobacteria. Culture work demonstrates that many Cyanobacteria do not synthesize cobalamin but rather produce pseudocobalamin, challenging the connection between the occurrence of cobalamin biosynthesis genes and production of the compound in marine ecosystems. Here we show that cobalamin and pseudocobalamin coexist in the surface ocean, have distinct microbial sources, and support different enzymatic demands. Even in the presence of cobalamin, Cyanobacteria synthesize pseudocobalamin-likely reflecting their retention of an oxygen-independent pathway to produce pseudocobalamin, which is used as a cofactor in their specialized methionine synthase (MetH). This contrasts a model diatom, Thalassiosira pseudonana, which transported pseudocobalamin into the cell but was unable to use pseudocobalamin in its homolog of MetH. Our genomic and culture analyses showed that marine Thaumarchaeota and select heterotrophic bacteria produce cobalamin. This indicates that cobalamin in the surface ocean is a result of de novo synthesis by heterotrophic bacteria or via modification of closely related compounds like cyanobacterially produced pseudocobalamin. Deeper in the water column, our study implicates Thaumarchaeota as major producers of cobalamin based on genomic potential, cobalamin cell quotas, and abundance. Together, these findings establish the distinctive roles played by abundant prokaryotes in cobalamin-based microbial interdependencies that sustain community structure and function in the ocean.

Membrane Lipid Composition of the Moderately Thermophilic Ammonia-Oxidizing Archaeon "Candidatus Nitrosotenuis uzonensis" at Different Growth Temperatures.

"Candidatus Nitrosotenuis uzonensis" is the only cultured moderately thermophilic member of the thaumarchaeotal order Nitrosopumilales (NP) that contains many mesophilic marine strains. We examined its membrane lipid composition at different growth temperatures (37°C, 46°C, and 50°C). Its lipids were all membrane-spanning glycerol dialkyl glycerol tetraethers (GDGTs), with 0 to 4 cyclopentane moieties. Crenarchaeol (cren), the characteristic thaumarchaeotal GDGT, and its isomer (cren') were present in high abundance (30 to 70%). The GDGT polar headgroups were mono-, di-, and trihexoses and hexose/phosphohexose. The ratio of glycolipid to phospholipid GDGTs was highest in the cultures grown at 50°C. With increasing growth temperatures, the relative contributions of cren and cren' increased, while those of GDGT-0 to GDGT-4 (including isomers) decreased. TEX86 (tetraether index of tetraethers consisting of 86 carbons)-derived temperatures were much lower than the actual growth temperatures, further demonstrating that TEX86 does not accurately reflect the membrane lipid adaptation of thermophilic Thaumarchaeota As the temperature increased, specific GDGTs changed relative to their isomers, possibly representing temperature adaption-induced changes in cyclopentane ring stereochemistry. Comparison of a wide range of thaumarchaeotal core lipid compositions revealed that the "Ca Nitrosotenuis uzonensis" cultures clustered separately from other members of the NP order and the Nitrososphaerales (NS) order. While phylogeny generally seems to have a strong influence on GDGT distribution, our analysis of "Ca Nitrosotenuis uzonensis" demonstrates that its terrestrial, higher-temperature niche has led to a lipid composition that clearly differentiates it from other NP members and that this difference is mostly driven by its high cren' content.IMPORTANCE For Thaumarchaeota, the ratio of their glycerol dialkyl glycerol tetraether (GDGT) lipids depends on growth temperature, a premise that forms the basis of the widely applied TEX86 paleotemperature proxy. A thorough understanding of which GDGTs are produced by which Thaumarchaeota and what the effect of temperature is on their GDGT composition is essential for constraining the TEX86 proxy. "Ca Nitrosotenuis uzonensis" is a moderately thermophilic thaumarchaeote enriched from a thermal spring, setting it apart in its environmental niche from the other marine mesophilic members of its order. Indeed, we found that the GDGT composition of "Ca Nitrosotenuis uzonensis" cultures was distinct from those of other members of its order and was more similar to those of other thermophilic, terrestrial Thaumarchaeota This suggests that while phylogeny has a strong influence on GDGT distribution, the environmental niche that a thaumarchaeote inhabits also shapes its GDGT composition.

Vertical distribution of particle-associated and free-living ammonia-oxidizing archaea in Suruga Bay, a deep coastal embayment of Japan.

We analyzed the vertical distributions of ammonia-oxidizing archaea (AOA) in terms of abundance in Suruga Bay, Japan. We distinguished particle-associated (PA) from free-living (FL) assemblages. According to quantitative PCR measurements of the ammonia monooxygenase subunit A gene (amoA), most marine AOA were in an FL state. The vertical distributions of PA AOA ecotypes differed from the general trend; the Shallow Marine clade was dominant in both the surface and deep layers. Thus, although PA AOA account for a small percentage of AOA abundance, they have a community structure distinct from that of FL AOA in planktonic environments. Marine particles should be investigated further as an unexplored niche of AOA in the ocean.

Influence of ammonia oxidation rate on thaumarchaeal lipid composition and the TEX86 temperature proxy.

Archaeal membrane lipids known as glycerol dibiphytanyl glycerol tetraethers (GDGTs) are the basis of the TEX86 paleotemperature proxy. Because GDGTs preserved in marine sediments are thought to originate mainly from planktonic, ammonia-oxidizing Thaumarchaeota, the basis of the correlation between TEX86 and sea surface temperature (SST) remains unresolved: How does TEX86 predict surface temperatures, when maximum thaumarchaeal activity occurs below the surface mixed layer and TEX86 does not covary with in situ growth temperatures? Here we used isothermal studies of the model thaumarchaeon Nitrosopumilus maritimus SCM1 to investigate how GDGT composition changes in response to ammonia oxidation rate. We used continuous culture methods to avoid potential confounding variables that can be associated with experiments in batch cultures. The results show that the ring index scales inversely (R(2) = 0.82) with ammonia oxidation rate (ϕ), indicating that GDGT cyclization depends on available reducing power. Correspondingly, the TEX86 ratio decreases by an equivalent of 5.4 °C of calculated temperature over a 5.5 fmol·cell(-1)·d(-1) increase in ϕ. This finding reconciles other recent experiments that have identified growth stage and oxygen availability as variables affecting TEX86 Depth profiles from the marine water column show minimum TEX86 values at the depth of maximum nitrification rates, consistent with our chemostat results. Our findings suggest that the TEX86 signal exported from the water column is influenced by the dynamics of ammonia oxidation. Thus, the global TEX86-SST calibration potentially represents a composite of regional correlations based on nutrient dynamics and global correlations based on archaeal community composition and temperature.

"Candidatus Nitrosotenuis aquarius," an Ammonia-Oxidizing Archaeon from a Freshwater Aquarium Biofilter.

Ammonia is a metabolic waste product excreted by aquatic organisms that causes toxicity when it accumulates. Aquaria and aquaculture systems therefore use biological filters that promote the growth of nitrifiers to convert ammonia to nitrate. Ammonia-oxidizing bacteria (AOB) have been isolated from aquarium biofilters and are available as commercial supplements, but recent evidence suggests that ammonia-oxidizing archaea (AOA) are abundant in aquarium biofilters. In this study, we report the cultivation and closed genome sequence of the novel AOA representative "Candidatus Nitrosotenuis aquarius," which was enriched from a freshwater aquarium biofilter. "Ca Nitrosotenuis aquarius" oxidizes ammonia stoichiometrically to nitrite with a concomitant increase in thaumarchaeotal cells and a generation time of 34.9 h. "Ca Nitrosotenuis aquarius" has an optimal growth temperature of 33°C, tolerates up to 3 mM NH4Cl, and grows optimally at 0.05% salinity. Transmission electron microscopy revealed that "Ca Nitrosotenuis aquarius" cells are rod shaped, with a diameter of ∼0.4 µm and length ranging from 0.6 to 3.6 µm. In addition, these cells possess surface layers (S-layers) and multiple proteinaceous appendages. Phylogenetically, "Ca Nitrosotenuis aquarius" belongs to the group I.1a Thaumarchaeota, clustering with environmental sequences from freshwater aquarium biofilters, aquaculture systems, and wastewater treatment plants. The complete 1.70-Mbp genome contains genes involved in ammonia oxidation, bicarbonate assimilation, flagellum synthesis, chemotaxis, S-layer production, defense, and protein glycosylation. Incubations with differential inhibitors indicate that "Ca Nitrosotenuis aquarius"-like AOA contribute to ammonia oxidation within the aquarium biofilter from which it originated.IMPORTANCE Nitrification is a critical process for preventing ammonia toxicity in engineered biofilter environments. This work describes the cultivation and complete genome sequence of a novel AOA representative enriched from a freshwater aquarium biofilter. In addition, despite the common belief in the aquarium industry that AOB mediate ammonia oxidation, the present study suggests an in situ role for "Ca Nitrosotenuis aquarius"-like AOA in freshwater aquarium biofilters.

Nitrosarchaeum koreense gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon member of the phylum Thaumarchaeota isolated from agricultural soil.

A mesophilic, chemolithoautotrophic, neutrophilic and aerobic ammonia-oxidizing archaeon, designated strain MY1T, was isolated from agricultural soil. Microscopic observation revealed short, rod-shaped cells with a diameter of 0.3-0.5 µm and length of 0.6-1.0 µm. The isolate had no flagella and pili, and possessed no genes associated with archaeal flagella synthesis. The major membrane lipids consisted mainly of the glycerol dibiphytanyl glycerol tetraether (GDGT) lipids GDGT-0 to GDGT-4 and crenarchaeol. The major intact polar lipids (IPLs) were determined as hexose plus phosphohexose IPL and dihexose IPL. Strain MY1T obtains energy by aerobically oxidizing ammonia and carbon by fixing CO2. An optimal growth was observed at 25 °C, at pH 7 and with 0.2-0.4 % (w/v) salinity that corresponds with its terrestrial habitat. The addition of α-keto acids was necessary to stimulate growth. The strain tolerated ammonium and nitrite concentrations up to 10 and 5 mM, respectively. The MY1T genome has a DNA G+C content of 32.7 mol%. Phylogenetic analysis based on the 16S rRNA gene showed that strain MY1T belongs to the family Nitrosopumilaceaeof the phylum Thaumarchaeota, sharing the highest 16S rRNA gene sequence similarity (96.6-97.1 %) with marine isolates of the genus Nitrosopumilus. The average nucleotide identity was 78 % between strain MY1T and Nitrosopumilus maritimus SCM1T, indicating distant relatedness. Based on the phenotypic, phylogenetic and genomic analyses, it was concluded that strain MY1T belongs to the novel genus Nitrosarchaeum, under which the name Nitrosarchaeum koreense sp. nov. is proposed as the type species. The type strain is MY1T (=JCM 31640T=KCTC 4249T).

Agelas Wasting Syndrome Alters Prokaryotic Symbiont Communities of the Caribbean Brown Tube Sponge, Agelas tubulata.

The brown tube sponge Agelas tubulata (cf. Agelas conifera) is an abundant and long-lived sponge on Caribbean reefs. Recently, a disease-like condition, Agelas wasting syndrome (AWS), was described from A. tubulata in the Florida Keys, where prevalence of the syndrome increased from 7 to 35% of the sponge population between 2010 and 2015. In this study, we characterized the prokaryotic symbiont community of A. tubulata for the first time from individuals collected within the same monitoring plots where AWS was described. We also sampled tissue from A. tubulata exhibiting symptoms of AWS to determine its effect on the diversity and structure of prokaryotic symbiont communities. Bacteria from the phyla Chloroflexi and Proteobacteria, particularly the class Gammaproteobacteria, dominated the sponge microbiome in tissue samples of both healthy sponges and those exhibiting AWS. Prokaryotic community structure differed significantly between the diseased and healthy sponge samples, with greater variability among communities in diseased samples compared to healthy samples. These differences in prokaryotic community structure included a shift in relative abundance of the dominant, ammonia-oxidizing (Thaumarchaeota) symbionts present in diseased and healthy sponge samples. Further research is required to determine the functional consequences of this shift in microbial community structure and the causal relationship of dysbiosis and sponge disease in A. tubulata.

Successive transitory distribution of Thaumarchaeota and partitioned distribution of Bathyarchaeota from the Pearl River estuary to the northern South China Sea.

Thaumarchaeota and Bathyarchaeota (formerly named Miscellaneous Crenarchaeotal Group, MCG) are globally occurring archaea playing potential roles in nitrogen and carbon cycling, especially in marine benthic biogeochemical cycle. Information on their distributional and compositional patterns could provide critical clues to further delineate their physiological and biochemical characteristics. Profiles of thaumarchaeotal and the total archaeal community in the northern South China Sea surface sediments revealed a successively transitional pattern of Thaumarchaeota composition using MiSeq sequencing. Shallow-sea sediment enriched phylotypes decreased gradually along the slope from estuarine and coastal marine region to the deep-sea, while deep-sea sediment enriched phylotypes showed a trend of increasing. Proportion of Thaumarchaeota within the total archaea increased with seawater depth. Phylotypes enriched in shallow- and deep-sea sediments were affiliated to OTUs originated from similar niches, suggesting that physiological adaption not geographical distance shaped the distribution of Thaumarchaeota lineages. Quantitative PCR also depicted a successive decrease of thaumarchaeotal 16S rRNA gene abundance from the highest at shallow-sea sites E708S and E709S (2.57 × 106 and 2.73 × 106 gene copies/g of dry sediment) to the lowest at deep-sea sites E525S and E407S (1.97 × 106 and 2.14 × 106 gene copies/g of dry sediment). Both of the abundance fractions of Bathyarchaeota subgroups (including subgroups 1, 6, 8, 10, 13, 15, 17, and ungrouped Bathyarchaeota) and the total Bathyarchaeota in the total archaea showed a negative distribution to seawater depth. Partitioned distribution of Bathyarchaeota fraction in the total archaea is documented for the first time in this study, and the shallow- and deep-sea Bathyarchaeota could account for 17.8 and 0.8%, respectively, on average. Subgroups 6 and 8, enriched subgroups in shallow-sea sediments, largely explained this partitioned distribution pattern according to seawater depth. Their prevalence in shallow-sea and suboxic estuarine sediments rather than deep-sea sediments hints that their metabolic properties of carbon metabolism are adapted to carbon substrates in these environments.

The Tubulin Superfamily in Archaea.

In comparison with bacteria and eukaryotes, the large and diverse group of microorganisms known as archaea possess a great diversity of cytoskeletal proteins, including members of the tubulin superfamily. Many species contain FtsZ, CetZ and even possible tubulins; however, some major taxonomic groups do not contain any member of the tubulin superfamily. Studies using the model archaeon, Halferax volcanii have recently been instrumental in defining the fundamental roles of FtsZ and CetZ in archaeal cell division and cell shape regulation. Structural studies of archaeal tubulin superfamily proteins provide a definitive contribution to the cytoskeletal field, showing which protein-types must have developed prior to the divergence of archaea and eukaryotes. Several regions of the globular core domain - the "signature" motifs - combine in the 3D structure of the common molecular fold to form the GTP-binding site. They are the most conserved sequence elements and provide the primary basis for identification of new superfamily members through homology searches. The currently well-characterised proteins also all share a common mechanism of GTP-dependent polymerisation, in which GTP molecules are sandwiched between successive subunits that are arranged in a head-to-tail manner. However, some poorly-characterised archaeal protein families retain only some of the signature motifs and are unlikely to be capable of dynamic polymerisation, since the promotion of depolymerisation by hydrolysis to GDP depends on contributions from both subunits that sandwich the nucleotide in the polymer.

Confounding effects of oxygen and temperature on the TEX86 signature of marine Thaumarchaeota.

Marine ammonia-oxidizing archaea (AOA) are among the most abundant of marine microorganisms, spanning nearly the entire water column of diverse oceanic provinces. Historical patterns of abundance are preserved in sediments in the form of their distinctive glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids. The correlation between the composition of GDGTs in surface sediment and the overlying annual average sea surface temperature forms the basis for a paleotemperature proxy (TEX86) that is used to reconstruct surface ocean temperature as far back as the Middle Jurassic. However, mounting evidence suggests that factors other than temperature could also play an important role in determining GDGT distributions. We here use a study set of four marine AOA isolates to demonstrate that these closely related strains generate different TEX86-temperature relationships and that oxygen (O2) concentration is at least as important as temperature in controlling TEX86 values in culture. All of the four strains characterized showed a unique membrane compositional response to temperature, with TEX86-inferred temperatures varying as much as 12 °C from the incubation temperatures. In addition, both linear and nonlinear TEX86-temperature relationships were characteristic of individual strains. Increasing relative abundance of GDGT-2 and GDGT-3 with increasing O2 limitation, at the expense of GDGT-1, led to significant elevations in TEX86-derived temperature. Although the adaptive significance of GDGT compositional changes in response to both temperature and O2 is unclear, this observation necessitates a reassessment of archaeal lipid-based paleotemperature proxies, particularly in records that span low-oxygen events or underlie oxygen minimum zones.

Coupling of diversification and pH adaptation during the evolution of terrestrial Thaumarchaeota.

The Thaumarchaeota is an abundant and ubiquitous phylum of archaea that plays a major role in the global nitrogen cycle. Previous analyses of the ammonia monooxygenase gene amoA suggest that pH is an important driver of niche specialization in these organisms. Although the ecological distribution and ecophysiology of extant Thaumarchaeota have been studied extensively, the evolutionary rise of these prokaryotes to ecological dominance in many habitats remains poorly understood. To characterize processes leading to their diversification, we investigated coevolutionary relationships between amoA, a conserved marker gene for Thaumarchaeota, and soil characteristics, by using deep sequencing and comprehensive environmental data in Bayesian comparative phylogenetics. These analyses reveal a large and rapid increase in diversification rates during early thaumarchaeotal evolution; this finding was verified by independent analyses of 16S rRNA. Our findings suggest that the entire Thaumarchaeota diversification regime was strikingly coupled to pH adaptation but less clearly correlated with several other tested environmental factors. Interestingly, the early radiation event coincided with a period of pH adaptation that enabled the terrestrial Thaumarchaeota ancestor to initially move from neutral to more acidic and alkaline conditions. In contrast to classic evolutionary models, whereby niches become rapidly filled after adaptive radiation, global diversification rates have remained stably high in Thaumarchaeota during the past 400-700 million years, suggesting an ongoing high rate of niche formation or switching for these microbes. Our study highlights the enduring importance of environmental adaptation during thaumarchaeotal evolution and, to our knowledge, is the first to link evolutionary diversification to environmental adaptation in a prokaryotic phylum.

Nitrosopumilus maritimus gen. nov., sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., four marine ammonia-oxidizing archaea of the phylum Thaumarchaeota.

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 Nitrosopumilusmaritimus 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.

NmPin from the marine thaumarchaeote Nitrosopumilus maritimus is an active membrane associated prolyl isomerase.

BACKGROUND: Peptidyl-prolyl isomerases (PPIases) are present in all forms of life and play a crucial role in protein folding and regulation. They catalyze the cis-trans isomerization of the peptide bond that precedes proline residues in numerous proteins. The parvulins, which is one family of PPIases, have been extensively investigated in several eukaryotes. However, nothing is known about their expression, function and localization in archaea. RESULTS: Here, we describe the endogenous expression, molecular structure, function and cellular localization of NmPin, a single-domain parvulin-type PPIase from Nitrosopumilus maritimus. This marine chemolithoautotrophic archaeon belongs to the globally abundant phylum Thaumarchaeota. Using high resolution NMR spectroscopy we demonstrate that the 3D structure of NmPin adopts a parvulin fold and confirmed its peptidyl-prolyl isomerase activity by protease-coupled assays and mutagenesis studies. A detailed topological analysis revealed a positively charged lysine-rich patch on the protein surface, which is conserved in all known parvulin sequences of thaumarchaeotes and targets NmPin to lipids in vitro. Immunofluorescence microscopy confirms that the protein is attached to the outer archaeal cell membrane in vivo. Transmission electron microscopy uncovered that NmPin has a uniform distribution at the membrane surface, which is correlated with a native cell shape of the prokaryote. CONCLUSION: We present a novel solution structure of a catalytically active thaumarchaeal parvulin. Our results reveal that a lysine-rich patch in NmPin mediates membrane localization. These findings provide a model whereby NmPin is located between the archaeal membrane and the surface layer and hence suggest proteins of the S-layer as the key target substrates of this parvulin.

Distinctive non-methanogen archaeal populations in anaerobic digestion.

Methanogens define the archaeal communities involved in anaerobic digestion. Recently, non-methanogen archaeal populations have been unexpectedly identified in anaerobic digestion processes. To gain insight into the ecophysiology of these uncharacterized archaeal populations, for the first time, a phylogenetic analysis was performed on a collection of non-methanogen archaeal 16S rRNA gene sequences from anaerobic digesters of broad geographic distribution, revealing a distinct clade formed by these sequences in subgroup 6 of the Miscellaneous Crenarchaeotal Group in the newly proposed archaeal phylum Bathyarchaeota. This exclusive phylogenetic assemblage enabled the development of a real-time quantitative PCR (qPCR) assay specifically targeting these non-methanogen archaeal populations in anaerobic digestion. Application of the qPCR assay in continuous anaerobic digesters indicated that these archaeal populations were minor constituents of the archaeal communities, and the abundance of these populations remained relatively constant irrespective of process perturbations. Analysis of the archaeal populations in methanogenic communities further revealed the co-occurrence of these non-methanogen archaea with acetoclastic methanogens. Nevertheless, the low abundance of non-methanogen archaea as compared with acetoclastic methanogens suggests that the non-methanogen archaeal populations were not major players in animal waste-fed methanogenic processes investigated in this study and the functions of these archaeal populations remain to be identified.

Differential distribution patterns of ammonia-oxidizing archaea and bacteria in acidic soils of Nanling National Nature Reserve forests in subtropical China.

In addition to ammonia-oxidizing bacteria (AOB) the more recently discovered ammonia-oxidizing archaea (AOA) can also oxidize ammonia, but little is known about AOA community structure and abundance in subtropical forest soils. In this study, both AOA and AOB were investigated with molecular techniques in eight types of forests at surface soils (0-2 cm) and deep layers (18-20 cm) in Nanling National Nature Reserve in subtropical China. The results showed that the forest soils, all acidic (pH 4.24-5.10), harbored a wide range of AOA phylotypes, including the genera Nitrosotalea, Nitrososphaera, and another 6 clusters, one of which was reported for the first time. For AOB, only members of Nitrosospira were retrieved. Moreover, the abundance of the ammonia monooxygenase gene (amoA) from AOA dominated over AOB in most soil samples (13/16). Soil depth, rather than forest type, was an important factor shaping the community structure of AOA and AOB. The distribution patterns of AOA and AOB in soil layers were reversed: AOA diversity and abundances in the deep layers were higher than those in the surface layers; on the contrary, AOB diversity and abundances in the deep layers were lower than those in the surface layers. Interestingly, the diversity of AOA was positively correlated with pH, but negatively correlated with organic carbon, total nitrogen and total phosphorus, and the abundance of AOA was negatively correlated with available phosphorus. Our results demonstrated that AOA and AOB were differentially distributed in acidic soils in subtropical forests and affected differently by soil characteristics.