Martin Könneke

Isolation of an autotrophic ammonia-oxidizing marine archaeon

For years, microbiologists characterized the Archaea as obligate extremophiles that thrive in environments too harsh for other organisms. The limited physiological diversity among cultivated Archaea suggested that these organisms were metabolically constrained to a few environmental niches. For instance, all Crenarchaeota that are currently cultivated are sulphur-metabolizing thermophiles. However, landmark studies using cultivation-independent methods uncovered vast numbers of Crenarchaeota in cold oxic ocean waters. Subsequent molecular surveys demonstrated the ubiquity of these low-temperature Crenarchaeota in aquatic and terrestrial environments. The numerical dominance of marine Crenarchaeota—estimated at 1028 cells in the worlds oceans—suggests that they have a major role in global biogeochemical cycles. Indeed, isotopic analyses of marine crenarchaeal lipids suggest that these planktonic Archaea fix inorganic carbon. Here we report the isolation of a marine crenarchaeote that grows chemolithoautotrophically by aerobically oxidizing ammonia to nitrite—the first observation of nitrification in the Archaea. The autotrophic metabolism of this isolate, and its close phylogenetic relationship to environmental marine crenarchaeal sequences, suggests that nitrifying marine Crenarchaeota may be important to global carbon and nitrogen cycles.

Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea

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.

Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil

Genes of archaea encoding homologues of ammonia monooxygenases have been found on a widespread basis and in large amounts in almost all terrestrial and marine environments, indicating that ammonia oxidizing archaea (AOA) might play a major role in nitrification on Earth. However, only one pure isolate of this group from a marine environment has so far been obtained, demonstrating archaeal ammonia oxidation coupled with autotrophic growth similar to the bacterial counterparts. Here we describe the cultivation and isolation of an AOA from soil. It grows on ammonia or urea as an energy source and is capable of using higher ammonia concentrations than the marine isolate, Nitrosopumilus maritimus. Surprisingly, although it is able to grow chemolithoautotrophically, considerable growth rates of this strain are obtained only upon addition of low amounts of pyruvate or when grown in coculture with bacteria. Our findings expand the recognized metabolic spectrum of AOA and help explain controversial results obtained in the past on the activity and carbon assimilation of these globally distributed organisms.

Intact Membrane Lipids of "Candidatus Nitrosopumilus maritimus," a Cultivated Representative of the Cosmopolitan Mesophilic Group I Crenarchaeota

ABSTRACT In this study we analyzed the membrane lipid composition of “Candidatus Nitrosopumilus maritimus,” the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota. The core lipids of “Ca. Nitrosopumilus maritimus” consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.

Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation

Significance CO2 fixation is the most important biosynthesis process on Earth, enabling autotrophic organisms to synthesize their entire biomass from inorganic carbon at the expense of energy generated by photo- or chemotrophic processes. In the present study we demonstrate an autotrophy pathway that represents the most energy-efficient mechanism for fixing inorganic carbon in the presence of oxygen. This novel variant of the hydroxypropionate/hydroxybutyrate cycle appears to be common in a ubiquitous and abundant group of microorganisms that can thrive in nutrient-limited environments. This discovery offers a biochemical explanation for the remarkable ecological success of the ammonia-oxidizing archaea in extremely nutrient-limited environments typical of most of the open ocean. Archaea of the phylum Thaumarchaeota are among the most abundant prokaryotes on Earth and are widely distributed in marine, terrestrial, and geothermal environments. All studied Thaumarchaeota couple the oxidation of ammonia at extremely low concentrations with carbon fixation. As the predominant nitrifiers in the ocean and in various soils, ammonia-oxidizing archaea contribute significantly to the global nitrogen and carbon cycles. Here we provide biochemical evidence that thaumarchaeal ammonia oxidizers assimilate inorganic carbon via a modified version of the autotrophic hydroxypropionate/hydroxybutyrate cycle of Crenarchaeota that is far more energy efficient than any other aerobic autotrophic pathway. The identified genes of this cycle were found in the genomes of all sequenced representatives of the phylum Thaumarchaeota, indicating the environmental significance of this efficient CO2-fixation pathway. Comparative phylogenetic analysis of proteins of this pathway suggests that the hydroxypropionate/hydroxybutyrate cycle emerged independently in Crenarchaeota and Thaumarchaeota, thus supporting the hypothesis of an early evolutionary separation of both archaeal phyla. We conclude that high efficiency of anabolism exemplified by this autotrophic cycle perfectly suits the lifestyle of ammonia-oxidizing archaea, which thrive at a constantly low energy supply, thus offering a biochemical explanation for their ecological success in nutrient-limited environments.

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

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

Significance The membrane lipids of marine Archaea form the basis of the temperature proxy called TEX86, which is used for paleoclimate reconstructions from the Jurassic to the present. To date there remains no satisfactory explanation for how planktonic Archaea are able to record water column temperatures, because TEX86 does not correlate well with in situ growth temperatures in the modern ocean. Here we show that the TEX86 lipid ratio changes in response to cellular growth rate, which is controlled by the ammonia oxidation rate. This implies that variation in the TEX86 ratio with water depth is influenced by the metabolic activity of Thaumarchaeota in the water column. 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 (R2 = 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.

Respiratory quinones in Archaea: phylogenetic distribution and application as biomarkers in the marine environment.

The distribution of respiratory quinone electron carriers among cultivated organisms provides clues on both the taxonomy of their producers and the redox processes these are mediating. Our study of the quinone inventories of 25 archaeal species belonging to the phyla Eury-, Cren- and Thaumarchaeota facilitates their use as chemotaxonomic markers for ecologically important archaeal clades. Saturated and monounsaturated menaquinones with six isoprenoid units forming the alkyl chain may serve as chemotaxonomic markers for Thaumarchaeota. Other diagnostic biomarkers are thiophene-bearing quinones for Sulfolobales and methanophenazines as functional quinone analogues of the Methanosarcinales. The ubiquity of saturated menaquinones in the Archaea in comparison to Bacteria suggests that these compounds may represent an ancestral and diagnostic feature of the Archaea. Overlap between quinone compositions of distinct thermophilic and halophilic archaea and bacteria may indicate lateral gene transfer. The biomarker potential of thaumarchaeal quinones was exemplarily demonstrated on a water column profile of the Black Sea. Both, thaumarchaeal quinones and membrane lipids showed similar distributions with maxima at the chemocline. Quinone distributions indicate that Thaumarchaeota dominate respiratory activity at a narrow interval in the chemocline, while they contribute only 9% to the microbial biomass at this depth, as determined by membrane lipid analysis.

Chemotaxonomic characterisation of the thaumarchaeal lipidome

Thaumarchaeota are globally distributed and abundant microorganisms occurring in diverse habitats and thus represent a major source of archaeal lipids. The scope of lipids as taxonomic markers in microbial ecological studies is limited by the scarcity of comparative data on the membrane lipid composition of cultivated representatives, including the phylum Thaumarchaeota. Here, we comprehensively describe the core and intact polar lipid (IPL) inventory of ten ammonia-oxidising thaumarchaeal cultures representing all four characterized phylogenetic clades. IPLs of these thaumarchaeal strains are generally similar and consist of membrane-spanning, glycerol dibiphytanyl glycerol tetraethers with monoglycosyl, diglycosyl, phosphohexose and hexose-phosphohexose headgroups. However, the relative abundances of these IPLs and their core lipid compositions differ systematically between the phylogenetic subgroups, indicating high potential for chemotaxonomic distinction of thaumarchaeal clades. Comparative lipidomic analyses of 19 euryarchaeal and crenarchaeal strains suggested that the lipid methoxy archaeol is synthesized exclusively by Thaumarchaeota and may thus represent a diagnostic lipid biomarker for this phylum. The unprecedented diversity of the thaumarchaeal lipidome with 118 different lipids suggests that membrane lipid composition and adaptation mechanisms in Thaumarchaeota are more complex than previously thought and include unique lipids with as yet unresolved properties.

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.