Eva Spieck

A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring

The recent discovery of ammonia-oxidizing archaea (AOA) dramatically changed our perception of the diversity and evolutionary history of microbes involved in nitrification. In this study, a moderately thermophilic (46°C) ammonia-oxidizing enrichment culture, which had been seeded with biomass from a hot spring, was screened for ammonia oxidizers. Although gene sequences for crenarchaeotal 16S rRNA and two subunits of the ammonia monooxygenase (amoA and amoB) were detected via PCR, no hints for known ammonia-oxidizing bacteria were obtained. Comparative sequence analyses of these gene fragments demonstrated the presence of a single operational taxonomic unit and thus enabled the assignment of the amoA and amoB sequences to the respective 16S rRNA phylotype, which belongs to the widely distributed group I.1b (soil group) of the Crenarchaeota. Catalyzed reporter deposition (CARD)–FISH combined with microautoradiography (MAR) demonstrated metabolic activity of this archaeon in the presence of ammonium. This finding was corroborated by the detection of amoA gene transcripts in the enrichment. CARD-FISH/MAR showed that the moderately thermophilic AOA is highly active at 0.14 and 0.79 mM ammonium and is partially inhibited by a concentration of 3.08 mM. The enriched AOA, which is provisionally classified as “Candidatus Nitrososphaera gargensis,” is the first described thermophilic ammonia oxidizer and the first member of the crenarchaeotal group I.1b for which ammonium oxidation has been verified on a cellular level. Its preference for thermophilic conditions reinvigorates the debate on the thermophilic ancestry of AOA.

Distinct gene set in two different lineages of ammonia-oxidizing archaea supports the phylum Thaumarchaeota

Globally distributed archaea comprising ammonia oxidizers of moderate terrestrial and marine environments are considered the most abundant archaeal organisms on Earth. Based on 16S rRNA phylogeny, initial assignment of these archaea was to the Crenarchaeota. By contrast, features of the first genome sequence from a member of this group suggested that they belong to a novel phylum, the Thaumarchaeota. Here, we re-investigate the Thaumarchaeota hypothesis by including two newly available genomes, that of the marine ammonia oxidizer Nitrosopumilus maritimus and that of Nitrososphaera gargensis, a representative of another evolutionary lineage within this group predominantly detected in terrestrial environments. Phylogenetic studies based on r-proteins and other core genes, as well as comparative genomics, confirm the assignment of these organisms to a separate phylum and reveal a Thaumarchaeota-specific set of core informational processing genes, as well as potentially ancestral features of the archaea.

The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations.

The cohort of the ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota is a diverse, widespread and functionally important group of microorganisms in many ecosystems. However, our understanding of their biology is still very rudimentary in part because all available genome sequences of this phylum are from members of the Nitrosopumilus cluster. Here we report on the complete genome sequence of Candidatus Nitrososphaera gargensis obtained from an enrichment culture, representing a different evolutionary lineage of AOA frequently found in high numbers in many terrestrial environments. With its 2.83 Mb the genome is much larger than that of other AOA. The presence of a high number of (active) IS elements/transposases, genomic islands, gene duplications and a complete CRISPR/Cas defence system testifies to its dynamic evolution consistent with low degree of synteny with other thaumarchaeal genomes. As expected, the repertoire of conserved enzymes proposed to be required for archaeal ammonia oxidation is encoded by N. gargensis, but it can also use urea and possibly cyanate as alternative ammonia sources. Furthermore, its carbon metabolism is more flexible at the central pyruvate switch point, encompasses the ability to take up small organic compounds and might even include an oxidative pentose phosphate pathway. Furthermore, we show that thaumarchaeota produce cofactor F420 as well as polyhydroxyalkanoates. Lateral gene transfer from bacteria and euryarchaeota has contributed to the metabolic versatility of N. gargensis. This organisms is well adapted to its niche in a heavy metal-containing thermal spring by encoding a multitude of heavy metal resistance genes, chaperones and mannosylglycerate as compatible solute and has the genetic ability to respond to environmental changes by signal transduction via a large number of two-component systems, by chemotaxis and flagella-mediated motility and possibly even by gas vacuole formation. These findings extend our understanding of thaumarchaeal evolution and physiology and offer many testable hypotheses for future experimental research on these nitrifiers.

Cultivation of a novel cold-adapted nitrite oxidizing betaproteobacterium from the Siberian Arctic

Permafrost-affected soils of the Siberian Arctic were investigated with regard to identification of nitrite oxidizing bacteria active at low temperature. Analysis of the fatty acid profiles of enrichment cultures grown at 4°C, 10°C and 17°C revealed a pattern that was different from that of known nitrite oxidizers but was similar to fatty acid profiles of Betaproteobacteria. Electron microscopy of two enrichment cultures grown at 10°C showed prevalent cells with a conspicuous ultrastructure. Sequence analysis of the 16S rRNA genes allocated the organisms to a so far uncultivated cluster of the Betaproteobacteria, with Gallionella ferruginea as next related taxonomically described organism. The results demonstrate that a novel genus of chemolithoautotrophic nitrite oxidizing bacteria is present in polygonal tundra soils and can be enriched at low temperatures up to 17°C. Cloned sequences with high sequence similarities were previously reported from mesophilic habitats like activated sludge and therefore an involvement of this taxon in nitrite oxidation in nonarctic habitats is suggested. The presented culture will provide an opportunity to correlate nitrification with nonidentified environmental clones in moderate habitats and give insights into mechanisms of cold adaptation. We propose provisional classification of the novel nitrite oxidizing bacterium as ‘Candidatus Nitrotoga arctica’.

Crenarchaeol dominates the membrane lipids of Candidatus Nitrososphaera gargensis, a thermophilic Group I.1b Archaeon

Analyses of archaeal membrane lipids are increasingly being included in ecological studies as a comparatively unbiased complement to gene-based microbiological approaches. For example, crenarchaeol, a glycerol dialkyl glycerol tetraether (GDGT) with a unique cyclohexane moiety, has been postulated as biomarker for ammonia-oxidizing Archaea (AOA). Crenarchaeol has been detected in Nitrosopumilus maritimus and ‘Candidatus Nitrosocaldus yellowstonii’ representing two of the three lineages within the Crenarchaeota containing described AOA. In this paper we present the membrane GDGT composition of ‘Candidatus Nitrososphaera gargensis’, a moderately thermophilic AOA, and the only cultivated Group I.1b Crenarchaeon. At a cultivation temperature of 46 °C, GDGTs of this organism consisted primarily of crenarchaeol, its regioisomer, and a novel GDGT. Intriguingly, ‘Ca. N. gargensis’ is the first cultivated archaeon to synthesize substantial amounts of the crenarchaeol regioisomer, a compound found in large relative abundances in tropical ocean water and some soils, and an important component of the TEX86 paleothermometer. Intact polar lipid (IPL) analysis revealed that ‘Ca. N. gargensis’ synthesizes IPLs similar to those reported for the Goup I.1a AOA, Nitrosopumilus maritimus SCMI, in addition to IPLs containing uncharacterized headgroups. Overall, the unique GDGT composition of ‘Ca. N. gargensis’ extends the known taxonomic distribution of crenarchaeol synthesis to the Group I.1b Crenarchaeota, implicating this clade as a potentially important source of crenarchaeol in soils and moderately high temperature environments. Moreover, this work supports the hypothesis that crenarchaeol is specific to all AOA and highlights specific lipids, which may prove useful as biomarkers for ‘Ca. N. gargensis’-like AOA.

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira

Significance Nitrification, the sequential aerobic oxidation of ammonia via nitrite to nitrate, is a key process of the biogeochemical nitrogen cycle and catalyzed by two aerobic microbial guilds (nitrifiers): ammonia oxidizers and nitrite-oxidizing bacteria (NOB). NOB are generally considered as metabolically restricted and dependent on ammonia oxidizers. Here, we report that, surprisingly, key NOB of many ecosystems (Nitrospira) convert urea, an important ammonia source in nature, to ammonia and CO2. Thus, Nitrospira supply urease-negative ammonia oxidizers with ammonia and receive nitrite produced by ammonia oxidation in return, leading to a reciprocal feeding interaction of nitrifiers. Moreover, Nitrospira couple formate oxidation with nitrate reduction to remain active in anoxia. Accordingly, Nitrospira are unexpectedly flexible and contribute to nitrogen cycling beyond nitrite oxidation. Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

Comparison of Oxidation Kinetics of Nitrite-Oxidizing Bacteria: Nitrite Availability as a Key Factor in Niche Differentiation

ABSTRACT Nitrification has an immense impact on nitrogen cycling in natural ecosystems and in wastewater treatment plants. Mathematical models function as tools to capture the complexity of these biological systems, but kinetic parameters especially of nitrite-oxidizing bacteria (NOB) are lacking because of a limited number of pure cultures until recently. In this study, we compared the nitrite oxidation kinetics of six pure cultures and one enrichment culture representing three genera of NOB (Nitrobacter, Nitrospira, Nitrotoga). With half-saturation constants (Km ) between 9 and 27 μM nitrite, Nitrospira bacteria are adapted to live under significant substrate limitation. Nitrobacter showed a wide range of lower substrate affinities, with Km values between 49 and 544 μM nitrite. However, the advantage of Nitrobacter emerged under excess nitrite supply, sustaining high maximum specific activities (V max) of 64 to 164 μmol nitrite/mg protein/h, contrary to the lower activities of Nitrospira of 18 to 48 μmol nitrite/mg protein/h. The V max (26 μmol nitrite/mg protein/h) and Km (58 μM nitrite) of “Candidatus Nitrotoga arctica” measured at a low temperature of 17°C suggest that Nitrotoga can advantageously compete with other NOB, especially in cold habitats. The kinetic parameters determined represent improved basis values for nitrifying models and will support predictions of community structure and nitrification rates in natural and engineered ecosystems.

Immunological Detection of Nitrospira-like Bacteria in Various Soils

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.

NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite‐oxidizing Nitrospira

Nitrospira are the most widespread and diverse known nitrite-oxidizing bacteria and key nitrifiers in natural and engineered ecosystems. Nevertheless, their ecophysiology and environmental distribution are understudied because of the recalcitrance of Nitrospira to cultivation and the lack of a molecular functional marker, which would allow the detection of Nitrospira in the environment. Here we introduce nxrB, the gene encoding subunit beta of nitrite oxidoreductase, as a functional and phylogenetic marker for Nitrospira. Phylogenetic trees based on nxrB of Nitrospira were largely congruent to 16S ribosomal RNA-based phylogenies. By using new nxrB-selective polymerase chain reaction primers, we obtained almost full-length nxrB sequences from Nitrospira cultures, two activated sludge samples, and several geographically and climatically distinct soils. Amplicon pyrosequencing of nxrB fragments from 16 soils revealed a previously unrecognized diversity of terrestrial Nitrospira with 1801 detected species-level operational taxonomic units (OTUs) (using an inferred species threshold of 95% nxrB identity). Richness estimates ranged from 10 to 946 coexisting Nitrospira species per soil. Comparison with an archaeal amoA dataset obtained from the same soils [Environ. Microbiol. 14: 525-539 (2012)] uncovered that ammonia-oxidizing archaea and Nitrospira communities were highly correlated across the soil samples, possibly indicating shared habitat preferences or specific biological interactions among members of these nitrifier groups.

Isolation and characterization of a moderately thermophilic nitrite‐oxidizing bacterium from a geothermal spring

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.