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Eva Spieck - One of the best experts on this subject based on the ideXlab platform.
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The draft genome sequence of "Nitrospira lenta" strain BS10, a nitrite oxidizing bacterium isolated from activated sludge.
Standards in genomic sciences, 2018Co-Authors: Dimitra Sakoula, Holger Daims, Eva Spieck, Boris Nowka, Sebastian LückerAbstract:The genus Nitrospira is considered to be the most widespread and abundant group of nitrite-oxidizing bacteria in many natural and man-made ecosystems. However, the ecophysiological versatility within this phylogenetic group remains highly understudied, mainly due to the lack of pure cultures and genomic data. To further expand our understanding of this biotechnologically important genus, we analyzed the high quality draft genome of “Nitrospira lenta” strain BS10, a sublineage II Nitrospira that was isolated from a municipal wastewater treatment plant in Hamburg, Germany. The genome of “N. lenta” has a size of 3,756,190 bp and contains 3968 genomic objects, of which 3907 are predicted protein-coding sequences. Thorough genome annotation allowed the reconstruction of the “N. lenta” core metabolism for energy conservation and carbon fixation. Comparative analyses indicated that most metabolic features are shared with N. moscoviensis and “N. defluvii”, despite their ecological niche differentiation and phylogenetic distance. In conclusion, the genome of “N. lenta” provides important insights into the genomic diversity of the genus Nitrospira and provides a foundation for future comparative genomic studies that will generate a better understanding of the nitrification process.
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expanded metabolic versatility of ubiquitous nitrite oxidizing bacteria from the genus Nitrospira
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Hanna Koch, Craig W. Herbold, Mads Albertsen, Michael Wagner, Eva Spieck, Katharina Kitzinger, Per Halkjaer Nielsen, Holger DaimsAbstract: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.
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improved isolation strategies allowed the phenotypic differentiation of two Nitrospira strains from widespread phylogenetic lineages
FEMS Microbiology Ecology, 2015Co-Authors: Boris Nowka, Holger Daims, Sandra Off, Eva SpieckAbstract:The second step of nitrification, the oxidation of nitrite to nitrate, is vital for the functioning of the nitrogen cycle, but our understanding of the ecological roles of the involved microorganisms is still limited. The known diversity of Nitrospira , the most widely distributed nitrite-oxidizing bacteria, has increased remarkably by analyses of 16S rRNA and functional gene sequences. However, only few representatives could be brought into laboratory cultures so far. In this study, two Nitrospira from activated sludge were isolated using novel approaches together with established methods. Highly enriched ‘ Candidatus Nitrospira defluvii ’ was separated from concomitant heterotrophs by taking advantage of its resistance against ampicillin and acriflavine. Beside this member of lineage I, a novel species of lineage II, named N. lenta , was initially enriched at 10°C and finally purified by using optical tweezers. The tolerance to elevated nitrite levels was much higher in N. defluvii than in the more fastidious N. lenta and was accompanied by pronounced biofilm formation. Phylogenetic classification of 12 additional enrichments indicated that Nitrospira lineage I is common in extreme and moderate ecosystems like lineage II. The new cultures will help to explore physiological and genomic differences affecting niche separation between members of this highly diverse genus.
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nxrb encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite oxidizing Nitrospira
Environmental Microbiology, 2014Co-Authors: Michael Peste, Eva Spieck, Thomas Rattei, Hanna Koch, Sebastia Lucke, Frank Maixne, David Erry, Oris Nowka, Andreas Richte, E LebedevaAbstract:Summary 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.
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relevance and diversity of Nitrospira populations in biofilters of brackish ras
PLOS ONE, 2013Co-Authors: Myriam Kruse, Eva Spieck, Sabine Keuter, Evert P Bakker, Till Eggers, André LipskiAbstract:Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with 13C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of Nitrospira-related labeled lipid markers were found confirming that Nitrospira is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as Nitrobacter or Nitrotoga were functionally not relevant in the investigated biofilters. Nitrospira-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of Nitrospira, though most sequences clustered with the type strain of Nitrospira marina and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of Nitrospira sublineage IV organisms. The membranes included two marker lipids (16∶1 cis7 and 16∶1 cis11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine Nitrospira sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems.
Holger Daims - One of the best experts on this subject based on the ideXlab platform.
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The draft genome sequence of "Nitrospira lenta" strain BS10, a nitrite oxidizing bacterium isolated from activated sludge.
Standards in genomic sciences, 2018Co-Authors: Dimitra Sakoula, Holger Daims, Eva Spieck, Boris Nowka, Sebastian LückerAbstract:The genus Nitrospira is considered to be the most widespread and abundant group of nitrite-oxidizing bacteria in many natural and man-made ecosystems. However, the ecophysiological versatility within this phylogenetic group remains highly understudied, mainly due to the lack of pure cultures and genomic data. To further expand our understanding of this biotechnologically important genus, we analyzed the high quality draft genome of “Nitrospira lenta” strain BS10, a sublineage II Nitrospira that was isolated from a municipal wastewater treatment plant in Hamburg, Germany. The genome of “N. lenta” has a size of 3,756,190 bp and contains 3968 genomic objects, of which 3907 are predicted protein-coding sequences. Thorough genome annotation allowed the reconstruction of the “N. lenta” core metabolism for energy conservation and carbon fixation. Comparative analyses indicated that most metabolic features are shared with N. moscoviensis and “N. defluvii”, despite their ecological niche differentiation and phylogenetic distance. In conclusion, the genome of “N. lenta” provides important insights into the genomic diversity of the genus Nitrospira and provides a foundation for future comparative genomic studies that will generate a better understanding of the nitrification process.
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Complete nitrification by Nitrospira bacteria
Nature, 2015Co-Authors: Holger Daims, Julia Vierheilig, Márton Palatinszky, Petra Pjevac, Craig W. Herbold, Nico Jehmlich, Mads Albertsen, Elena V Lebedeva, Aleksandr G. BulaevAbstract:Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.
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expanded metabolic versatility of ubiquitous nitrite oxidizing bacteria from the genus Nitrospira
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Hanna Koch, Craig W. Herbold, Mads Albertsen, Michael Wagner, Eva Spieck, Katharina Kitzinger, Per Halkjaer Nielsen, Holger DaimsAbstract: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.
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Functionally relevant diversity of closely related Nitrospira in activated sludge
The ISME Journal, 2015Co-Authors: Christiane Gruber-dorninger, Michael Wagner, Thomas Rattei, Katharina Kitzinger, Michael Pester, Domenico F Savio, Holger DaimsAbstract:Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira -specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira , revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized high diversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability.
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improved isolation strategies allowed the phenotypic differentiation of two Nitrospira strains from widespread phylogenetic lineages
FEMS Microbiology Ecology, 2015Co-Authors: Boris Nowka, Holger Daims, Sandra Off, Eva SpieckAbstract:The second step of nitrification, the oxidation of nitrite to nitrate, is vital for the functioning of the nitrogen cycle, but our understanding of the ecological roles of the involved microorganisms is still limited. The known diversity of Nitrospira , the most widely distributed nitrite-oxidizing bacteria, has increased remarkably by analyses of 16S rRNA and functional gene sequences. However, only few representatives could be brought into laboratory cultures so far. In this study, two Nitrospira from activated sludge were isolated using novel approaches together with established methods. Highly enriched ‘ Candidatus Nitrospira defluvii ’ was separated from concomitant heterotrophs by taking advantage of its resistance against ampicillin and acriflavine. Beside this member of lineage I, a novel species of lineage II, named N. lenta , was initially enriched at 10°C and finally purified by using optical tweezers. The tolerance to elevated nitrite levels was much higher in N. defluvii than in the more fastidious N. lenta and was accompanied by pronounced biofilm formation. Phylogenetic classification of 12 additional enrichments indicated that Nitrospira lineage I is common in extreme and moderate ecosystems like lineage II. The new cultures will help to explore physiological and genomic differences affecting niche separation between members of this highly diverse genus.
Barth F. Smets - One of the best experts on this subject based on the ideXlab platform.
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genomic profiling of Nitrospira species reveals ecological success of comammox Nitrospira
bioRxiv, 2019Co-Authors: Alejandro Palomo, Arnaud Dechesne, Barth F. SmetsAbstract:Abstract Nitrification was long thought to consist in the stepwise oxidation of ammonia to nitrite and of nitrite to nitrate by ammonia oxidizing and nitrite oxidizing microorganisms, respectively. Recently, single microorganisms capable of complete ammonia to nitrate oxidation (comammox) were identified in the Nitrospira genus. This genus, previously considered to only contain canonical nitrite oxidizers is diverse and has a broad environmental distribution. Yet, a global insight into the abundance, niche preference, and genomic diversity of Nitrospira is missing. Here, we established the largest Nitrospira genome database to date, revealing 68 putative species, most without cultivated representatives. We performed a global survey through read recruitment of metagenomic data from various environments against this database that identified that environmental filtering structures species distribution, without large scale biogeographical signal. The ecological success of comammox Nitrospira is evident as they outnumber and are more diverse than canonical Nitrospira in communities from all environments but wastewater treatment plants. We detect a phylogenetic signal in Nitrospira species habitat preference, that is strongest for canonical Nitrospira species. Comammox Nitrospira eco-evolutionary history is more complex with subclades achieving rapid niche divergence via horizontal transfer of genes, including that encoding the hydroxylamine oxidoreductase, one of the key enzymes involved in nitrification.
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Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira
The ISME Journal, 2018Co-Authors: Alejandro Palomo, Anders G. Pedersen, S. Jane Fowler, Arnaud Dechesne, Thomas Sicheritz-pontén, Barth F. SmetsAbstract:The description of comammox Nitrospira spp., performing complete ammonia-to-nitrate oxidation, and their co-occurrence with canonical β-proteobacterial ammonia oxidizing bacteria (β-AOB) in the environment, calls into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonia oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira . Unlike canonical Nitrospira , comammox Nitrospira genomes lack genes for assimilatory nitrite reduction, suggesting that they have lost the potential to use external nitrite nitrogen sources. By contrast, compared to canonical Nitrospira , comammox Nitrospira harbor a higher diversity of urea transporters and copper homeostasis genes and lack cyanate hydratase genes. Additionally, the two comammox clades differ in their ammonium uptake systems. Contrary to β-AOB, comammox Nitrospira genomes have single copies of the two central ammonia oxidation pathway operons. Similar to ammonia oxidizing archaea and some oligotrophic AOB strains, they lack genes involved in nitric oxide reduction. Furthermore, comammox Nitrospira genomes encode genes that might allow efficient growth at low oxygen concentrations. Regarding the evolutionary history of comammox Nitrospira , our analyses indicate that several genes belonging to the ammonia oxidation pathway could have been laterally transferred from β-AOB to comammox Nitrospira . We postulate that the absence of comammox genes in other sublineage II Nitrospira genomes is the result of subsequent loss.
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comammox Nitrospira are abundant ammonia oxidizers in diverse groundwater fed rapid sand filter communities
Environmental Microbiology, 2018Co-Authors: Susan Jane Fowler, Alejandro Palomo, Arnaud Dechesne, Paul D Mines, Barth F. SmetsAbstract:: The recent discovery of completely nitrifying Nitrospira demands a re-examination of nitrifying environments to evaluate their contribution to nitrogen cycling. To approach this challenge, tools are needed to detect and quantify comammox Nitrospira. We present primers for the simultaneous quantification and diversity assessement of both comammox Nitrospira clades. The primers cover a wide range of comammox diversity, spanning all available high quality sequences. We applied these primers to 12 groundwater-fed rapid sand filters, and found comammox Nitrospira to be abundant in all filters. Clade B comammox comprise the majority (∼75%) of comammox abundance in all filters. Nitrosomonadaceae were present in all filters, although at low abundance (mean = 1.8%). Ordination suggests that temperature impacts the structure of nitrifying communities, and in particular that increasing temperature favours Nitrospira. The nitrogen content of the filter material, sulfate concentration and surface ammonium loading rates shape the structure of the comammox guild in the filters. This work provides an assay for simultaneous detection and diversity assessment of clades A and B comammox Nitrospira, expands our current knowledge of comammox Nitrospira diversity and demonstrates a key role for comammox Nitrospira in nitrification in groundwater-fed biofilters.
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comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira
bioRxiv, 2017Co-Authors: Alejandro Palomo, Anders G. Pedersen, Arnaud Dechesne, Jane Fowler, Thomas Sicheritzponten, Barth F. SmetsAbstract:The description of comammox Nitrospira spp., performing complete ammonium-to-nitrate oxidation, and their co-occurrence with canonical betaproteobacterial ammonium oxidizing bacteria (β-AOB) in the environment, call into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonium oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira . Comammox Nitrospira has lost the potential to use external nitrite as energy and nitrogen source: compared to strictly nitrite oxidizing Nitrospira ; they lack genes for assimilative nitrite reduction and reverse electron transport from nitrite. By contrast, compared to other Nitrospira , their ammonium oxidizer physiology is exemplified by genes for ammonium and urea transporters and copper homeostasis and the lack of cyanate hydratase genes. Two comammox clades are different in their ammonium uptake systems. Contrary to β-AOB, comammox Nitrospira genomes have single copies of the two central ammonium oxidation pathway genes, lack genes involved in nitric oxide reduction, and encode genes that would allow efficient growth at low oxygen concentrations. Hence, comammox Nitrospira seems attuned to oligotrophy and hypoxia compared to β-AOB. β-AOBs are the clear origin of the ammonium oxidation pathway in comammox Nitrospira : reconciliation analysis indicates two separate early amoA gene transfer events from β-AOB to an ancestor of comammox Nitrospira , followed by clade specific losses. For haoA , one early transfer from β-AOB to comammox Nitrospira is predicted - followed by intra-clade transfers. We postulate that the absence of comammox genes in most Nitrospira genomes is the result of subsequent loss.
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Metagenomic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp.
The ISME Journal, 2016Co-Authors: Alejandro Palomo, Arda Gulay, Thomas Sicheritz-pontén, S Jane Fowler, Simon Rasmussen, Barth F. SmetsAbstract:Rapid gravity sand filtration is a drinking water production technology widely used around the world. Microbially catalyzed processes dominate the oxidative transformation of ammonia, reduced manganese and iron, methane and hydrogen sulfide, which may all be present at millimolar concentrations when groundwater is the source water. In this study, six metagenomes from various locations within a groundwater-fed rapid sand filter (RSF) were analyzed. The community gene catalog contained most genes of the nitrogen cycle, with particular abundance in genes of the nitrification pathway. Genes involved in different carbon fixation pathways were also abundant, with the reverse tricarboxylic acid cycle pathway most abundant, consistent with an observed Nitrospira dominance. From the metagenomic data set, 14 near-complete genomes were reconstructed and functionally characterized. On the basis of their genetic content, a metabolic and geochemical model was proposed. The organisms represented by draft genomes had the capability to oxidize ammonium, nitrite, hydrogen sulfide, methane, potentially iron and manganese as well as to assimilate organic compounds. A composite Nitrospira genome was recovered, and amo -containing Nitrospira genome contigs were identified. This finding, together with the high Nitrospira abundance, and the abundance of atypical amo and hao genes, suggests the potential for complete ammonium oxidation by Nitrospira , and a major role of Nitrospira in the investigated RSFs and potentially other nitrifying environments.
Satoshi Tsuneda - One of the best experts on this subject based on the ideXlab platform.
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genomic and physiological characteristics of a novel nitrite oxidizing Nitrospira strain isolated from a drinking water treatment plant
Frontiers in Microbiology, 2020Co-Authors: Hirotsugu Fujitani, Norisuke Ushiki, Kengo Momiuchi, Kento Ishii, Manami Nomachi, Shuta Kikuchi, Yuji Sekiguchi, Satoshi TsunedaAbstract:Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genus Nitrospira are often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox) Nitrospira support the ecological importance of the genus Nitrospira. However, niche differentiation between nitrite-oxidizing Nitrospira and comammox Nitrospira remains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizing Nitrospira strain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment of Nitrospira-like cells, as well as members of ammonia-oxidizing genus Nitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizing Nitrospira strain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity with Nitrospira japonica (98%) at the 16S rRNA gene level. The two other Nitrospira OTUs shared over 99% sequence similarities with N. japonica and Nitrospira sp. strain GC86. Only one strain identified as Nitrospira was successfully subcultivated and designated as Nitrospira sp. strain KM1 with high sequence similarity with N. japonica (98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other known Nitrospira strains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolated Nitrospira strains. Therefore, the new Nitrospira strain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known cultured Nitrospira genome sequences are 76.7-78.4%, suggesting at least species-level novelty of the strain in the Nitrospira lineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizing Nitrospira.
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enrichment of comammox and nitrite oxidizing Nitrospira from acidic soils
Frontiers in Microbiology, 2020Co-Authors: Yu Takahashi, Hirotsugu Fujitani, Yuhei Hirono, Kanako Tago, Yong Wang, Masahito Hayatsu, Satoshi TsunedaAbstract:In agricultural soils fertilized with a high amount of ammonium nitrogen, the pH decreases because of the oxidation of ammonia by nitrifiers. Molecular-based analyses have revealed that members of the genus Nitrospira dominate over other nitrifiers in some acidic soils. However, terrestrial Nitrospira are rarely cultivated and little is known about their ecophysiology. In addition, recent studies discovered a single microbe with the potential to oxidize both ammonia and nitrite (complete ammonia oxidizer; comammox) within Nitrospira, which had been previously recognized as a nitrite oxidizer. Despite their broad distribution, there are no enrichment samples of comammox from terrestrial or acidic environments. Here, we report the selective enrichment of both comammox and nitrite-oxidizing Nitrospira from the acidic soil of a heavily fertilized tea field. Long-term enrichment was performed with two individual continuous-feeding bioreactors capable of controlling ammonia or nitrite concentration and pH. We found that excessive ammonium supply was a key factor to enhance the growth of comammox Nitrospira under acidic conditions. Additionally, a low concentration of nitrite was fed to prevent the accumulation of free nitrous acid and inhibition of cell growth under low pH, resulting in the selective enrichment of nitrite-oxidizing Nitrospira. Based on 16S rRNA gene analysis, Nitrospira accounting for only 1.2% in an initial soil increased to approximately 80% of the total microorganisms in both ammonia- and nitrite-fed bioreactors. Furthermore, amoA amplicon sequencing revealed that two phylotypes belonging to comammox clade A were enriched in an ammonia-fed bioreactor. One group was closely related to previously cultivated strains, and the other was classified into a different cluster consisting of only uncultivated representatives. These two groups coexisted in the bioreactor controlled at pH 6.0, but the latter became dominant after the pH decreased to 5.5. Additionally, a physiological experiment revealed that the enrichment sample oxidizes ammonia at pH <4, which is in accordance with the strongly acidic tea field soil; this value is lower than the active pH range of isolated acid-adapted nitrifiers. In conclusion, we successfully enriched multiple phylotypes of comammox and nitrite-oxidizing Nitrospira and revealed that the pH and concentrations of protonated N-compounds were potential niche determinants.
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successful enrichment of low abundant comammox Nitrospira from nitrifying granules under ammonia limited conditions
Fems Microbiology Letters, 2020Co-Authors: Hirotsugu Fujitani, Manami Nomachi, Yu Takahashi, Yoshiaki Hasebe, Masahiro Eguchi, Satoshi TsunedaAbstract:In artificial engineered systems, nitrification is a key reaction that accounts for the removal of biological nitrogen. Recently, a single microbe capable of oxidizing ammonia to nitrate, known as a complete ammonia oxidizer (comammox), has been discovered. Although the abundance and diversity of comammox Nitrospira in engineered systems have been identified through molecular-based approaches, the enrichment and isolation of comammox Nitrospira remains a challenge. Therefore, the aim of this study was to enrich comammox Nitrospira from nitrifying granules, which were used to increase the efficiency of biological nitrogen removal in wastewater treatment plants. We sought to accomplish this through the use of a fixed-bed continuous feeding bioreactor. Fluorescence in situ hybridization, 16S rRNA gene amplicon sequencing and qPCR of functional genes were utilized to monitor the growth of nitrifiers including comammox Nitrospira. Cloning of comammox amoA genes identified amoA phylogeny of enriched comammox Nitrospira. This work is an example demonstrating that continuous supply of low ammonium concentrations alongside biomass carriers is effective in cultivating comammox Nitrospira from engineered systems.
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genomic analysis of two phylogenetically distinct Nitrospira species reveals their genomic plasticity and functional diversity
Frontiers in Microbiology, 2018Co-Authors: Norisuke Ushiki, Hirotsugu Fujitani, Yuji Sekiguchi, Yu Shimada, Tomohiro Morohoshi, Satoshi TsunedaAbstract:The genus Nitrospira represents a dominant group of nitrite-oxidizing bacteria in natural and engineered ecosystems. This genus is phylogenetically divided into six lineages, for which vast phylogenetic and functional diversity has been revealed by recent molecular ecophysiological analyses. However, the genetic basis underlying these phenotypic differences remains largely unknown because of the lack of genome sequences representing their diversity. To gain a more comprehensive understanding of Nitrospira, we performed genomic comparisons between two Nitrospira strains (ND1 and NJ1 belonging to lineages I and II, respectively) previously isolated from activated sludge. In addition, the genomes of these strains were systematically compared with previously reported six Nitrospira genomes to reveal their similarity and presence/absence of several functional genes/operons. Comparisons of Nitrospira genomes indicated that their genomic diversity reflects phenotypic differences and versatile nitrogen metabolisms. Although most genes involved in key metabolic pathways were conserved between strains ND1 and NJ1, assimilatory nitrite reduction pathways of the two Nitrospira strains were different. In addition, the genomes of both strains contain a phylogenetically different urease locus and we confirmed their ureolytic activity. During gene annotation of strain NJ1, we found a gene cluster encoding a quorum-sensing system. From the enriched supernatant of strain NJ1, we successfully identified seven types of acyl-homoserine lactones with a range of C10-C14. In addition, the genome of strain NJ1 lacks genes relevant to flagella and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated genes) systems, whereas most nitrifying bacteria including strain ND1 possess these genomic elements. These findings enhance our understanding of genomic plasticity and functional diversity among members of the genus Nitrospira.
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nitrite oxidation kinetics of two Nitrospira strains the quest for competition and ecological niche differentiation
Journal of Bioscience and Bioengineering, 2017Co-Authors: Norisuke Ushiki, Hirotsugu Fujitani, Masaru Jinno, Toshikazu Suenaga, Akihiko Terada, Satoshi TsunedaAbstract:Nitrite oxidation is an aerobic process of the nitrogen cycle in natural ecosystems, and is performed by nitrite-oxidizing bacteria (NOB). Also, nitrite oxidation is a rate-limiting step of nitrogen removal in wastewater treatment plants (WWTPs). Although Nitrospira is known as dominant NOB in WWTPs, information on their physiological properties and kinetic parameters is limited. Here, we report the kinetic parameters and inhibition of nitrite oxidation by free ammonia in pure cultures of Nitrospira sp. strain ND1 and Nitrospira japonica strain NJ1, which were previously isolated from activated sludge in a WWTP. The maximum nitrite uptake rate ( V max _ NO 2 ) and the half-saturation constant for nitrite uptake ( K m _ NO 2 ) of strains ND1 and NJ1 were 45 ± 7 and 31 ± 5 (μmol NO 2 − /mg protein/h), and 6 ± 1 and 10 ± 2 (μM NO 2 − ), respectively. The V max _ NO 2 and K m _ NO 2 of two strains indicated that they adapt to low-nitrite-concentration environments like activated sludge. The half-saturation constants for oxygen uptake ( K m _ O 2 ) of the two strains were 4.0 ± 2.5 and 2.6 ± 1.1 (μM O 2 ), respectively. The K m _ O 2 values of the two strains were lower than those of other NOB, suggesting that Nitrospira in activated sludge could oxidize nitrite in the hypoxic environments often found in the interiors of biofilms and flocs. The inhibition thresholds of the two strains by free ammonia were 0.85 and 4.3 (mg-NH 3 l −1 ), respectively. Comparing the physiological properties of the two strains, we suggest that tolerance for free ammonia determines competition and partitioning into ecological niches among Nitrospira populations.
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enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification
The ISME Journal, 2021Co-Authors: Mike S. M. Jetten, Hanna Koch, Dimitra Sakoula, Jeroen Frank, Maartje A H J Van Kessel, Sebastian LückerAbstract:The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Furthermore, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.036 µM), but a higher nitrite affinity (Km(app)_NO2- ≈ 13.8 µM), indicating an adaptation to highly oligotrophic environments. Counterintuitively for a nitrifying microorganism, we also observed an inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This substrate inhibition of “Ca. N. kreftii” indicate that differences in ammonium tolerance rather than affinity can be a niche determining factor for different comammox Nitrospira.Competing Interest StatementThe authors have declared no competing interest.
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selective enrichment and metagenomic analysis of three novel comammox Nitrospira in a urine fed membrane bioreactor
ISME Communications, 2021Co-Authors: Zhengshuang Hua, Sebastian Lücker, Mike S. M. Jetten, Tao Liu, Chengwen Wang, Ge Bai, Min Zheng, Jianhua GuoAbstract:The discovery of complete ammonia-oxidizing (comammox) Nitrospira has added an important new process to the microbial nitrogen cycle. While comammox Nitrospira have been detected in various ecosystems, only few studies have achieved their enrichment over other canonical nitrifiers. Here, we obtained a selective enrichment of comammox Nitrospira in a urine-fed membrane bioreactor in less than 200 days. By using 16S rRNA gene amplicon sequencing and quantitative PCR of the functional marker gene amoA, we observed a dominance (up to 30% relative abundance) of comammox Nitrospira over ammonia-oxidizing bacteria and archaea. Furthermore, the complete genomes of three new clade A comammox Nitrospira were recovered by metagenomics. These three strains were divergent from previously reported comammox species according to comparative genome and amoA-based analyses. In addition to the key genes for ammonia and nitrite oxidation, the three recovered genomes contained a complete urea utilization pathway. Our findings suggest that the urea present in the urine media played a significant role in the selective enrichment of these novel comammox Nitrospira, and support the diversity and versatility of their metabolism.
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investigating the chemolithoautotrophic and formate metabolism of Nitrospira moscoviensis by constraint based metabolic modeling and 13c tracer analysis
bioRxiv, 2021Co-Authors: Mike S. M. Jetten, Hanna Koch, Christopher E Lawson, Aniela B Mundinger, Tyler B Jacobson, Coty A Weathersby, Martin Pabst, Daniel AmadornoguezAbstract:Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used constraint-based analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and 13C-tracer experiments with bicarbonate and formate coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings support that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation. We also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospiras in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. ImportanceNitrospira are globally abundant nitrifying bacteria in soil and aquatic ecosystems and wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of N. moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite- oxidizing bacteria.
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enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification
bioRxiv, 2020Co-Authors: Dimitra Sakoula, Mike S. M. Jetten, Hanna Koch, Jeroen Frank, Maartje A H J Van Kessel, Sebastian LückerAbstract:Abstract The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Furthermore, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.036 µM), but a higher nitrite affinity (Km(app)_NO2- ≈ 13.8 µM), indicating an adaptation to highly oligotrophic environments. Counterintuitively for a nitrifying microorganism, we also observed an inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This substrate inhibition of “Ca. N. kreftii” indicate that differences in ammonium tolerance rather than affinity can be a niche determining factor for different comammox Nitrospira.
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cultivation and transcriptional analysis of a canonical Nitrospira under stable growth conditions
Frontiers in Microbiology, 2019Co-Authors: Aniela B Mundinger, Hanna Koch, Mike S. M. Jetten, Christopher E Lawson, Sebastian LückerAbstract:Nitrite-oxidizing bacteria (NOB) are vital players in the global nitrogen cycle that convert nitrite to nitrate during the second step of nitrification. Within this functional guild, members of the genus Nitrospira are most widespread, phylogenetically diverse, and physiologically versatile, and they drive nitrite oxidation in many natural and engineered ecosystems. Despite their ecological and biotechnological importance, our understanding of their energy metabolism is still limited. A major bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing and fastidious microorganisms. In this study, we cultivated Nitrospira moscoviensis under nitrite-oxidizing conditions in a continuous stirred tank reactor (CSTR) system. This cultivation setup enabled accurate control of physicochemical parameters and avoided fluctuating levels of their energy substrate nitrite, thus ensuring constant growth conditions and furthermore allowing continuous biomass harvesting. Transcriptomic analyses under these conditions supported the predicted core metabolism of N. moscoviensis, including expression of all proteins required for carbon fixation via the reductive tricarboxylic acid cycle, assimilatory nitrite reduction, and the complete respiratory chain. Here, simultaneous expression of multiple copies of respiratory complexes I and III suggested functional differentiation. The transcriptome also indicated that the previously assumed membrane-bound nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation, is formed by three soluble subunits. Overall, the transcriptomic data greatly refined our understanding of the metabolism of Nitrospira. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of these fascinating microorganisms.