Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical spongeIanthella basta

SummaryMarine sponges represent one of the few eukaryotic groups that frequently harbor symbiotic members of theThaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However, in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of ammonia-oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine spongeIanthella bastausing metaproteogenomics, fluorescencein situhybridization, qPCR and isotope-based functional assays. “CandidatusNitrosospongia bastadiensis” is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia inI. bastathat surprisingly does not harbor nitrite-oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system, represent important adaptations of AOA to life within these ancient filter-feeding animals.Originality-Significance StatementMany marine sponges harbor symbiotic members of theThaumarchaeota, but there is generally only indirect evidence available about their functional role within these filter-feeding animals. Furthermore, the specific adaptations of thaumarchaeal symbionts to their sponge hosts are incompletely understood. In this study, we thoroughly characterized a thaumarchaeal symbiont residing in the reef spongeIanthella bastaand demonstrate by using a combination of molecular tools and isotope techniques, that it is the only ammonia-oxidizer in its host. In contrast to other sponges,I. bastadoes not contain nitrite-oxidizing microbes and thus excretes considerable amounts of nitrite. Furthermore, using metagenomics and metaproteomics we reveal important adaptations of this symbiont, that represents a new genus within theThaumarchaeota, and conclude that it most likely lives as a mixotroph in its sponge host.

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Transcriptional Activity of Aerobic and Anaerobic Ammonia-Oxidizing community in the Intertidal Sponge Cinachyrella Australiensis, Ambient Seawater, and Sediment

10.21203/rs.3.rs-382482/v1 ◽

2021 ◽

Author(s):

Guofang Feng ◽

Shaofeng Li ◽

Lijuan Zhang ◽

Huchun Tao

Keyword(s):

Nitrogen Cycling ◽

Ammonia Oxidation ◽

Niche Differentiation ◽

Marine Sponges ◽

Ammonia Oxidizing Bacteria ◽

Ambient Seawater ◽

Ammonia Oxidizing Archaea ◽

Intertidal Sponge ◽

Aerobic And Anaerobic

Abstract Microbial ammonia oxidation plays a central role in nitrogen cycling. Hitherto, four types of autotrophic ammonia-oxidizing microorganisms are identified, including aerobic ammonia-oxidizing archaea (AOA), aerobic partial-nitrification ammonia-oxidizing bacteria (parAOB), aerobic complete-nitrification AOB (comAOB), and anaerobic AOB (AnAOB). However, revelation and comparison of the active ammonia-oxidizing community in the marine sponges and their ambient environments is scarce. Here, transcribed ammonia oxidation phylomarker gene amoA of AOA, parAOB, and comAOB and hzsB of AnAOB were amplified to investigate the active ammonia-oxidizing populations in a representative marine sponge Cinachyrella australiensis, ambient seawater, and sediment niches. Ammonia-oxidizing population in C. australiensis consists of AOA, parAOB, and AnAOB, significantly different from that in seawaters comprising of AOA and in sediments containing AOA, parAOB, comAOB, and AnAOB. The quantitative assay demonstrates that AOA amoA transcripts are exclusively detectable or higher in abundance than parAOB amoA, comAOB amoA, or AnAOB hzsB transcripts by orders of magnitude in C. australiensis, seawater, and sediment niches. This transcript-based analysis clarifies the remarkable niche differentiation of putatively active ammonia-oxidizing microbiota in C. australiensis and the ambient environments. Such a work further contributes to the understanding of in situ active ecological functions of sponge microsymbionts in nitrogen cycling.

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Transcriptional Response of the Archaeal Ammonia Oxidizer Nitrosopumilus maritimus to Low and Environmentally Relevant Ammonia Concentrations

Applied and Environmental Microbiology ◽

10.1128/aem.02028-13 ◽

2013 ◽

Vol 79 (22) ◽

pp. 6911-6916 ◽

Cited By ~ 28

Author(s):

Tatsunori Nakagawa ◽

David A. Stahl

Keyword(s):

Ammonia Oxidation ◽

Physiological State ◽

Transcriptional Response ◽

Ammonia Oxidizer ◽

Mrna Levels ◽

Marine Microorganisms ◽

Transcript Levels ◽

Content Type ◽

Ammonia Oxidizing Archaea ◽

Ammonia Transport

ABSTRACTThe ability of chemoautotrophic ammonia-oxidizing archaea to compete for ammonia among marine microorganisms at low ambient concentrations has been in part attributed to their extremely high affinity for ammonia, but as yet there is no mechanistic understanding of supporting metabolism. We examined transcription of selected genes for anabolic functions (CO2fixation, ammonia transport, and cell wall synthesis) and a central catabolic function (ammonia oxidation) in the thaumarchaeonNitrosopumilus maritimusSCM1 growing at two ammonia concentrations, as measured by combined ammonia and ammonium, one well above theKmfor ammonia oxidation (∼500 μM) and the other well below theKm(<10 nM). Transcript levels were generally immediately and differentially repressed when cells transitioned from ammonia-replete to ammonia-limiting conditions. Transcript levels for ammonia oxidation, CO2fixation, and one of the ammonia transport genes were approximately the same at high and low ammonia availability. Transcripts for all analyzed genes decreased with time in the complete absence of ammonia, but with various rates of decay. The new steady-state mRNA levels established are presumably more reflective of the natural physiological state of ammonia-oxidizing archaea and offer a reference for interpreting message abundance patterns in the natural environment.

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Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream

Applied and Environmental Microbiology ◽

10.1128/aem.00250-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4492-4504 ◽

Cited By ~ 14

Author(s):

Manabu Nishizawa ◽

Sanae Sakai ◽

Uta Konno ◽

Nozomi Nakahara ◽

Yoshihiro Takaki ◽

...

Keyword(s):

Oxygen Isotope ◽

Ammonia Oxidation ◽

Isotope Effects ◽

Isotope Ratios ◽

Oxidation Activity ◽

Content Type ◽

Ammonia Oxidizing Archaea ◽

Nitrogen Pools ◽

Archaeal Ammonia Oxidation

ABSTRACTAmmonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in thein situquantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ15NNO2−and δ18ONO2−, respectively) are geochemical tracers for evaluating the sources and thein siturate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilicThaumarchaeotapopulations composed almost entirely of “CandidatusNitrosocaldus.” The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ18O value of nitrite produced from ammonia oxidation varied with the δ18O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilicThaumarchaeotacould be estimated using δ18ONO2−in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments.IMPORTANCEBecause ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilicThaumarchaeotaand elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

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Molecular investigation of the microbial community associated with the fire sponge, Tedania ignis, in Bermuda

10.7287/peerj.preprints.905v1 ◽

2015 ◽

Author(s):

Nicholas J Jouett ◽

Meredith E Bibbings ◽

Clarisse E Sullivan ◽

Rachel J Parsons

Keyword(s):

Microbial Communities ◽

Fragment Length ◽

Ammonia Oxidation ◽

Marine Sponges ◽

Dapi Staining ◽

Ambient Seawater ◽

Microbial Associations ◽

The Common ◽

Peak Abundance

The complex, phylogenetically diverse, and specific microbial communities associated with marine sponges are a key aspect of the ecology and evolution of the metazoan host and the endosymbiotic microbes. Using fluorescence in situ hybridization (FISH) methods, terminal restriction fragment length polymorphism (T RFLP), and functional gene probing via PCR, the current study investigates the microbial associations in the common Caribbean fire sponge, Tedania ignis. Sponge and water samples were collected from different sites around Bermuda from 2012 to 2014 in order to assess their respective microbial communities. Using FISH, SAR202 (Chloroflexi) (5.82% ± 0.59%) and Crenarchaea (7.97% ± 1.08%) were identified as the most abundant contributors to the microbial assemblage of T. ignis while the Alphaproteobacterium SAR11 (30.68% ± 1.68%) was identified as the most dominant species in the surrounding seawater. Due to the presence of Crenarchaea, the Archaeal gene for ammonia oxidation (amoA) was probed via PCR and found to be present. T RFLP identified the most abundant fragment length present in the sponge as 336 bp (>60% of T RFLP peak abundance). The sponge community was consistent and markedly distinct from that of the ambient seawater as identified by both FISH and T RFLP. Epifluorescent microscopy with DAPI staining also identified T. ignis as a high microbial abundance (HMA) sponge, in contrast to previous studies. Together, these data characterize the microbiome of T. ignis in much further detail than has previously been described.

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Molecular investigation of the microbial community associated with the fire sponge, Tedania ignis, in Bermuda

10.7287/peerj.preprints.905 ◽

2015 ◽

Author(s):

Nicholas J Jouett ◽

Meredith E Bibbings ◽

Clarisse E Sullivan ◽

Rachel J Parsons

Keyword(s):

Microbial Communities ◽

Fragment Length ◽

Ammonia Oxidation ◽

Marine Sponges ◽

Dapi Staining ◽

Ambient Seawater ◽

Microbial Associations ◽

The Common ◽

Peak Abundance

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The Response of Estuarine Ammonia-Oxidizing Communities to Constant and Fluctuating Salinity Regimes

Frontiers in Microbiology ◽

10.3389/fmicb.2020.574815 ◽

2020 ◽

Vol 11 ◽

Author(s):

João Pereira Santos ◽

António G. G. Sousa ◽

Hugo Ribeiro ◽

Catarina Magalhães

Keyword(s):

Ammonia Oxidation ◽

Estuarine Sediments ◽

Ammonia Oxidizer ◽

Rrna Gene ◽

Ammonia Oxidizing Bacteria ◽

Salinity Regime ◽

Salinity Fluctuation ◽

Ammonia Oxidizing Archaea ◽

Negative Effect ◽

The Impact

Aerobic nitrification is a fundamental nitrogen biogeochemical process that links the oxidation of ammonia to the removal of fixed nitrogen in eutrophicated water bodies. However, in estuarine environments there is an enormous variability of water physicochemical parameters that can affect the ammonia oxidation biological process. For instance, it is known that salinity can affect nitrification performance, yet there is still a lack of information on the ammonia-oxidizing communities behavior facing daily salinity fluctuations. In this work, laboratory experiments using upstream and downstream estuarine sediments were performed to address this missing gap by comparing the effect of daily salinity fluctuations with constant salinity on the activity and diversity of ammonia-oxidizing microorganisms (AOM). Activity and composition of AOM were assessed, respectively by using nitrogen stable isotope technique and 16S rRNA gene metabarcoding analysis. Nitrification activity was negatively affected by daily salinity fluctuations in upstream sediments while no effect was observed in downstream sediments. Constant salinity regime showed clearly higher rates of nitrification in upstream sediments while a similar nitrification performance between the two salinity regimes was registered in the downstream sediments. Results also indicated that daily salinity fluctuation regime had a negative effect on both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) community’s diversity. Phylogenetically, the estuarine downstream AOM were dominated by AOA (0.92–2.09%) followed by NOB (0.99–2%), and then AOB (0.2–0.32%); whereas NOB dominated estuarine upstream sediment samples (1.4–9.5%), followed by AOA (0.27–0.51%) and AOB (0.01–0.23%). Analysis of variance identified the spatial difference between samples (downstream and upstream) as the main drivers of AOA and AOB diversity. Our study indicates that benthic AOM inhabiting different estuarine sites presented distinct plasticity toward the salinity regimes tested. These findings help to improve our understanding in the dynamics of the nitrogen cycle of estuarine systems by showing the resilience and consequently the impact of different salinity regimes on the diversity and activity of ammonia oxidizer communities.

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Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities

10.1101/2021.03.02.433310 ◽

2021 ◽

Author(s):

Man-Young Jung ◽

Christopher J. Sedlacek ◽

K. Dimitri Kits ◽

Anna J. Mueller ◽

Sung-Keun Rhee ◽

...

Keyword(s):

Ammonia Oxidation ◽

Oxidation Kinetic ◽

Ammonia Oxidizer ◽

Substrate Affinity ◽

Kinetic Properties ◽

Ammonia Oxidizing Bacteria ◽

Ammonia Oxidizers ◽

Kinetic Measurements ◽

Ammonia Oxidizing Archaea ◽

Wide Range

AbstractNitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co- occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest substrate affinity of any characterized AOA, which are similar to previously determined affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values strongly supports the hypothesis that – like for AOB – ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

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Competition of Ammonia-Oxidizing Archaea and Bacteria from Freshwater Environments

Applied and Environmental Microbiology ◽

10.1128/aem.01038-21 ◽

2021 ◽

Author(s):

Elizabeth French ◽

Jessica A. Kozlowski ◽

Annette Bollmann

Keyword(s):

Ammonia Oxidation ◽

Heterotrophic Bacteria ◽

Ammonia Oxidizer ◽

Ammonium Concentration ◽

Natural Environments ◽

Ammonia Oxidizing Bacteria ◽

Ammonia Oxidizers ◽

Batch Cultures ◽

Freshwater Systems ◽

Ammonia Oxidizing Archaea

In the environment, nutrients are rarely available in constant supply. Therefore, microorganisms require strategies to compete for limiting nutrients. In freshwater systems, ammonia-oxidizing archaea (AOA) and bacteria (AOB) compete with heterotrophic bacteria, photosynthetic microorganisms, and each other for ammonium, which AOA and AOB utilize as their sole source of energy and nitrogen. We investigated the competition between highly enriched cultures of an AOA (AOA-AC1) and an AOB (AOB-G5-7) for ammonium. Based on the amoA gene, the newly enriched archaeal ammonia oxidizer in AOA-AC1 was closely related to Nitrosotenuis spp. and the bacterial ammonia oxidizer in AOB-G5-7, Nitrosomonas sp. Is79, belonged to the Nitrosomonas oligotropha group ( Nitrosomonas cluster 6a). Growth experiments in batch cultures showed that AOB-G5-7 had higher growth rates than AOA-AC1 at higher ammonium concentrations. During chemostat competition experiments under ammonium-limiting conditions, AOA-AC1 dominated the cultures, while AOB-G5-7 decreased in abundance. In batch cultures, the outcome of the competition between AOA and AOB was determined by the initial ammonium concentrations. AOA-AC1 was the dominant ammonia oxidizer at an initial ammonium concentration of 50 μM and AOB-G5-7 at 500 μM. These findings indicate that, during direct competition, AOA-AC1 was able to use ammonium that was unavailable to AOB-G5-7, while AOB-G5-7 dominated at higher ammonium concentrations. The results are in strong accordance with environmental survey data suggesting that AOA are mainly responsible for ammonia oxidation under more oligotrophic conditions, whereas AOB dominate under eutrophic conditions. Importance Nitrification is an important process in the global nitrogen cycle. The first step - ammonia oxidation to nitrite – can be carried out by Ammonia-oxidizing Archaea (AOA) and Ammonia-oxidizing Bacteria (AOB). In many natural environments, these ammonia oxidizers coexist. Therefore, it is important to understand the population dynamics in response to increasing ammonium concentrations. Here, we study the competition between AOA and AOB enriched from freshwater systems. The results demonstrate that AOA are more abundant in systems with low ammonium availabilities and AOB when the ammonium availability increases. These results will help to predict potential shifts in community composition of ammonia oxidizers in the environment due to changes in ammonium availability.

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DNA- and RNA-SIP Reveal Nitrospira spp. as Key Drivers of Nitrification in Groundwater-Fed Biofilters

mBio ◽

10.1128/mbio.01870-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 6

Author(s):

Arda Gülay ◽

S. Jane Fowler ◽

Karolina Tatari ◽

Bo Thamdrup ◽

Hans-Jørgen Albrechtsen ◽

...

Keyword(s):

16S Rrna ◽

Ammonia Oxidation ◽

Sequence Similarity ◽

Sodium Chlorate ◽

Amplicon Sequencing ◽

Stable Isotope Probing ◽

Ammonia Oxidizer ◽

16S Rrna Sequence ◽

Dna And Rna

ABSTRACT Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira. Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4+ or NO2− in the presence of 13C-HCO3− (labeled) or 12C-HCO3− (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3− uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome. IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3− uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

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Production of oceanic nitrous oxide by ammonia-oxidizing archaea

Biogeosciences ◽

10.5194/bg-9-2419-2012 ◽

2012 ◽

Vol 9 (7) ◽

pp. 2419-2429 ◽

Cited By ~ 143

Author(s):

C. R. Löscher ◽

A. Kock ◽

M. Könneke ◽

J. LaRoche ◽

H. W. Bange ◽

...

Keyword(s):

Nitrous Oxide ◽

Ammonia Oxidation ◽

Selective Inhibition ◽

Ammonia Oxidizer ◽

Low Oxygen ◽

Tropical Ocean ◽

N2o Production ◽

Ammonia Oxidizing Archaea ◽

Tropical North Atlantic ◽

Oxygen Minimum

Abstract. The recent finding that microbial ammonia oxidation in the ocean is performed by archaea to a greater extent than by bacteria has drastically changed the view on oceanic nitrification. The numerical dominance of archaeal ammonia-oxidizers (AOA) over their bacterial counterparts (AOB) in large parts of the ocean leads to the hypothesis that AOA rather than AOB could be the key organisms for the oceanic production of the strong greenhouse gas nitrous oxide (N2O) that occurs as a by-product of nitrification. Very recently, enrichment cultures of marine ammonia-oxidizing archaea have been reported to produce N2O. Here, we demonstrate that archaeal ammonia monooxygenase genes (amoA) were detectable throughout the water column of the eastern tropical North Atlantic (ETNA) and eastern tropical South Pacific (ETSP) Oceans. Particularly in the ETNA, comparable patterns of abundance and expression of archaeal amoA genes and N2O co-occurred in the oxygen minimum, whereas the abundances of bacterial amoA genes were negligible. Moreover, selective inhibition of archaea in seawater incubations from the ETNA decreased the N2O production significantly. In studies with the only cultivated marine archaeal ammonia-oxidizer Nitrosopumilus maritimus SCM1, we provide the first direct evidence for N2O production in a pure culture of AOA, excluding the involvement of other microorganisms as possibly present in enrichments. N. maritimus showed high N2O production rates under low oxygen concentrations comparable to concentrations existing in the oxycline of the ETNA, whereas the N2O production from two AOB cultures was comparably low under similar conditions. Based on our findings, we hypothesize that the production of N2O in tropical ocean areas results mainly from archaeal nitrification and will be affected by the predicted decrease in dissolved oxygen in the ocean.

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