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      Deep amoA amplicon sequencing reveals community partitioning within ammonia-oxidizing bacteria in the environmentally dynamic estuary of the River Elbe

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          Abstract

          The community composition of betaproteobacterial ammonia-oxidizing bacteria (ß-AOB) in the River Elbe Estuary was investigated by high throughput sequencing of ammonia monooxygenase subunit A gene ( amoA) amplicons. In the course of the seasons surface sediment samples from seven sites along the longitudinal profile of the upper Estuary of the Elbe were investigated. We observed striking shifts of the ß-AOB community composition according to space and time. Members of the Nitrosomonas oligotropha-lineage and the genus Nitrosospira were found to be the dominant ß-AOB within the river transect, investigated. However, continuous shifts of balance between members of both lineages along the longitudinal profile were determined. A noticeable feature was a substantial increase of proportion of Nitrosospira-like sequences in autumn and of sequences affiliated with the Nitrosomonas marina-lineage at downstream sites in spring and summer. Slightly raised relative abundances of sequences affiliated with the Nitrosomonas europaea/Nitrosomonas mobilis-lineage and the Nitrosomonas communis-lineage were found at sampling sites located in the port of Hamburg. Comparisons between environmental parameters and AOB-lineage (ecotype) composition revealed promising clues that processes happening in the fluvial to marine transition zone of the Elbe estuary are reflected by shifts in the relative proportion of ammonia monooxygenase sequence abundance, and hence, we propose ß-AOB as appropriate indicators for environmental dynamics and the ecological condition of the Elbe Estuary.

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          Complete nitrification by Nitrospira bacteria

          Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered as a two-step process catalyzed 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 both the pathways for ammonia and nitrite oxidation, which are concomitantly expressed 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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            Complete nitrification by a single microorganism

            Summary Nitrification is a two-step process where ammonia is considered to first be oxidized to nitrite by ammonia-oxidizing bacteria (AOB) and/or archaea (AOA), and subsequently to nitrate by nitrite-oxidizing bacteria (NOB). Described by Winogradsky already in 18901, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle2. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible and it was postulated that this process could occur under conditions selecting for species with lower growth-rates but higher growth-yields than canonical ammonia-oxidizing microorganisms3. Still, organisms catalysing this process have not yet been discovered. Here, we report the enrichment and initial characterization of two Nitrospira species that encode all enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding on the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.
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              Archaea predominate among ammonia-oxidizing prokaryotes in soils.

              Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.
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                Author and article information

                Contributors
                andreas.pommerening@uni-hamburg.de
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                13 October 2020
                13 October 2020
                2020
                : 10
                : 17165
                Affiliations
                [1 ]GRID grid.9026.d, ISNI 0000 0001 2287 2617, Department of Microbiology and Biotechnology, , University of Hamburg, ; Ohnhorststr. 18, 22609 Hamburg, Germany
                [2 ]GRID grid.23731.34, ISNI 0000 0000 9195 2461, Section Geomicrobiology, , GFZ German Research Centre for Geosciences, ; Telegrafenberg, 14473 Potsdam, Germany
                [3 ]GRID grid.418481.0, ISNI 0000 0001 0665 103X, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, AG96 Technology-Platform, ; Martinistr. 52, 20246 Hamburg, Germany
                [4 ]Hamburg Port Authority AöR, Neuer Wandrahm 4, 20457 Hamburg, Germany
                [5 ]GRID grid.13648.38, ISNI 0000 0001 2180 3484, Bioinformatics Core, , University Medical Center Hamburg-Eppendorf, ; Martinistr. 52, 20246 Hamburg, Germany
                Author information
                http://orcid.org/0000-0003-0911-3637
                http://orcid.org/0000-0002-5993-7709
                http://orcid.org/0000-0001-7617-7396
                Article
                74163
                10.1038/s41598-020-74163-0
                7555866
                d9658db5-922a-410e-bbac-ce3e7dba4ffc
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 21 May 2019
                : 24 September 2020
                Funding
                Funded by: Projekt DEAL
                Categories
                Article
                Custom metadata
                © The Author(s) 2020

                Uncategorized
                molecular ecology,water microbiology,freshwater ecology,environmental monitoring,hydrology

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