Background. Biogeochemical Role. Archaeal Nitrification in the Ocean by Cornelia Wutcher et al Presenters: Brian Drupieski and Megan McCurdy

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1 Archaeal Nitrification in the Ocean by Cornelia Wutcher et al Presenters: Brian Drupieski and Megan McCurdy Background Crenarchaeota From the kingdom Archaea Most abundant oceanic prokaryote Limited knowledge of: Physiology Biogeochemical role Biogeochemical Role Possible significant role in ocean nitrification 1

2 Biogeochemical Role Previously β and γ protebacteria were seen as sole operators in marine nitrification. β proteobacteria γ proteobacteria Biogeochemical Role cont d New studies have shown autotrophic and heterotrophic characteristics. The recent isolation of Nitrosopumilus maritimus presented autotrophic abilities Oxidized ammonium to nitrite The encoding gene ammonia monooxygenase (amoa) found in a fosmid sequence of Cenarchaeum symbiosum may provide the link to Crenarchaeota and the oceanic nitrogen cycle Objectives Presenting evidence that Crenarchaeota in seawater are capable of nitrification (Wuchter et al. 2006) Show through analysis of archaeal and bacterial amoa in coastal and open oceans that Crenarchaeota plays an important role in the marine nitrogen cycle 2

3 Sampling Site: Texel Island, Netherlands Source: Results and Discussion Enrichment Culture of a Nitrifying Crenarchaeote Enriched Crenarchaeota from the North Sea depleted nearly all ammonia in 850 liter tank over 6 months Did Crenarchaeota oxidize the ammonia? Replicate conditions, but monitor Crenarchaeota with CARD-FISH (catalyzed reporter depositionfluorescence in situ hybridization) Fig. 4. Epifluorescence microscopy of CARD- FISH signal of the Cren537 probe from the crenarchaeotal enrichment culture revealing the dominance of the marine Crenarchaeote. 3

4 Ammonium Nitrite Crenarchaea Proteobacteria DAPI-stainable % Crenarchaeota - <5% bacteria - <1% β- and γ- proteobacteria ~4 fmol of NH 3 cell -1 day -1 (estimated) (Candidatus N. maritimus ) Isolation of an autotrophic ammonia-oxidizing marine archaeon Martin Könneke et. al Nature 437, (22 September 2005) amoa Characterization Only one amoa identified 91% nucleotide, 98% amino acid identity to Candidatus N. maritimus amoa 90% nucleotide, 95% amino acid identity to Sargasso Sea sequences amoa vs. 16S rrna abundance at day 7 = 0.9/1 Suggests enriched crenarchaeota has 1 copy of amoa 4

5 Fig. 2. Phylogenetic analyses of archaeal amoa recovered from the enrichment culture (red) the North Sea (blue) the Atlantic Ocean (green). Fig. 5. Phylogenetic analyses of crenarchaeotal 16S rrna genes recovered from the enrichment culture (B) Neighbor-joining tree (1,324 bp) of Archaea showing the affiliation of crenarchaeotal 16S rrna gene sequences and the almost-complete 16S rrna sequence of the enriched Crenarchaeote (red). Objective 1 Complete To present evidence that crenarchaeota in seawater are capable of nitrification Changes in nutrient concentrations Single amoa Single phylotype of marine crenarchaeotal 16S rrna gene 5

6 Importance of Archaeal Nitrification in an Ocean Margin System Resampled the North Sea over 1 year DGGE revealed Crenarchaeotal domination from early fall to late spring 16S rrna sequences >96% similar to each other and to the enriched sample Fig. 5. Phylogenetic analyses of crenarchaeotal 16S rrna genes recovered from the enrichment culture and the North Sea time series. (A) Neighbor-joining tree (400 bp) of Group I.1a Crenarchaeota showing the affiliation of crenarchaeotal 16S rrna recovered from the enrichment culture (red) and North Sea waters (blue). Ammonia 12.7 to 8.5 µm Nitrate 0.8 to 2.5 µm (Nov. to Dec.) Crenarchaeota, 2 orders of magnitude β- and γ-proteobacteria Archaeal amoa 1-2 orders higher than bacteria amoa during consumption Fig. 3. Crenarchaeotal abundance in the North Sea between August 2002 and July 2003 as a response to changing nutrient concentrations. Coastal amoa Characterization Only one dominant archaeal amoa 92% nucleotide, 97% amino acid identity to Candidatus N. maritimus amoa 2-3 copies of amoa per cell for North Sea vs. 1 copy for enriched crenarchaeota Rate of nitrification also ~2x higher 6

7 Fig. 2. Phylogenetic analyses of archaeal amoa recovered from the enrichment culture (red) the North Sea (blue) the Atlantic Ocean (green). Archaeal Nitrification in the Open Ocean Crenarchaeota found to perform chemolithoautotrophic nitrification Chemolithoautotrophic means that these organisms obtain the necessary carbon for metabolic processes from carbon dioxide in their environment. They also use inorganic compounds such as nitrogen, iron, or sulfur for the energy to power these processes. (Rice. 2005) Archaeal Nitrification in the Open Ocean Highest absolute cell numbers in the photic zone, but as depths increase the cell numbers change minimally Dominate prokaryote community below photic zone Nitrogen level are 20-40µM in the meso-and bathypelagic zones of the Pacific and Atlantic Oceans. 7

8 Archaeal Nitrification in the Open Ocean Marine snow descending from upper ocean zones allows for particulate organic nitrogen to be decomposed, and regenerate ammonium. Analysis of the amoa Gene in the Open Ocean Examination revealed: β proteobacteria had highest abundance of the gene Archaeal amoa was 1-3 magnitudes less γ-proteobacteria was below the detection limit Conclusion Marine crenarchaeota in the mesopelagic layer of the open ocean are involved in ocean nitrification more than bacterial nitrifiers, and indeed may be responsible for the majority of ocean nitrification during the winter. 8

9 What s next? Determine causes for changes in crenarchaeota abundance throughout the year (e.g. in December of time series) Explore differences in amoa per cell nitrification rates present between enriched sample and North Sea sample Could differences in expression affect global ocean inorganic nitrogen content? 9