Enrichment and characterization of ammonia-oxidizing archaea from the open ocean : phylogeny, physiology and stable isotope fractionation

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 5 (2011): 1796–1808, doi:10.1038/ismej.2011.58.Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the north Pacific Ocean that have been maintained in laboratory culture for over three years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S rRNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98-99% identity) to those recovered in DNA and cDNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonium (NH4 +) to nitrite (NO2 -). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (15εNH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal 15εNH3 ranged from 13- 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by 15εNH3 is similar.This work was supported by a Woods Hole Oceanographic Institution (WHOI) Postdoctoral Scholar fellowship to AES and the WHOI Ocean Life Institute

Nitrogen isotope fractionation in 12 species of marine phytoplankton during growth on nitrate

The nitrogen isotopic composition of 12 species of marine phytoplankton were determined by isotope ratio mass spectrometry in order to investigate isotope fractionation associated with growth on nitrate. The species, representing diatoms, coccolithophores, dinoflagellates, green algae, and cyanobacteria, were grown in batch cultures in artificial seawater under the same laboratory conditions of constant light and temperature. The species (with isotope fractionation values in parenthesis) were: Thalassiosira weissflogii (6.2 +/- 0.4parts per thousand); Chaetoceros simplex (2.7 +/- 0.3parts per thousand); Ditylum brightwellii (3.3 +/- 0.4parts per thousand); Skeletonema costatum (2.7 +/- 0.3parts per thousand); Phaeodactylum tricornutum (4.8 +/- 0.3parts per thousand); Emiliania huxleyi (4.5 +/- 0.2parts per thousand), Isochrysis galbana (3.2 +/- 0.4parts per thousand); Pavlova lutheri, (3.6 +/- 0.5parts per thousand); Amphidinium carterae (2.2 +/- 0.3parts per thousand); Prorocentrum minimum (2.5 +/- 0.3parts per thousand); Dunaliella tertiolecta (2.2 +/- 0.2parts per thousand); and Synechococcus sp. (5.4 +/- 0.6parts per thousand). There was no relationship between isotope fractionation and organism group, nor was there a direct effect of cell size or growth rate on the degree of isotope fractionation among all the groups, Overall, the results show that isotope fractionation during growth on nitrate is lower than values obtained from field samples (i.e. 4 to 9parts per thousand). These results indicate that there is no simple mechanism for describing differences in isotope fractionation between groups of phytoplankton, and that a physiological understanding of isotope fractionation during uptake and assimilation of nitrate is needed to properly understand the delta(15)N signal generated by phytoplankton in the ocean

Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria

Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed ‘diazotrophs') are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats

The Contamination of Commercial 15N2 Gas Stocks with 15N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

We report on the contamination of commercial 15-nitrogen (N-15) N-2 gas stocks with N-15-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. N-15(2) gas is used to estimate N-2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled N-15(2) into organic matter. However, the microbial assimilation of bioavailable N-15-labeled N-2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N-2 fixation rates. N-15(2) gas procured from three major suppliers was analyzed for the presence of these N-15-contaminants. Substantial concentrations of N-15-contaminants were detected in four Sigma-Aldrich N-15(2) lecture bottles from two discrete batch syntheses. Per mole of N-15(2) gas, 34 to 1900 mmoles of N-15-ammonium, 1.8 to 420 mmoles of (15)Nnitrate/nitrite, and 0.01 nmoles N L-1 d(-1), to 530 nmoles N L-1 d(-1), contingent on experimental conditions. These rates are comparable to, or greater than, N-2 fixation rates commonly detected in field assays. These results indicate that past reports of N-2 fixation should be interpreted with caution, and demonstrate that the purity of commercial N-15(2) gas must be ensured prior to use in future N-2 fixation rate determinations