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. 2011 May 17;108(20):8420-5.
doi: 10.1073/pnas.1013488108. Epub 2011 Apr 27.

Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil

Maria Tourna  1 , Michaela Stieglmeier, Anja Spang, Martin Könneke, Arno Schintlmeister, Tim Urich, Marion Engel, Michael Schloter, Michael Wagner, Andreas Richter, Christa Schleper
Affiliations

Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil

Maria Tourna et al. Proc Natl Acad Sci U S A. .

Abstract

Genes of archaea encoding homologues of ammonia monooxygenases have been found on a widespread basis and in large amounts in almost all terrestrial and marine environments, indicating that ammonia oxidizing archaea (AOA) might play a major role in nitrification on Earth. However, only one pure isolate of this group from a marine environment has so far been obtained, demonstrating archaeal ammonia oxidation coupled with autotrophic growth similar to the bacterial counterparts. Here we describe the cultivation and isolation of an AOA from soil. It grows on ammonia or urea as an energy source and is capable of using higher ammonia concentrations than the marine isolate, Nitrosopumilus maritimus. Surprisingly, although it is able to grow chemolithoautotrophically, considerable growth rates of this strain are obtained only upon addition of low amounts of pyruvate or when grown in coculture with bacteria. Our findings expand the recognized metabolic spectrum of AOA and help explain controversial results obtained in the past on the activity and carbon assimilation of these globally distributed organisms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Growth of enrichments EN76 and EN123. (A) Correlation between ammonia oxidation and growth of EN76 in inorganic medium containing 1 mM NH4+, incubated at 37 °C. Near-stoichiometric conversion of ammonium (dotted lines) to nitrite (solid lines) was observed. Growth was assessed by qPCR for archaeal amoA genes and bacterial 16S rRNA genes. Numbers on top of the graph represent the percentage of archaeal enrichment at each time point. (B–D) Growth of enrichments EN76 (C) and EN123 (D) estimated by nitrite production in inorganic medium containing 1 mM NH4+ and incubated at five different temperatures. (B) Optimal temperature of enrichment EN76. Generation time was estimated from the slope of the log-transformed nitrite production measurements during exponential growth (as in C). All plotted data represent means of measurements from triplicate incubations. Error bars represent SE (in the case of ammonium and nitrite measurements, sometimes smaller than symbol size).
Fig. 2.
Fig. 2.
Phylogenetic relationships between archaeal 16S rRNA gene sequences of strains EN76 and EN123 (N. viennensis) and all described AOA isolates or cultures, as well as relevant environmental clone sequences. Both EN76 and EN123 belong to the group 1.1b of Thaumarchaeota (formerly Crenarchaeota). The tree (1,272 nucleotide positions) was constructed by using maximum-likelihood analysis with PhyML (HKY85 with four categories). Bootstrap support was calculated 100 times. (Scale bar: 0.05 nucleotide changes per position.)
Fig. 3.
Fig. 3.
(A) Growth of the pure culture EN76 (N. viennensis) as estimated by measurements of nitrite production (solid lines) and ammonia consumption (dotted lines) when grown in duplicate either on 1 mM NH4+ (open squares) or 1 mM NH4+ supplemented with pyruvate (A+P, closed squares). Three different concentrations of added pyruvate were used (0.25, 0.5, and 1 mM) without causing significant differences in growth. Therefore, data were combined and the average from the six incubations is presented. (B) Correlation between cell density (solid bars), ammonia consumption (dotted line), and nitrite production (solid line) during growth of strain EN76 in medium with 1 mM NH4+ and supplemented with 1 mM pyruvate. Plotted data represent means of triplicate incubations. Error bars represent SEs. (C) Nitrite production of strain EN76 incubated at different initial ammonium concentrations and (D) generation times (estimated as in Fig. 1) of strain EN76 when grown on different initial pyruvate concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 3, 5, and 10 mM).
Fig. 4.
Fig. 4.
Incorporation of 13C-labeled carbon from pyruvate into cells of strain EN76 grown for 10 d with 0.5 mM 13C-labeled pyruvate, in mineral medium supplemented with 2 mM unlabeled sodium bicarbonate and 1 mM NH4+. Incorporation of 13C-labeled carbon was analyzed by NanoSIMS. (B and D) CN− secondary ion intensity distribution visualizing individual cells. (E) Box plot represents the relative isotopic composition [13C / (13C + 12C), given in at%] of the biomass as obtained from analysis of particular regions of interest (ROIs; n = 100). (A and C) Relative deviation of the 13C/12C isotope ratio (given in per mill) from the natural abundance level for each individual pixel of B and D. Image size is 40 × 40 μm; d, delta.
Fig. 5.
Fig. 5.
Microscopy pictures of strain EN76 (N. viennensis) in pure culture. (A) Phase contrast-light micrograph. (Scale bar: 5 μm.) (B) Transmission EM image of negative stained cells. (Scale bar: 1 μm.) (C and D) Scanning EM images of cells. (Scale bar: 1 μm.) The arrow in D points to a cell appendage.
See this image and copyright information in PMC

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