Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil

Abstract

Ammonia oxidation is an essential part of the global nitrogen cycling and was long thought to be driven only by bacteria. Recent findings expanded this pathway also to the archaea. However, most questions concerning the metabolism of ammonia-oxidizing archaea, such as ammonia oxidation and potential CO(2) fixation, remain open, especially for terrestrial environments. Here, we investigated the activity of ammonia-oxidizing archaea and bacteria in an agricultural soil by comparison of RNA- and DNA-stable isotope probing (SIP). RNA-SIP demonstrated a highly dynamic and diverse community involved in CO(2) fixation and carbon assimilation coupled to ammonia oxidation. DNA-SIP showed growth of the ammonia-oxidizing bacteria but not of archaea. Furthermore, the analysis of labeled RNA found transcripts of the archaeal acetyl-CoA/propionyl-CoA carboxylase (accA/pccB) to be expressed and labeled. These findings strongly suggest that ammonia-oxidizing archaeal groups in soil autotrophically fix CO(2) using the 3-hydroxypropionate-4-hydroxybutyrate cycle, one of the two pathways recently identified for CO(2) fixation in Crenarchaeota. Catalyzed reporter deposition (CARD)-FISH targeting the gene encoding subunit A of ammonia monooxygenase (amoA) mRNA and 16S rRNA of archaea also revealed ammonia-oxidizing archaea to be numerically relevant among the archaea in this soil. Our results demonstrate a diverse and dynamic contribution of ammonia-oxidizing archaea in soil to nitrification and CO(2) assimilation and that their importance to the overall archaeal community might be larger than previously thought.

Fig. 1.

Distribution of amoA transcripts from archaea (A–D) and bacteria (E–H) in RNA-SIP gradients after incubation for 8 wk (A, C, E, and G) or 12 wk(B, D, F, and H) with ¹³CO₂ and fertilization with 15 μg (A, B, E, and F) or 100 μg (C, D, G, and H) (NH₄)₂SO₄-N·g⁻¹ d.w.s. Distribution of amoA transcripts was measured by qPCR of cDNA from gradient fractions.

Fig. 2.

Distribution of amoA genes from archaea (A–D) and bacteria (E–H) in DNA-SIP gradients after incubation for 8 wk (A, C, E, and G) and 12 wk (B, D, F, and H) with ¹³CO₂ and fertilization with 15 μg (A, B, E, and F) or 100 μg (C, D, G, and H) (NH₄)₂SO₄-N·g⁻¹ d.w.s. Distribution of amoA gene abundance was measured by qPCR of DNA from gradient fractions.

Fig. 3.

Phylogenetic affiliation of putative amoA (A) and accA (B) sequences derived from SIP gradient fractions of soil after incubation and fertilization with 15 μg N·g⁻¹ d.w.s. (A) amoA transcript and gene clones from ¹³CO₂ RNA- and DNA-SIP gradient fractions of soil after 8 and 12 wk of incubation. The amoA clones are shown as clusters 1–4 (GenBank accession nos. HQ685759–HQ685837), and relative abundances of respective cluster sequences in the clone libraries of the different RNA- and DNA-SIP gradient fractions are included as bar charts with percentages. The tree is rooted with the amoA gene of Nitrosospira briensis (U76553). (B) Putative AccA/PccB transcript clones derived from ¹³CO₂ RNA-SIP gradient fractions of soil after 12 wk of incubation. The accA clones from ¹³C-labeled heavy RNA are shown as cluster RH_HF (GenBank accession nos. HM996921–HM996934). The tree is rooted with accA gene of Haloquadratum walsbyi DSM 16790 (YP_658717). Neighbor-joining analysis using 1,000 bootstrap replicates was used to infer tree topology, and the nodes with the percentage of bootstrap resampling above 90% are indicated by filled circles. (Scale bars: 10% amino acid sequence divergence.)

Fig. 4.

Detection of amoA mRNA transcripts by application of CARD-FISH with archaeal amoA antisense probe in agricultural soil after 12 wk incubation with 5% CO₂ and fertilization with 15 μg N·g⁻¹ d.w.s. Fluorescence images for amoA CARD-FISH (A and D) and respective phase-contrast images (C and F) are shown. Archaeal cells in the soil incubation also were detected by 16S rRNA CARD-FISH using the HRP-labeled probe Arch915 (B and E). (Scale bars:10 μm.)