A mesophilic, autotrophic, ammonia-oxidizing archaeon of thaumarchaeal group I.1a cultivated from a deep oligotrophic soil horizon

Abstract

Soil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia-oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautotrophic ammonia oxidizer that belongs to a distinct clade of nonmarine thaumarchaeal group I.1a, which is widespread in terrestrial environments. The archaeal strain MY2 was cultivated from a deep oligotrophic soil horizon. The similarity of the 16S rRNA gene sequence of strain MY2 to those of other cultivated group I.1a thaumarchaeota members, i.e., Nitrosopumilus maritimus and "Candidatus Nitrosoarchaeum koreensis," is 92.9% for both species. Extensive growth assays showed that strain MY2 is chemolithoautotrophic, mesophilic (optimum temperature, 30°C), and neutrophilic (optimum pH, 7 to 7.5). The accumulation of nitrite above 1 mM inhibited ammonia oxidation, while ammonia oxidation itself was not inhibited in the presence of up to 5mM ammonia. The genome size of strain MY2 was 1.76 Mb, similar to those of N. maritimus and "Ca. Nitrosoarchaeum koreensis," and the repertoire of genes required for ammonia oxidation and carbon fixation in thaumarchaeal group I.1a was conserved. A high level of representation of conserved orthologous genes for signal transduction and motility in the noncore genome might be implicated in niche adaptation by strain MY2. On the basis of phenotypic, phylogenetic, and genomic characteristics, we propose the name "Candidatus Nitrosotenuis chungbukensis" for the ammonia-oxidizing archaeal strain MY2.

FIG 1

Phylogenetic analysis of the archaeal amoA gene sequence (ca. 560 bp) obtained from strain MY2. The archaeal amoA genes were amplified using primers AamoAF and AamoAR. Branching patterns supported by >50% bootstrap values (1,000 iterations) according to the neighbor-joining method are denoted by their respective bootstrap values. The cluster groups are shown on the right, based on the origins of the reference sequences. ThAOA, thermophilic AOA lineage. The scale bar represents 5% estimated sequence divergence. Enriched or isolated AOA among the reference sequences are indicated in bold.

FIG 2

Morphology of strain MY2, based on FISH, TEM, and SEM analyses. (A) Merged image with a Cy3-labeled Archaea-specific probe (Arc915; red) and a FAM-labeled Bacteria-specific probe (EUB338; green). (B) Merged image with a Cy3-labeled Archaea-specific probe (Arc915) and DAPI. Magenta indicates the archaeal cells. (C) Transmission electron micrograph of strain MY2. The arrows indicate the archaeal flagella. (D) Scanning electron micrograph of strain MY2 cells. (E) Transmission electron micrograph of ultrathin section of strain MY2 cells.

FIG 3

Relationships between ammonia oxidation, nitrite production, and archaeal growth in the enrichment culture of strain MY2. The ammonia and nitrite concentrations were determined colorimetrically. Archaeal cells were counted by FISH. The cell density in the initial inoculum was ca. 8.7 × 10⁶ cells ml⁻¹. The error bars represent the standard deviations based on triplicate experiments.

FIG 4

Mass spectra of GDGTs showing distributions of MH⁺ ions in the ranges of m/z 1302 to 1312 and 1292 to 1302 for GDGT-0 (A to C) and crenarchaeol (D to F), respectively, measured by liquid chromatography-mass spectrometry (LC-MS) analysis of these restricted m/z ranges. Panels B and E show the spectra obtained for strain MY2 cultivated with exposure to ¹³C-labeled bicarbonate. For reference, panels A and D show the spectra obtained for “Ca. Nitrosoarchaeum koreensis” cultivated with nonlabeled bicarbonate (10). Panels C and F show the modeled distributions of MH⁺ ions obtained using ¹³C contents of 6.3% (GDGT-0) and 7.8% (crenarchaeol), respectively. The ¹³C enrichment in the bicarbonate was 8.0%.

FIG 5

Effects of pH (A), temperature (B), ammonia concentration (C), and nitrite concentration (D) on the ammonia oxidation activity of strain MY2. For the experiments shown in panels A, B, and C, the ammonia oxidation activity was determined by nitrite accumulation. For the experiments shown in panel D, ammonia consumption was determined to study nitrite inhibition. The cell density in the initial inoculum was ca. 3.2 × 10⁶ cells ml⁻¹. The error bars represent the standard deviations based on triplicate experiments.