Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats

Abstract

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.

Figure 1. Near-stoichiometric oxidation of ammonia and production of nitrite by culture N4 over 15 days.

Two replicate culture flasks (indicated by red and green symbols) were inoculated with 10 vol% of the parent culture, leading to an initial nitrite concentration of ~0.5 mM. Nitrate was not detectable during the whole experiment. Axis scaling is identical for the ammonia (triangles) and nitrite (squares) concentrations.

Figure 2. Phylogenetic trees inferred from the 16S rRNA (A), AmoA (B), AmoB (C) and Hcd (D) sequences of “Ca. Nitrosotenuis uzonensis“ and related archaea.

All trees show only Nitrosopumilus cluster (also called group I.1a) sequences, while the respective sequences from the Nitrososphaera cluster (also called group I.1b), Nitrosotalea cluster (group I.1a-affiliated) and Nitrosocaldus cluster (ThAOA group) are contained within the outgroups. Circles on tree nodes indicate parsimony bootstrap support ≥90%. Numbers in parentheses indicate the number of sequences within a group. Dotted lines were used for short sequences that were added after construction of the overall tree. Scale bars show 10% estimated sequence divergence. SAGM, South African Gold Mine. For an extended AmoA tree please refer to Figure S1.

Figure 3. Findings supporting flagellation of “Ca. Nitrosotenuis uzonensis”

(A) Intact cell stained with uranyl acetate, showing a subpolar tail-like cell projection, possibly a flagellum, and a network of hexagonal structures on the cell surface. (B) Organization of flagellar- and chemotaxis-associated genes in its genome. Genes encoding parts of the archaeal flagellum (fla genes) and chemotaxis apparatus (che genes) are given in red and green, respectively, whereas other genes are shown in grey. For details on these genes please refer to Table S8 in File S1. Black double bars indicate separate genomic regions, while blue bars display contig ends. MCP, putative methyl-accepting chemotaxis protein.