Proteomics and comparative genomics of Nitrososphaera viennensis reveal the core genome and adaptations of archaeal ammonia oxidizers

Abstract

Ammonia-oxidizing archaea (AOA) are among the most abundant microorganisms and key players in the global nitrogen and carbon cycles. They share a common energy metabolism but represent a heterogeneous group with respect to their environmental distribution and adaptions, growth requirements, and genome contents. We report here the genome and proteome of Nitrososphaera viennensis EN76, the type species of the archaeal class Nitrososphaeria of the phylum Thaumarchaeota encompassing all known AOA. N. viennensis is a soil organism with a 2.52-Mb genome and 3,123 predicted protein-coding genes. Proteomic analysis revealed that nearly 50% of the predicted genes were translated under standard laboratory growth conditions. Comparison with genomes of closely related species of the predominantly terrestrial Nitrososphaerales as well as the more streamlined marine Nitrosopumilales [Candidatus (Ca.) order] and the acidophile "Ca. Nitrosotalea devanaterra" revealed a core genome of AOA comprising 860 genes, which allowed for the reconstruction of central metabolic pathways common to all known AOA and expressed in the N. viennensis and "Ca Nitrosopelagicus brevis" proteomes. Concomitantly, we were able to identify candidate proteins for as yet unidentified crucial steps in central metabolisms. In addition to unraveling aspects of core AOA metabolism, we identified specific metabolic innovations associated with the Nitrososphaerales mediating growth and survival in the soil milieu, including the capacity for biofilm formation, cell surface modifications and cell adhesion, and carbohydrate conversions as well as detoxification of aromatic compounds and drugs.

Keywords: ammonia oxidation; archaea; biofilm; comparative genomics; proteomics.

Fig. 1.

Simplified Venn diagram illustrating the numbers of COGs shared between marine Ca. Nitrosopumilales (COGs shared between N. maritimus and N. brevis and between either of them and the other categories), soil Ca. Nitsosopumilales (COGs shared between N. koreensis and the other categories), Ca. Nitrosotaleales (COGs shared between N. devanaterra and the other categories), and Nitrososphaerales [COGs shared between N. viennensis, N. gargensis, and N. evergladensis (either among all three or pairwise) and between either of them and the other categories]. Proteins that were not grouped into COGs are represented as specific proteins for each organism. Not all combinations of shared COGs are represented in this Venn diagram. Numbers in parentheses indicate the numbers of clusters detected in the experimentally determined proteomes of N. viennensis and N. brevis, respectively. Genome abbreviations are Nbrev, N. brevis; Ndev, N. devanaterra; Never, N. evergladensis; Ngar, N. gargensis; Nkor, N. koreensis; Nmar, N. maritimus; and Nvie, N. viennensis.

Fig. S1.

Distribution of the arCOG functional categories assigned to the defined core COGs.

Fig. 2.

Reconstruction of the predicted central carbon metabolism modules in AOA emphasizing the conservation of the core pathway enzymes and carriers and features of the Nitrososphaerales. Canonical enzymes belonging to the core COGs are depicted in light gray boxes, whereas candidate enzymes according to the work by Spang et al. (28) are in dark gray boxes. COGs conserved among Nitrososphaerales are depicted in orange boxes, and COGs present in some of the analyzed genomes are in blue (refer to the color key in Fig. 3). Proteins catalyzing all displayed reactions were detected in the proteome of N. viennensis by proteotypic peptides. Gene accession numbers and enzyme abbreviations are listed in Dataset S2.

Fig. 3.

Reconstruction of putative ammonia oxidation and primary nitrogen assimilation in AOA emphasizing the conservation of the core pathway enzymes and carriers and features of the Nitrososphaerales. The ammonia oxidation module was adapted from ref. . Dark gray indicates candidate enzyme [in this case, the unidentified HURM (21)]. Dashed lines indicate binding/regulation. Dotted lines indicate absent reaction, which is included here for clarity. All depicted proteins except the urea transporters were detected in the proteome of N. viennensis. Gene accession numbers are in Dataset S2. The presence and function of urea transporters (UT and SSS) and urease (Ure) as well as the two Amt ammonium transporters have been presented earlier (refs. and , respectively) and are not discussed here.

Fig. 4.

Maximum likelihood tree (after automatic model selection with IQ-Tree, version 1.4.2) of multicopper proteins found in all available AOA genomes (25 in total) and Aigarchaeota genomes and selected genomes from AOB and nitrite-oxidizing bacteria. NirK is as identified in ref. . The two uncollapsed clades contain exclusively genes from AOA and are represented in almost all 25 species (MCO1 is not in N. devanaterra, and NirK is not in C. symbiosum and N. yellowstonii). More details and strain abbreviations are in the text and SI Materials and Methods. Values at nodes represent ultrafast bootstraps («UF-boot »). BC, additional blue copper domain.

Fig. S2.

Maximum likelihood tree (after automatic model selection with IQ-Tree, version 1.4.2) of multicopper proteins found in all available AOA genomes (25 in total) and Aigarchaeota genomes and selected genomes from AOB and nitrite-oxidizing bacteria. NirK was as identified in the work by Bartossek et al. (49). This tree is an uncollapsed version of Fig. 4. Values at nodes represent ultrafast bootstraps («UF-boot »). BC, additional blue copper domain.

Fig. S3.

Maximum likelihood tree (after automatic model selection with IQ-Tree, version 1.4.2) of the core ZIP family permease associated with MCO1. More details and strain abbreviations are in the text and SI Materials and Methods. Values at nodes represent ultrafast bootstraps («UF-boot »).

Fig. 5.

Distribution of polysaccharide biosynthesis and adhesion-related domains in the analyzed AOA genomes. Shading reflects the expansion of the respective gene family/domain: darker shades of red represent an increasing number of homologs, and white represents absence of the respective category in the genome. The number of homologs detected in the proteome of N. viennensis, out of the total number of homologs in the N. viennensis genome, are listed in the last column. An extended version of this table, including the numbers and the protein family accession numbers, can be found in Dataset S2. Nbrev, N. brevis; Ndev, N. devanaterra; Never, N. evergladensis; Ngar, N. gargensis; Nkor, N. koreensis; Nmar, N. maritimus; and Nvie, N. viennensis.

Fig. S4.

Gene clusters of N. viennensis putatively involved in EPS production/modification and/or N-glycosylation.

Fig. 6.

N. viennensis biofilm grown on a glass coverslip: (A) stained with DAPI (blue) and FITC-conjugated WGA (green), the latter binding to N-acetylglucosamine and/or N-acetylneuraminic acid residues; (B) phase-contrast image of the biofilm. (Scale bar: 5 μm.)