Salt tolerance-based niche differentiation of soil ammonia oxidizers

Abstract

Ammonia oxidizers are key players in the global nitrogen cycle, yet little is known about their ecological performances and adaptation strategies for growth in saline terrestrial ecosystems. This study combined ¹³C-DNA stable-isotope probing (SIP) microcosms with amplicon and shotgun sequencing to reveal the composition and genomic adaptations of active ammonia oxidizers in a saline-sodic (solonetz) soil with high salinity and pH (20.9 cmol_c exchangeable Na⁺ kg^-1 soil and pH 9.64). Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) exhibited strong nitrification activities, although AOB performed most of the ammonia oxidation observed in the solonetz soil and in the farmland soil converted from solonetz soil. Members of the Nitrosococcus, which are more often associated with aquatic habitats, were identified as the dominant ammonia oxidizers in the solonetz soil with the first direct labeling evidence, while members of the Nitrosospira were the dominant ammonia oxidizers in the farmland soil, which had much lower salinity and pH. Metagenomic analysis of "Candidatus Nitrosococcus sp. Sol14", a new species within the Nitrosococcus lineage, revealed multiple genomic adaptations predicted to facilitate osmotic and pH homeostasis in this extreme habitat, including direct Na⁺ extrusion/H⁺ import and the ability to increase intracellular osmotic pressure by accumulating compatible solutes. Comparative genomic analysis revealed that variation in salt-tolerance mechanisms was the primary driver for the niche differentiation of ammonia oxidizers in saline-sodic soils. These results demonstrate how ammonia oxidizers can adapt to saline-sodic soil with excessive Na⁺ content and provide new insights on the nitrogen cycle in extreme terrestrial ecosystems.

Fig. 1. Schematic diagram showing the sampling sites of agricultural soil that was reclaimed from typical saline-sodic soils in Northeastern China.

The area in brown yellow represents the Songnen Plain, one of the three major regions with saline-sodic soil in the world. The solid circle in red within the Songnen Plain refers to the long-term reclamation experiment field of the Da’an Sodic Land Ecological Experiment Station of Northeast, Chinese Academy of Sciences. Soil samples were collected from the Solonetz saline-sodic field (upright panel) that contains no vegetation cover for >40 years as a control, and farmland (downright panel) that has been maintained for maize-watermelon rotation system more than 40 years.

Fig. 2. Stable-isotope probing (SIP) of active ammonia oxidizers in solonetz and farmland soil.

Changes in the concentration of soil nitrite- plus nitrate-N (NO_x⁻-N) (a), N₂O emissions (b), and amoA gene abundances (c, d) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in SIP microcosms over an incubation period of 56 days. The ¹³C-DNA of AOA and AOB was revealed by quantitative analysis of archaeal and bacterial amoA gene abundances across the entire buoyant density gradient of the fractionated DNA from SIP microcosms at day 56 (e). SIP microcosms were incubated with either ¹³C (CO₂ and urea) or ¹²C (CO₂ and urea), and an equal volume of H₂O instead of urea solution was amended as a control to monitor nitrification activity due to ammonia released from soil mineralization. The designations “0 d” and “56 d” denote days 0 and 56, respectively. “56 d-H₂O” and “56 d-urea” indicate samples from microcosms that received water or urea every seven days, respectively. The designation “56d-Urea+C₂H₂” represents the sample at day 56 from SIP microcosms incubated with both urea and 100 Pa C₂H₂. Different letters above the columns indicate significant differences (p < 0.05) (a, b). Different letters above the columns in each soil indicate significant differences (p < 0.05) (c, d). The data are normalized units (e) using the ratio of the amoA gene copy number in each DNA gradient fraction to the maximum quantity of two soils, and the “¹³C-DNA” (“heavy DNA”) and “¹²C-DNA” (“light DNA”) fractions are indicated by the shaded rectangles in red and blue, respectively.

Fig. 3. Population dynamics of active ammonia oxidizers in solonetz and farmland soil.

Total DNA (day 0 and 56) and ¹³C-DNA (day 56) were sequenced for phylogenetic identification of ammonia oxidizers in soils based on amoA genes of AOB (a) and AOA (b). Numbers in black, blue, and red represent the relative abundance of each operational taxonomic unit (OTU) sequences to the total amoA gene sequences in the total DNA at day 0, total DNA at day 56, and ¹³C-DNA, respectively. “S” and “F” in parentheses refer to solonetz and farmland soil, respectively. For instance, the designation of AOB-amoA-OTU-1 (S: 47.4, 52.0, 35.3; F:0, 0, 0) indicates that OTU-1 of AOB accounts for 47.4, 52.0, and 35.3% of the total AOB amoA gene sequences in the total DNA at day 0, total DNA at day 56 and ¹³C-DNA from solonetz soils, respectively. The population size of AOB (c) and AOB (d) was determined by multiplying the relative abundance of different lineages/clusters by the total AOA or AOB abundance (Table S2). OTUs were clustered at 93% identity. The phylogeny of AOA and AOB was generated using IQtree 1.6.12 with the best fit SYM+I+G4 and TPM2u+F+G4 model selected using the BIC. Bootstraps are based on 1000 replicated trees. Methylovulum psychrotolerans Sph1 and Methylosoma difficile LC 2 were included as an outgroup within the class Gammaproteobacteria. The results of distance-based linear modeling (DISTLM) analysis of the ammonia oxidizer compositions using the physicochemical properties of soils as predictor variables (e), where the explanatory proportion of each variable is shown beside the arrow line. The significance level is *p < 0.05. MBN microbial biomass nitrogen, SBD soil bulk density, E_Na exchangeable Na⁺ content, MBC microbial carbon, HCO₃⁻ HCO₃⁻ content.

Fig. 4. Genome-wide, pairwise comparisons of the average nucleotide identity (gANI) and average amino acid identity (gAAI) values between MAGs (highlighted in bold) and known genomes of ammonia oxidizers.

a Symmetrical matrix of pairwise gANI and gAAI between AOA MAGs (Ca. Nitrososphaera sp. Far3, Ca. Nitrososphaera sp. Far49 and Ca. Nitrososphaera sp. Far68) and known AOA genomes (Table S6). The gANI is presented in the lower left triangle and values ≥70. The gAAI is presented in the upper right triangle and values ≥60 are provided. b Symmetrical matrix of pairwise gANI and gAAI between AOB MAG (Ca. Nitrosococcus sp. Sol14) and known AOB genomes (Table S6). The gANI is presented in the lower left triangle and values ≥70. The gAAI is presented in the upper right triangle and values ≥60 are provided.

Fig. 5. Metabolic reconstruction of active ammonia-oxidizing bacteria and archaea in response to agricultural reclamation of Solonetz saline-sodic soil.

Cell metabolism diagrams of AOB (a) and AOA (b) were constructed from the genome annotation of Ca. Nitrosococcus sp. Sol14, Ca. Nitrososphaera sp. FarX and Nitrososphaera bin12, 62 and the scaffold annotation of the genus Nitrosospira. Putative adaptations to high salinity and selected core metabolic pathways of ammonia oxidizers are shown. NhaA NhaA Na⁺/H⁺ antiporter, NhaD NhaD Na⁺/H⁺ antiporter, NhaP NhaP Na⁺/H⁺ antiporter, Mrp Mrp Na⁺/H⁺ antiporter, Trk Trk K⁺ uptake system, MgtE/CorA magnesium uptake mediated by facilitated diffusion, Opu glycine betaine uptake transporter, TreS trehalose synthase, EctA diaminobutyrate acetyl transferase, EctB diaminobutyrate transaminase, EctC ectoine synthase, EctD ectoine hydroxylase, GDH2 and GdhA glutamate dehydrogenase, GlnA glutamine synthetase, GltDB glutamate synthase, ProA glutamate-5-semialdehyde dehydrogenase, ProB glutamate-5-kinase, ProC pyrroline-5-carboxylate reductase, YrbG Ca^2+/Na⁺ antiporters, PRODH proline dehydrogenase, E1.2.1.88 1-pyrroline-5-carboxylate dehydrogenase, MpgS mannosyl-3-phosphoglycerate synthase, question mark (?) uncharacterized phosphatase, GB glycine betaine. See Table S7 for detailed gene presence/absence.

Fig. 6. Meta-analysis of salt resistance genes in phylogenetically distinct ammonia-oxidizing bacteria and archaea.

Phylogenetic tree of amoA genes from typical AOA and AOB species, with known genomes (see Table S6), including those from the MAGs in this study, highlighted in bold. Representative amoA sequences were phylogenetically analyzed with MEGA version 7.0 using the neighbor-joining method and the maximum composite likelihood model with 1000 replicates to generate bootstrap values. Outside the tree, the phylogenetic grouping of AOA and AOB is shown in the first internal ring with the colored strip in red and blue, respectively. The salt tolerance proteins identified in active ammonia oxidizers in this study (Fig. 5) are shown from the internal 2nd ring to the 20th ring including those encoding proteins of NhaA; NhaD; NhaP; Mrp; YrbG; Trk; MgtE; CorA; Opu; TreS; EctABCD; GltDB; GDH2; GdhA; GudB; GlnA; ProABC; PRODH-PCD; and MpgS. The presence and absence of these proteins in ammonia oxidizers are indicated in green and white, respectively. GudB, glutamate dehydrogenase (K00260); PCD, E1.2.1.88/1-pyrroline-5-carboxylate dehydrogenase.