Truncated denitrifiers dominate the denitrification 1 pathway in tundra soil metagenomes 2

11 In contrast to earlier assumptions, there is now mounting evidence for the role of 12 tundra soils as important sources of the greenhouse gas nitrous oxide (N2O). 13 However, the microorganisms involved in the cycling of N2O in these soils remain 14 largely uncharacterized. In this study, we manually binned and curated 541 15 metagenome-assembled genomes (MAGs) from tundra soils in northern Finland. We 16 then searched for MAGs encoding enzymes involved in denitrification, the main 17 biotic process driving N2O emissions. Denitrifying communities were dominated by 18 poorly characterized taxa with truncated denitrification pathways, i.e. lacking one 19 or more denitrification genes. Among these, MAGs with the metabolic potential to 20 produce N2O comprised the most diverse functional group. Re-analysis of a 21 previously published metagenomic dataset from soils in northern Sweden supported 22 these results, suggesting that truncated denitrifiers are dominant throughout the 23 tundra biome. 24

collections. In contrast with earlier estimates based on genomes from cultured 64 microorganisms 11 , our genome-resolved metagenomics survey allowed access to the genomes of 65 poorly characterized taxa and revealed that, within a defined ecosystem, the proportion of 66 genomes encoding the complete denitrification pathway can be as low as 1%. 67

68
A manually curated genomic database from tundra soil metagenomes 69 We analysed more than 9 billion Illumina (1.4 Tb) and 7 million Nanopore reads (21.5 Gb) from  Fig. S2). 83 Two Illumina co-assemblies and two individual Nanopore assemblies yielded nearly 20 million 84 contigs longer than 1,000 bp, with a total assembly size of 50.0 Gb. Using anvi'o 30 , we obtained 85 3,257 genomic bins and manually curated these to a set of 539 unique metagenome assembled 86 genomes (MAGs) (Fig. 1, Suppl. Fig. S3, Suppl. Fig. S4, Suppl. Table S2). On average, 8.6% 87 of the reads from each sample were recruited by the MAGs (minimum: 2.7%, maximum: 22.4%). 88 According to estimates based on domain-specific single-copy genes, the obtained MAGs are on 89 average 67.7% complete (50.7-100.0%) and 2.5% redundant (0.0-9.9%) (Suppl . Table S2). 90 Phylogenomic analyses based on 122 archaeal and 120 bacterial single-copy genes in the context and Verrucomicrobiota (n = 31) (Fig. 1, Suppl. Fig. S3). Of the 541 MAGs, only 78 were 95 4 assigned to a validly described genera (Suppl . Table S2). Most MAGs (n = 463) belong to 96 genera that do not comprise formally described species. These include 183 MAGs that were 97 placed outside genus-level lineages currently described in GTDB and thus likely represent novel 98 genera. given sample if ≥ 50% of its nucleotides had ≥ 1x coverage. Phylum-level taxonomic assignments 103 are shown only for the major groups found. A complete representation of all phyla can be found 104 in Suppl. Fig. S3 and additional information about the MAGs is provided in Suppl. Table S2. 105 The highest number of MAGs (n = 341) was detected in the fen soils (Suppl. Fig. S4). Although 106 barren and fen soils had similar taxonomic richness according to gene-centric estimates (Suppl.  Table S1). The number of detected MAGs in heathland and meadow 110 soils was similar (n = 223 and n = 217, respectively) (Suppl. Fig. S4). In agreement with the 111 gene-centric assessment (Suppl. Fig. S2), we observed differences in MAG composition across 112 the soil ecosystems (Suppl. Fig. S4). Only 41 MAGs (7.6%) were shared between the heathland 113 and fen soils. On the other hand, meadow communities represent an intermediate state between 114 heathland and fens, sharing 115 and 125 MAGs with these ecosystems, respectively. This is 115 likely a reflection of edaphic similarities between the ecosystems (Suppl. Fig. S1 with a metabolic potential for denitrification, the main process controlling N2O production in 123 soils 9 . The complete denitrification pathway is performed by microorganisms containing the 124 narG/napA, nirK/nirS, norB, and nosZ genes, which encode the nitrate (Nar), nitrite (Nir), nitric 125 oxide (Nor), and nitrous oxide (Nos) reductases, respectively 10 . In the Kilpisjärvi soils, the 126 metabolic potential for denitrification was exclusively restricted to MAGs with truncated 127 denitrification pathways, i.e. MAGs missing one or more denitrifying reductases (Fig. 2). Of the as Gamma-and Alphaproteobacteria, Acidobacteriota, Bacteroidota, Actinobacteriota, and 134 Chloroflexota (Fig. 2). Only 19 MAGs were assigned to a validly described genera (Suppl. 135 Table S2). 136 Truncated pathways do not appear to be a methodological artifact arising from the metabolic  Fig. S6). 144 The distribution of denitrifying reductases was remarkably similar in MAGs from both systems, 145 6 and MAGs with truncated denitrification pathways were also the norm in Stordalen Mire soils.

146
Of the 396 Stordalen Mire MAGs encoding denitrifying reductases, only six harbour all the Nir, 147 Nor, and Nos enzymes required for complete denitrification. Altogether, the analysis of these 148 two comprehensive datasets suggest that microorganisms with truncated denitrification 149 pathways are widespread throughout tundra ecosystems. Alpha-and Gammaproteobacteria, Chloroflexota, and Actinobacteriota (Fig. 2). Denitrifying 169 stricto sensu communities differed between the meadow and fen soils (Suppl. Figure S7a). 170 Alphaproteobacteria comprised half of the Nir-encoding MAGs in the meadow soils, while 171 Chloroflexota and Gammaproteobacteria were the majority in the fens. Interestingly, the amino 172 acid sequence composition of the Nir enzymes from Chloroflexota MAGs were quite divergent 173 (Suppl. Figure S7b). These had lower identity with reference sequences from RefSeq 174 (31.6-66.3%) than the sequences of MAGs from better characterized phyla such as the Alpha- 175 and Gammaproteobacteria (67.5-88.8% and 76.3-91.7%, respectively). Indeed, most Nir-176 encoding Chloroflexota MAGs were assigned to the class-level lineage Ellin6529 (Suppl. Table   177 S2), a major group in soils worldwide that at present does not include cultured 178 representatives 38 .

179
The potential for N2O production is widespread among the Acidobacteriota 180 The stepwise reduction of NO to N2O and N2 catalysed by the Nor and Nos enzymes represents  MAGs with the potential to reduce NO to N2O were particularly prominent among the phylum 193 Acidobacteriota (n = 18 MAGs) (Fig. 2), an ubiquitous group in tundra and other soil ecosystems The 539 MAGs obtained in the present study by a manual binning and curation effort represent 216 one of the largest genomic catalogues of microorganisms from tundra soils to date (Fig. 1,   217 Suppl. Fig. S3, Suppl. Fig. S4, Suppl. Table S2). Our dataset is comparable to a previous  However, gene-centric approaches fail to reveal the wider genomic context in which these genes 248 are inserted. By applying the genome-resolved metagenomics approach, we traced 249 denitrification genes to specific microbial populations, thereby allowing a detailed investigation 250 of the genomic makeup of potential denitrifiers in tundra soils. In addition to their 251 predominance in genomic databases 11 , our genome-resolved metagenomics survey revealed that 252 truncated denitrifiers are also dominant within a defined ecosystem.

253
Microorganisms harbouring the Nor enzyme -and thus with the potential to reduce NO to N2O 254 -were the most diverse functional group and were particularly prominent among the phylum 255 Acidobacteriota (Fig. 3). Members of this phylum, which comprise mostly oligotrophic (k- The quality of the raw Illumina data was verified with fastQC 55 v0.11.9 and multiQC 56 v1.8.