Draft Metagenome-Assembled Genome Sequences of Three Novel Ammonia-Oxidizing Nitrososphaera Strains Recovered from Agricultural Soils in Western Colorado

Ammonium- or urea-based fertilizers are the dominant form of applied nitrogen in agricultural systems (1). Ammonia-oxidizing bacteria and archaea (AOA) are recognized as partially controlling the fate of this nitrogen through nitrification, rapidly converting ammonium to nitrate, which is more mobile and can lead to substantial nitrous oxide (N₂O) production (1–3). The AOA are classified as Thermoproteota (formerly Thaumarchaeota) and have been primarily tracked by marker gene sequencing in soil (4). Here, we report three metagenome-assembled genomes (MAGs) for a novel Nitrososphaera species recovered from agricultural soil in western Colorado.

Soil samples were collected from the Western Colorado Research Center (Fruita, CO, USA; 39°10′47.9994″, −108°42′0″) in February 2021. Surface (0- to 5-cm) soil samples were taken from fallow agricultural plots managed under conventional tillage (n = 1) and an untilled system (n = 1). DNA was extracted from 0.4 g of each soil using the Zymo Quick-DNA fecal/soil microbe microprep kit, following the soil protocol. Metagenomic libraries were prepared using the Tecan Ovation Ultralow v2 system and were sequenced on the NovaSeq 6000 platform on a S4 flow cell at Genomics Shared Resource, Colorado Cancer Center (Denver, CO, USA). The untilled and tilled metagenomes comprised 37.9 Gbp and 28.5 Gbp of 150-bp paired-end reads, respectively. For each metagenome, the read quality was determined using FastQC v0.11.2 (5), the reads were trimmed using Sickle v1.33 (pe -t sanger) (6) and assembled using MEGAHIT v1.2.9 (–k-min 31 –k-max 121 –k-step 10 –mem-flag 1) (7), and the contigs were binned using MetaBat2 v2.12.1 (8). The MAG quality was assessed using CheckM v1.1.2 (9), and the taxonomy was assigned using GTDB-tk v1.5.0 (r202) (10). MAG annotation was performed using DRAM (11) within KBase (12). Default parameters were used unless noted. Two of the Nitrososphaera MAGs (WCRC_1 and WCRC_3) were recovered from the conventional tilled soil metagenome and the other (WCRC_2) from the untilled soil metagenome.

The three MAGs were assigned to a new species in the genus Nitrososphaera using GTDB-tk, where there are currently 14 Nitrososphaera genomic representatives across seven species (GTDB-tk r202) (Fig. 1). The pairwise amino acid identity is 97.67% between the three MAGs, suggesting that they are members of the same species (13). The statistics of these three MAGs are presented in Table 1.

Genome annotation of the Nitrososphaera MAGs supported their roles as AOA. All MAGs encoded the B and C subunits of ammonia monooxygenase (EC 1.14.99.39). While the A subunit is missing across the MAGs, this is likely due to the known challenge of assembling this gene and the incomplete nature of these MAGs (Table 1). In accordance with other observed Thaumarchaeota, hydroxylamine oxidase was absent across the MAGs, while WCRC_1 encoded nitrite reductase (14). Collectively, these MAGs provide genomic context for a novel species of ammonia-oxidizing Nitrososphaera derived from agricultural soils.

We thank Katrina Deiner at the Genomics Core at the University of Colorado Anschutz Medical Campus for sequencing expertise. We also thank the students of the Soil Microbiome Research Experience course (SOCR 481A-5) for their support during data analysis. Finally, we thank the DOE Systems Biology Knowledgebase (KBase) educational team, specifically Elisha Wood-Charlson and Ellen Dow, for assistance with data ingestion into KBase.

KBase is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under award numbers DE-AC02-05CH11231, DE-AC02-06CH11357, DE-AC05-00OR22725, and DE-AC02-98CH10886. B.B.M. and K.C.W. were fully or partially supported by an Early Career Award to K.C.W. from the National Science Foundation under award number 1912915.