Draft genome sequences of Arthrobacter sp. AZCC_0090 and Mycobacterium sp. AZCC_0083 isolated from oligotrophic subsurface forest soil in the Santa Catalina mountains of Southern Arizona

Deep soil horizons contain microbes crucial for subsurface biogeochemistry and the global carbon cycle. While microbial communities have been extensively studied in surface horizons, we know far less about the microbial communities residing in deeper soil horizons despite their important roles in the mineralization of nutrients necessary for plant life (1–3).

Arizona Culture Collection (AZCC) cultures Arthrobacter sp. AZCC_0090 and Mycobacterium sp. AZCC_0083 were isolated from 50 cm subsurface soil at Oracle Ridge in Tucson, AZ, USA as described elsewhere (3) (Table 1). The cultures were propagated from isolated colonies on yeast mannitol medium (YM) consisting of (L⁻¹) 1.0 g yeast extract, 10.0 g mannitol, 0.5 g K₂HPO₄, 0.2 g MgSO₄, and 0.1 g NaCl. Solid YM medium was prepared by including 15 g L⁻¹ Noble agar. The cultures were incubated at 20°C. Broth cultures were shaken at 180 RPM. Growth appeared within 4 days.

Genomic DNA (gDNA) was isolated from a broth culture originating from a single colony using the Joint Genome Institute CTAB protocol for bacterial DNA isolation (6). For AZCC_0083, 100 ng of gDNA was sheared to 511 bp using the Covaris LE220 and size selected with SPRI using TotalPure NGS beads (Omega Bio-tek) to enrich for 200–500 bp fragments. For AZCC_0090, 200 ng of gDNA was sheared to 461 bp. After size selection with double-SPRI to enrich for 200–500 bp fragments, DNA fragments were end-repaired, A-tailed, and ligated with Illumina-compatible sequencing adapters containing a unique molecular index barcode for each sample library using the KAPA-HyperPrep kit (KAPA biosystems).

The libraries were quantified using KAPA Biosystems’ next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. Genomes were generated from Illumina 2 × 151 bp libraries using the Illumina NovaSeq XP V1 reagent kits and the S4 flowcell. Illumina sequences were quality filtered using BBTools: BBDuk (version 38.75) and BBMap and assembled with SPAdes (version v3.13.0; –phred-offset 33 –cov-cutoff auto -t 16 -m 64 –careful -k 25,55,95) (7, 8). Contigs < 1 kb were discarded (BBTools reformat.sh: minlength = 1,000 ow = t). Annotations were made using the prokaryotic genome annotation pipeline (9) and are available in IMG/MER (10). Both genomes are nearly complete, with minimal contamination via CheckM2 (5) (Table 1). Strain AZCC_0083 codes for 40 predicted biosynthetic gene clusters, and AZCC_0090 codes for two putative CRISPR genes (Table 1).

Funding from this work came from startup funds provided to P.C. from the University of Arizona’s Technology and Research Initiative Fund (the Water, Environmental, and Energy Solutions initiative), seed grants from the University of Arizona Center for Environmentally Sustainable Mining, and College of Agriculture and Life Sciences. With outside funding from the National Science Foundation (awards IOS-2141605 to P.C. and DGE-2137419 to I.A.V.), and a Simons Foundation Early Career Investigator in Marine Microbial Ecology and Evolution Award to P.C. The work (proposal: 10.46936/10.25585/60001177) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231.

M.K., I.A.V., and P.C. wrote the announcement and analyzed genomes, and B.S. and R.B. performed the experiments.