Complete Genome Sequence of Desulfomicrobium sp. Strain ZS1 from Zodletone Spring in Oklahoma, USA

The anoxic sediments and air-exposed water of Zodletone Spring (Kiowa County, OK, global positioning system [GPS] coordinates 35.002444 N, 98.688167 W) host some of the most highly diverse microbial communities known, encompassing over 60 phyla and candidate phyla of Bacteria and Archaea (1). The interplay of anaerobic and aerobic sulfur-based metabolism in Zodletone may be reminiscent of physiological adaptations characteristic of microbial life during Archean Earth leading to the great oxygenation event (2). Desulfomicrobium sp. strain ZS1 was isolated from a Zodletone sediment sample (1) as a strict anaerobic, motile chemolithoheterotroph, reducing sulfate with lactate as electron donor. A pure culture was obtained following streaking to single colonies on DSMZ medium 63 at 25°C under 85% N₂, 10% CO₂, and 5% H₂.

For genomic DNA isolation, strain ZS1 was grown in 50 mL liquid DSMZ medium 63 for 5 days at 25°C. All subsequent protocols followed manufacturers’ instructions. DNA was isolated using the Promega Wizard HMW DNA extraction kit. A short insert library was prepared using the Illumina Nextera XT DNA library preparation kit, followed by sequencing (2 × 250-nucleotide reads) on a MiSeq instrument (Illumina, Inc., San Diego, CA), yielding 1 million paired reads. Trimmomatic v0.36 (3) was used for quality-based trimming. Long-read sequencing was performed using the Oxford Nanopore ligation sequencing kit followed by sequencing on a MinION R9.4.1 device (Oxford Nanopore Technologies [ONT], Inc., Cambridge MA), yielding 1.9 Gb with an N₅₀ of 8.2 kbp. All subsequent data analyses were performed using software defaults. Base calling and long-read polishing were performed using ONT’s Guppy and Medaka v1.5, respectively. First-pass assemblies of the long reads were generated using the Trycycler v0.5.0 pipeline (4) with Miniasm/Minpolish v0.3-r179 (5), Flye v2.9 (6), Raven v1.5.3 (7), and wtdbg2 v2.5 (8) assemblers, followed by polishing of the consensus single contig with the Illumina short reads using Polypolish v0.4.3 (9) and POLCA v4.0.5 (10) in tandem. Circularity was confirmed as part of Trycycler pipeline assembly and further verified by back mapping of the Illumina reads. The genome is 3,867,579 bp long, with an average coverage of 479× and a G+C% of 58.9.

To predict and annotate the genes, we used the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.1 (11). The genome encodes 3,364 proteins and 81 RNAs, which include three rRNA operons. The gene encoding the chromosomal replication initiator protein DnaA was set as the first gene. Comparative genomic analyses were performed using software implemented in KBase (12) as follows. A phylogenetic tree constructed using SpeciesTree v2.2.0, using a set of 49 core, universal bacterial genes, placed Desulfomicrobium sp. ZS1 closest to Desulfomicrobium baculatum strain X^T, the genus type species (NCBI accession number PRJNA29527) (13, 14) (Fig. 1). Whole-genome average nucleotide identity (ANI) between the two genomes, calculated using FastANI v0.1.3 (15), is 94%, suggesting that ZS1 is a species closely related to D. baculatum. Desulfomicrobium sp. ZS1 will facilitate studies on the evolution of microbial sulfur metabolism and adaptation to anoxic and microoxic environments.

This research was funded by the National Science Foundation grants 2016371 (to M.P.) and 2016423 (to N.H.Y. and M.S.E.). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.

For a companion article on this topic, see https://doi.org/10.1128/mBio.00016-22.