Draft Genome Sequence of Schaalia odontolytica Strain ORNL0103, a Basibiont of “Candidatus Saccharibacteria” HMT352

There are 36 known genera of actinobacteria in the human microbiota, some of which can cause infections (actinomycoses) at different body sites (1). Actinomyces is one the most diverse genera, although some of its species have recently been reclassified under a new genus, Schaalia (2, 3). In the oral cavity, various actinobacteria have been shown to serve as basibionts (“hosts”) for ectoparasitic saccharibacteria (previously known as candidate division TM7) (4–7). Using labeling of a human saliva sample with a fluorescent antibody against saccharibacteria and single-particle cell sorting on nutrient agar plates, we isolated an actinobacterium (strain ORNL0103) with >99% 16S rRNA identity to Schaalia sp. strain HMT180 and Schaalia odontolytica HMT701 (7). ORNL0103 was identified as the basibiont of coisolated “Candidatus Saccharibacteria” HMT352 strain ORNL0105 (7) and can also serve as host for other saccharibacteria (HMT952). Streaking of the ORNL0103-ORNL0105 coculture on plates led to identification of colonies of pure ORNL0103 that had lost the saccharibacterium ectobiont.

S. odontolytica ORNL0103 was grown anaerobically (90% N₂ and 10% CO₂) in 10-ml aliquots of brain heart infusion (BHI) medium (Difco) for 2 days at 37°C. Genomic DNA (gDNA) was isolated using standard proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation, as described previously (8). DNA sizing using a Femto Pulse system (Agilent) revealed that >80% of the fragments were between 14 and 20 kbp. For Pacific Biosciences (PacBio) sequencing, a library was prepared directly from unsheared gDNA using the SMRTbell template preparation kit v1.0 (PacBio, Menlo Park, CA) and was sequenced on a PacBio Sequel instrument using a Sequel II 8M single-molecule real-time (SMRT) cell. All data analyses were performed using software defaults unless stated otherwise. The 1.6 million raw reads (N₅₀, 10.9 kb; maximum, 206 kb) were filtered based on quality values and assembled using HGAP4 in PacBio SMRT Link v7. The polished assembly had 10 contigs and included 61,246 polymerase reads (mean length, 49.7 kb), with 1,045-fold mean coverage. A separate library was prepared using the RhinoSeq high-throughput library preparation kit (SeqOnce Biosciences, Inc.), followed by sequencing (2 × 250-nucleotide reads) on a MiSeq instrument (Illumina, Inc., San Diego, CA). Following quality trimming using Trimmomatic v0.36 in KBase (9), 4.4 million paired-end reads were coassembled with the PacBio reads using hybridSPAdes v3.13.0 (10), resulting in 20 contigs (total length, 2,348,695 bp). The PacBio and hybrid assemblies were annotated using Prokka v1.14.5 (11) and then were coassembled and manually curated in Geneious Prime v2021.1 (12), resulting in 4 contigs with a total length of 2,351,375 bp and a G+C content of 65.6%. Based on the presence of conserved unique genes, as analyzed with CheckM v1.0.18 (13), the genome is 100% complete but not closed.

To predict and annotate the genes, we used the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.0 (14). The genome encodes 1,948 proteins, 48 tRNAs, 3 rRNA operons (with 23S, 16S, and 5.8S genes), and 3 other small/regulatory RNAs and was compared with other Schaalia genomes using FastANI v0.1.2 and SpeciesTreeBuilder v2.2.0 in KBase (9). The genome average nucleotide identity (ANI) between strain ORNL103 and its closest relatives, i.e., S. odontolytica XH001 (6, 15) and S. odontolytica ATCC 17892 (HMT701), is 94.7% and 93.7%, respectively. The ANI of ORNL0103 with Schaalia sp. HMT180 strain F0310 is only 84%, and their phylogenetic relationship is shown in Fig. 1. The genome will facilitate study of the diversity of oral actinobacteria and their association with saccharibacteria.

We thank the Genomic Resource Center at the University of Maryland School of Medicine for genomic library preparation and sequencing.

This research was funded by grants R01DE024463 (M.P.) and R01DE016937 (F.E.D.) from the National Institute of Dental and Craniofacial Research (NIDCR) of the U.S. National Institutes of Health. Oak Ridge National Laboratory is managed by UT-Battelle, LLC., for the U.S. Department of Energy under contract DE-AC05-00OR22725.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.