Complete Genome Sequences and Methylome Analysis of Two Environmental Spirochaetes

Here, we report the finished closed genomes of two environmental bacteria, Oceanispirochaeta crateria K2 and Thiospirochaeta perfilievii P (formally known as Spirochaeta perfilievii P). In addition, we provide methylation data and the associated enzymes predicted and confirmed to be responsible for each modified motif.

O ceanispirochaeta crateria K2 and Thiospirochaeta perfilievii P were previously described (1) and deposited into the DSMZ collection under DSM 16308 T and DSM 19205 T , respectively. They are particularly interesting because of their ability to metabolize sulfur, and ongoing biochemical studies are probing this feature of their metabolism. Methylome analysis was carried out as part of our ongoing studies of bacterial methylation. Genomic DNAs (10 g) of both strains were obtained directly from the DSMZ collection.
Single-molecule real-time (SMRT) libraries were sequenced using the Pacific Biosciences (PacBio) RS II sequencing platform. Briefly, SMRTbell libraries were constructed from a genomic DNA sample sheared to ϳ10 to 20 kb using the G-TUBE protocol (Covaris, Woburn, MA, USA), end repaired, and ligated to PacBio hairpin adapters. Incompletely formed SMRTbell templates and linear DNAs were digested with a combination of exonuclease III and exonuclease VII (New England Biolabs, Ipswich, MA, USA). DNA qualification and quantification were performed using the Qubit fluorometer (Invitrogen, Eugene, OR) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Two 8-kb SMRTbell libraries for each DNA species were prepared according to the PacBio sample preparation protocol, including additional separation on a BluePippin system (Sage Science, Beverly, MA), and were sequenced with C4-P6 chemistry on two SMRT cells, one with a non-size-selected (8-kb) library and one with a size-selected (10-kb) library, with a 360-minute collection time for each library. The total size-selected and non-size-selected sequencing reads (92,726 and 139,998 reads, respectively) with mean subread lengths of 10,188 and 9,090 bp, respectively, yielded 0.94 and 1.2 Gb of data for T. perfilievii P and O. crateria K2, respectively. They were de novo assembled using HGAP_Assembly.3 version 2.3.0 with default quality and read-length parameters and polished 3 times using Quiver (2). The polished assemblies generated 2 closed circular genome elements for T. perfilievii P with 32.62% GC content for the main chromosome (3,667,739 bp) and 29.80% GC content for the plasmid pSpeP (32,844 bp). The polished assembly generated one closed circular element for O. crateria K2 with 43.24% GC content for the main chromosome (4,012,482 bp). In all cases, direct repeats on the left and right flanks of the chromosomes and plasmid allowed closure as circular elements. The assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (3, 4).
One advantage of the PacBio sequencing platform is its ability to detect the epigenetic state of sequenced DNA (4-7). Six m6A-modified DNA motifs were detected using SMRT motif and modification analysis version 2.3.0 in T. perfilievii P, and four DNA modification motifs were detected in O. crateria K2, where three of them were m6A and one motif contained m4C. Results are presented in Table 1, showing that most of the motifs were matched with the responsible methyltransferases (MTases), and have been deposited in REBASE (8).
Data availability. The complete genome sequence of Thiospirochaeta perfilievii P is available in GenBank under the accession numbers CP035807 and CP035808. The original sequence reads have been deposited in NCBI under the SRA numbers SRR8551239 and SRR8551240. The BioProject number is PRJNA521375. The complete genome sequence of Oceanispirochaeta crateria K2 is available in GenBank under the accession number CP036150. The original sequence reads have been deposited in NCBI under the SRA numbers SRR8580034 and SRR8580035. The BioProject number is PRJNA521952.

ACKNOWLEDGMENTS
This project was supported by Russian Foundation of Fundamental Investigations (RFFI) grant 18-04-00556 to M.Y.G.
A.F., T.V., and R.J.R. work for New England Biolabs, a company that sells research reagents, including restriction enzymes and DNA methyltransferases, to the scientific community.