Complete Genome Sequence of Athalassotoga saccharophila Strain NAS-01, a Deep-Branching Thermophilic Lineage in the Phylum Thermotogae.

Athalassotoga saccharophila strain NAS-01 (=JCM 19762T) is an anaerobic thermoacidophilic bacterium which is one of the deepest branching lineages of the phylum Thermotogae Here, we report the complete genome sequence (1.96 Mbp) and two plasmid sequences (43.7 kbp and 43.5 kbp) of this strain.

ANNOUNCEMENT

Diverse thermophiles thrive in terrestrial hot springs. The thermoacidophilic bacterium Athalassotoga saccharophila strain NAS-01 was isolated from a terrestrial hot spring in Japan, and it belongs to the phylum Thermotogae (1). The order Mesoaciditogales, including A. saccharophila and Mesoaciditoga lauensis (2), is the deepest branching clade in the Thermotogae. Previous genome analyses indicated that the chimeric feature of genomes of the order Thermotogales are due to horizontal gene transfer from members of the phylum Firmicutes and even the domain Archaea (3, 4). Although the draft genome sequence of M. lauensis has been publicly released (Integrated Microbial Genomes [IMG] identification no. 2579779163), none of the complete genome sequences of members of the Mesoaciditogales have been reported so far.

Here, we report the complete genome sequence of A. saccharophila strain NAS-01. This strain was cultivated in an Fe(III)-containing medium (JCM medium no. 1014) under anaerobic conditions at 55°C and pH 6.0 as previously described (1). Total DNA was extracted from cells using a DNeasy UltraClean microbial kit (Qiagen, Germany) and purified as previously described (1). The purified DNA was applied to shotgun DNA sequencing using a MiSeq instrument (Illumina, USA) with a QIAseq FX DNA library kit (Qiagen), a MiSeq reagent kit version 3 (600 cycles, 300-bp paired-end reads; Illumina), and a MinION device (Oxford Nanopore Technologies [ONT], United Kingdom) with a flow cell (FLO-MIN107 R9 version; ONT) and an R9.5 1D2 sequencing kit (ONT). The MiSeq and MinION sequencing runs resulted in 4,325,178 reads with an average read length of 220 bases and 3,859,209 reads with an average read length of 1,051 bases, respectively. The MiSeq reads were quality trimmed and filtered using the read quality control pipeline of MetaWRAP version 1.0.5 (5) with the default settings. The low-quality MinION reads (<8 read quality score and <5,000 bases long) were filtered using NanoFilt version 2.2.0 (6). The trimming and filtering of the reads from the MiSeq and MinION runs resulted in 4,310,774 reads with an average read length of 217 bases and 14,710 reads with an average read length of 8,616 bases, respectively. The high-quality short and long sequences were coassembled using Unicycler version 0.4.7 (7) with the conservative mode, resulting in three circular contigs (1,957,762, 43,703, and 43,534 bp with G+C contents of 40.64%, 35.97%, and 35.16%, respectively). The gene prediction and annotation were performed using the Rapid Annotations using Subsystems Technology (RAST) server version 2.0 (8), Prokka version 1.13 (9), and the DDBJ Fast Annotation and Submission Tool (DFAST) (10) with the default settings. The annotation was manually curated based on the results from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (11), InterProScan version 5.33-72.0 (12), and eggNOG version 4.5.1 (13).

The longest circular contig corresponding to the chromosomal DNA contained 1,914 protein-coding regions (CDSs), 2 copies of rRNA gene operons (5S, 16S, and 23S), and 48 coding regions of tRNAs. CDSs for the complete set of the Embden-Meyerhof-Parnas pathway and those for the pentose phosphate pathway were found. In contrast, few CDSs involved in the tricarboxylic acid (TCA) cycle were found. Remarkably, the genome encodes CDSs for both F-type and V-type ATPases. The other two circular contigs, defined as pATS1 (43.7 kbp) and pATS2 (43.5 kbp), correspond to plasmid DNAs. pATS1 and pATS2 contained 43 and 47 CDSs, respectively. Further genome sequencing and analysis of members of the deepest clade, Mesoaciditogales, will provide insights into the unique evolutionary history of the phylum Thermotogae.

Data availability.

The sequences determined in the present study have been deposited in DDBJ/ENA/GenBank under the accession no. AP019551 for chromosome DNA, AP019552 for pATS1, and AP019553 for pATS2. The raw sequence data have been deposited under the accession no. DRA008891 for the MiSeq run and no. DRA009879 for the MinION run.