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Spiroplasma sp. NBRC 100390 was initially described as a duplicate of S. atrichopogonis GNAT3597T (=ATCC BAA-520T) but later found to be different in the 16S rDNA sequences. Here, we report the complete genome sequence of this bacterium to establish its identity and to facilitate future investigation.
The bacterial strain Spiroplasma sp. NBRC 100390 deposited in the National Institute of Technology and Evaluation–Biological Resource Center (NBRC) was initially described as a duplicate of S. atrichopogonis GNAT3597T (1). However, subsequent investigations revealed that the 16S rDNA sequence of this strain (GenBank no. AB681165.1) shows only 96% nucleotide sequence identity to that of S. atrichopogonis GNAT3597T maintained in Gail Gasparich’s laboratory at Towson University (GenBank no. KR349130.1) and another duplicate deposited in the American Type Culture Collection (ATCC BAA-520T; GenBank no. KR349131.1) (2). To establish the identity of this strain and to facilitate future comparative genomics of Spiroplasma species (3, 4), we determined the complete genome sequence of Spiroplasma sp. NBRC 100390.
The procedures for sequencing, assembly, and annotation were based on our previous studies of Spiroplasma genomes (2, 5,–7). We utilized the Illumina MiSeq platform to obtain 301-bp sequencing reads from one paired-end library with approximately 300-fold coverage. Our preliminary analysis revealed that the sequences are almost identical to Spiroplasma sp. TU-14 (7). Thus, we chose a resequencing approach using the Spiroplasma sp. TU-14 genome (GenBank no. CP017658.1) as the reference. The raw reads were mapped to the reference using the Burrows–Wheeler alignment (BWA) tool version 0.7.12 (8), programmatically checked using the MPILEUP program in SAMtools package version 1.2 (9), and visually inspected using the Integrative Genomics Viewer (IGV) version 2.3.67 (10). The programs RNAmmer (11), tRNAscan-SE (12), and Prodigal (13) were used for gene prediction. Additionally, the presence of putative clustered regularly interspaced short palindromic repeats (CRISPRs) was checked using CRISPRFinder (14).
A total of 18 polymorphic sites were found between Spiroplasma sp. NBRC 100390 and the reference, including 11 1-bp deletions, four 1-bp insertions, two single-nucleotide polymorphisms (SNPs), and one 12-bp deletion. All polymorphic sites were manually inspected using Artemis (15) to check if the polymorphisms affected genic regions. We found that all 15 1-bp indels were located in homopolymeric intergenic regions, while the two SNPs both resulted in a nonsynonymous substitution in the predicted functional domain of the respective genes (locus tags S100390_v1c06730 and S100390_v1c09590). However, the BLASTp (16) searches against the NCBI NR database (17) showed that these polymorphisms did not affect the protein domain identification. Finally, the 12-bp deletion was located outside of the predicted functional domain (locus tag S100390_v1c07060). In conclusion, the gene content of Spiroplasma sp. NBRC 100390 is identical to that of Spiroplasma sp. TU-14 (7), and we annotated the gene names and product descriptions entirely based on the reference genome.
The circular chromosome of Spiroplasma sp. NBRC 100390 is 1,199,621 bp in size and has a G+C content of 28.7%; no plasmid was found. The first version of annotation includes one set of 16S-23S-5S rRNA genes, 32 tRNA genes (covering all 20 amino acids), 1,036 protein-coding genes, and four pseudogenes. No putative plectrovirus-related sequence or CRISPR element was found.
The complete genome sequence of Spiroplasma sp. NBRC 100390 has been deposited at DDBJ/EMBL/GenBank under accession number CP018022.
The bacterial strain was provided by the Biological Resource Center, National Institute of Technology and Evaluation (Chiba, Japan). The Sanger sequencing service was provided by the DNA Analysis Core Laboratory, Institute of Plant and Microbial Biology, Academia Sinica (Taipei, Taiwan). The Illumina sequencing library preparation service was provided by the DNA Microarray Core Laboratory, Institute of Plant and Microbial Biology, Academia Sinica (Taipei, Taiwan). The Illumina MiSeq sequencing service was provided by the DNA Sequencing Core Facility, Institute of Molecular Biology, Academia Sinica (Taipei, Taiwan).
The funding for this project was provided by the Institute of Plant and Microbial Biology at Academia Sinica and the Ministry of Science and Technology of Taiwan (NSC 101-2621-B-001-004-MY3 and MOST 104-2311-B-001-019) to C.-H.K. Also, W.-S.L. was supported by the Taiwan International Graduate Program (TIGP) in Molecular and Biological Agricultural Sciences (MBAS). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Citation Haryono M, Lo W-S, Gasparich GE, Kuo C-H. 2017. Complete genome sequence of Spiroplasma sp. NBRC 100390. Genome Announc 5:e00008-17. https://doi.org/10.1128/genomeA.00008-17.