Abstract
Spiroplasma litorale TN-1T (DSM 21781) was isolated from the gut of a green-eyed horsefly (Tabanus nigrovittatus), collected at Ocracoke Island in North Carolina in 1983. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology.
GENOME ANNOUNCEMENT
Spiroplasma litorale is a bacterium known to be associated with tabanid flies belonging to the genus Tabanus (1). Strains assigned to this species have been isolated from Tabanus species T. americanus, T. atratus, T. gladiator, T. lineola, T. nigrovittatus, T. sulcifrons, and T. zythicolor. The restriction of S. litorale to the coastal areas of the southeastern United States makes it one of the best-known examples of a geographically limited insect symbiont within the genus Spiroplasma (1). To facilitate the future investigation on the biology of this bacterium and to improve the taxon sampling of available Spiroplasma sequences for comparative genomics and evolutionary studies, we determined the complete genome sequence of S. litorale TN-1T.
The strain was acquired from the German Collection of Microorganisms and Cell Cultures (catalogue no. DSM 21781). The freeze-dried sample was processed according to the manufacturer’s instructions and cultured in the M1D medium (2) prior to DNA extraction using the Wizard genomic DNA purification kit (Promega, USA). PCR and Sanger sequencing were performed to verify that the 16S rRNA gene sequence matched the reference record (GenBank accession no. NR_025708.1).
The procedures for sequencing, assembly, and annotation were based on those described in our previous studies on Spiroplasma genomes (3–8). Briefly, the Illumina MiSeq platform was used to generate 300-bp reads from one paired-end library (~570-bp insert, 2,155,514 reads) and one mate-pair library (~4,000-bp insert, 2,024,260 reads). The initial de novo assembly was performed using ALLPATHS-LG release 52188 (9). Subsequently, PAGIT version 1 (10) was used to assist an iterative process for improving the assembly. For each iteration, the raw reads were mapped to the assembly using Burrows-Wheeler Aligner (BWA) version 0.7.12 (11), programmatically checked using the MPILEUP program in the SAMTools package version 1.2 (12), and visually inspected using IGV version 2.3.57 (13). Polymorphic sites and gaps were corrected based on the mapped reads. The process was repeated until the complete genome sequence was obtained.
The programs RNAmmer (14), tRNAscan-SE (15), and Prodigal (16) were used for gene prediction. The gene names and product descriptions were first annotated based on the homologous genes in other Spiroplasma genomes (3–8), as identified by OrthoMCL (17). Subsequent manual curation was based on BLASTp (18) searches against the NCBI nonredundant database (19) and the KEGG database (20, 21).
The circular chromosome of S. litorale TN-1T is 1,225,519 bp in size and has a G+C content of 24.9%; no plasmid was found. The first version of annotation includes one set of 16S-23S-5S rRNA genes, 29 tRNA genes (covering all 20 amino acids), and 1,064 protein-coding genes.
Nucleotide sequence accession number.
The complete genome sequence of S. litorale TN-1T has been deposited at DDBJ/EMBL/GenBank under the accession no. CP012357.
ACKNOWLEDGMENTS
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 (grants NSC 101-2621-B-001-004-MY3 and MOST 104-2311-B-001-019) to C.-H.K. W.-S.L. was supported by the TIGP-MBAS program (Academia Sinica and National Chung Hsing University). Y.-C.L., Y.-W.L., and T.-H.W. were supported by the Summer Undergraduate Internship Program (Institute of Plant and Microbial Biology, Academia Sinica).
The bacterial strain was imported under permit no. 103-B-001 issued by the Council of Agriculture of Taiwan. The Sanger sequencing service was provided by the DNA Analysis Core Laboratory (Institute of Plant and Microbial Biology, Academia Sinica). The Illumina sequencing service was provided by the DNA Microarray Core Laboratory (Institute of Plant and Microbial Biology, Academia Sinica) and the DNA Sequencing Core Facility (Institute of Molecular Biology, Academia Sinica).
Footnotes
Citation Lo W-S, Lai Y-C, Lien Y-W, Wang T-H, Kuo C-H. 2015. Complete genome sequence of Spiroplasma litorale TN-1T (DSM 21781), a bacterium isolated from a green-eyed horsefly (Tabanus nigrovittatus). Genome Announc 3(5):e01116-15. doi:10.1128/genomeA.01116-15.
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