Complete Genome Sequence of Leptospira kobayashii Strain E30, Isolated from Soil in Japan

ABSTRACT The spirochete bacterium Leptospira kobayashii is a recently designated species of the genus Leptospira. Here, we report the complete genome sequence of L. kobayashii strain E30, consisting of two circular chromosomes and two plasmids.

L eptospira kobayashii strain E30 was isolated from soil in Japan (1,2). The bacterium has a long, thin spiral cell body with hook-shaped ends, which is a typical morphology for the members of the genus Leptospira, family Leptospiraceae, and order Spirochaetales (3). The strain is a saprophyte; it grows at 13°C and with 8-azaguanine (final concentration, 225 mg/ml) at 30°C and does not infect mice (4,5). The bacterium is motile, but its smooth swimming is induced by visible light exposure (6).
L. kobayashii strain E30 was cloned from the primary culture that had been cryopreserved at 280°C at Chiba Institute of Science (Chiba, Japan) by colony formation using a plate of Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (1% agar) and then was maintained using liquid EMJH medium at 30°C (3). Genomic DNA of L. kobayashii strain E30 was extracted using the Wizard genomic DNA purification kit (Promega, Madison, WI) and Genomic-tips (Qiagen, Germany) for Illumina and Nanopore sequencing, respectively. The Illumina sequencing library was prepared with 200 ng of total DNA using a TruSeq Nano DNA sample preparation kit (Illumina, San Diego, CA) following the manufacturer's instructions. A total of 5,615,056 paired-end sequencing reads, with an average length of 298.6 bp, were obtained on the MiSeq platform using the MiSeq reagent kit v3 (Illumina). The Nanopore sequencing library was prepared with 1,000 ng of total DNA, without shearing, using a native barcoding expansion kit (EXP-NBD104), and fragments of .3 kb were selected using a ligation sequence kit (SQK LSK 109) (Oxford Nanopore Technologies, Oxford, UK) following the manufacturer's instructions. The libraries were loaded onto R9.4.1 flow cells and run on a GridION system (Oxford Nanopore Technologies). The resulting fast5 reads were base called using Guppy v4.4.0 (Oxford Nanopore Technologies), and a total of 39,278 reads, with an average length of 10,007.3 bp, were obtained.
The genome of L. kobayashii strain E30 consists of two circular chromosomes (3,985,339 and 274,191 bp in length) and two plasmids (45,413 and 5,386 bp in length). Chromosome I contains 3,609 coding sequences, 6 rRNAs (two each of 5S, 16S, and 23S rRNAs), and 36 tRNAs, while chromosome II contains 250 protein-coding genes ( Table 1). Plasmids pE30-1 and pE30-2 contain 43 and 6 protein-coding genes, respectively. The data presented here will facilitate comparative genomic analyses of the genus Leptospira and expand our understanding of the genetic diversity of Leptospira species and the molecular mechanisms of the photoresponsive motility of L. kobayashii.
Data availability. The complete genome sequence was deposited in DDBJ/EMBL/ GenBank under accession numbers AP025028, AP025029, AP025030, and AP025031. The raw sequence data were deposited in the SRA under accession numbers DRR124442 (Illumina reads) and DRR315250 (Nanopore reads).

ACKNOWLEDGMENTS
Computations were partially performed on the supercomputer at the Research Organization of Information and Systems (ROIS) National Institute of Genetics.
This work was partly supported by JSPS KAKENHI (grant 19H03118 to R.N., grant 20H05524 to S.N., and grant 19K07571 to N.K.).