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Complete genome sequence of Bacillus velezensis 2F-NA isolation from the soil of a livestock farmhouse
Korean J. Microbiol. 2023;59(3):214-216
Published online September 30, 2023
© 2023 The Microbiological Society of Korea.

GyuDae Lee1, TaeHyung Park2, Min-Ji Kim1, and Jae-Ho Shin1,2,3*

1Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
2Department of Integrative Biology, Kyungpook National University, Daegu 41566, Republic of Korea
3NGS Core Facility, Kyungpook National University, Daegu 41566, Republic of Korea
Correspondence to: *E-mail: jhshin@knu.ac.kr; Tel.: +82-53-950-5716; Fax: +82-53-953-7233
Received July 3, 2023; Revised July 11, 2023; Accepted July 11, 2023.
Abstract
Bacillus velezensis 2F-NA was isolated from the soil of a livestock farmhouse in Republic of Korea. Long read sequencing was conducted using the Nanopore MinION device, resulting in the generation of a complete genome with a size of 3,933,901 bp through de novo assembly. The genome exhibits a G + C content of 46.5% and contains 3,004 protein-coding, 30 rRNA, 91 tRNA, and 5 ncRNA genes. Moreover, the NCBI Prokaryotic Genome Annotation was used to predict the presence of genes involved in plant growth promotion, such as urease subunit encoding, Indole-3-acetic acid (IAA) production pathway , and nitrate reductase related genes.
Keywords : Bacillus velezensis 2F-NA, complete genome, nanopore, whole genome sequencing
Body

Nutrient cycling plays a vital role in plant growth promoting by facilitating the transformation of diverse organic matter into a form that can be readily absorbed by crops (Hendrix et al., 1992; De Graaff et al., 2006). Within the organic-rich environment of livestock farm soil, microbes, including Bacillus species, have been reported to actively participate in utilizing organic matter and contributing to nutrient cycling (Maeda et al., 2011). Therefore, B. velezensis 2F-NA was isolated from the soil of a livestock farmhouse, located in Sangju-si, Gyeongsangbuk-do, Republic of Korea (36.379°E, 128.175°N) with the aim of obtaining a beneficial microbe capable of promoting plant growth. One gram of the collected soil was suspended in 0.85% NaCl solution, then serially diluted into 10-1–10-6. The diluted solutions were plated on LB agar medium. The B. velezensis 2F-NA selected from the plate is available in Korean Collection for Type Cultures under the accession number KCTC 13373BP.

Genomic DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega). Prior to sequencing library preparation, the quality and quantity of the genomic DNA were assessed using the Nanodrop One Spectrophotometer (Thermo Fisher Scientific) and Qubit 3.0 fluorometer (Thermo Fisher Scientific), respectively. The sequencing library was prepared using the Ligation Sequencing Kit SQK-LSK109 (ONT) and NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (NEB). The Library preparation was performed according to DNA repair, end-prep, adapter ligation, and priming, then the prepared library was loaded into the SpotON flow cell. Whole genome sequencing on the Nanopore MinION platform was conducted using the flow cell FLO-MIN111 (R10.3, ONT) for 70 h at NGS Core facility (Kyungpook National University). Fast5 files were generated after sequencing, and Guppy (v.4.4.1) was used to basecall the fast5 files. During the basecalling, the sequencing reads which have an average Phred quality score less than 7 were filtered. The sequencing quality of the reads in fastq files was confirmed using fastqc (v0.11.9). A total of 420,804,958 bp was obtained from basecalling by Guppy (v.4.4.1). De novo assembly was performed using Flye (v.2.9) with the following options: --nano-raw -genome-size 3.9 m.

A circular chromosome was successfully assembled, revealing a genome size of 3,933,901 bp with a G + C content of 46.5% (Table 1). To predict the functional genes, the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016) was utilized. The complete genome sequence of B. velezensis 2F-NA comprises a total of 3,004 protein-coding, 30 rRNA, 91 tRNA and 5 ncRNA genes. Further analysis was conducted to identify genes associated with plant growth promoting functions. Through the PGAP annotation, we successfully predicted the presence of several genes in the genome of Bacillus velezensis 2F-NA. By identifying urease encoding genes (ureA, ureB, and ureC) and nitrate reductase related genes (narJ, nirB, nirD), we have successfully identified functional genes associated with nitrogen source utilization and their contribution to plant growth promoting (Table 2) (Pothier et al., 2008; Goswami et al., 2015). Furthermore, our investigation of genes involved in the IAA production pathway including trpA, trpB, trpC, and trpD revealed their potential for direct impact on plant growth promotion (Table 2) (Idris et al., 2007). The complete genome data of B. velezensis 2F-NA not only contribute to expanding the environmental bacterial database but also hold potential benefits for the agriculture industry by providing functional information.

Features of B. velezensis 2F-NA complete genome
Genome feature Value
Genome length (bp) 3,933,901
G + C content (%) 46.5
Total number of genes 3,948
Number of protein-coding genes 3,004
Total number of RNA genes 126
rRNA genes (5S, 16S, 23S) 10, 10, 10
tRNA genes 91
ncRNA genes 5
Pseudo genes 818


The list of plant growth-promoting related genes in the genome of B. velezensis 2F-NA
Gene Size (bp) Protein Locus-tag
ureA 318 Urease subunit gamma NNL12_06630
ureB 375 Urease subunit beta NNL12_06635
ureC 1,709 Urease subunit alpha NNL12_06640
trpA 798 Tryptophan synthase subunit alpha NNL12_13480
trpB 1,203 Tryptophan synthase subunit beta NNL12_13475
trpC 750 Indole-3-glycerol phosphate synthase NNL12_13465
trpD 1,017 Anthranilate phosphoribosyltransferase NNL12_13460
narJ 558 Nitrate reductase molybdenum cofactor NNL12_06330
nirB 2,322 Nitrite reductase large subunit NNL12_02625
nirD 321 Nitrite reductase small subunit NNL12_02640


Nucleotide sequence accession numbers

The complete genome sequence data of B. velezensis 2F-NA was deposited in GenBank with accession number (CP101612.1).

적 요

한국의 축산 농가 토양으로부터 Bacillus velezensis 2F-NA를 분리하였다. Nanopore MinION 장비를 사용하여 long read sequencing을 수행한 결과, de novo assembly를 통해 3,933,901 bp 크기의 전장 유전체를 생성하였다. 이 유전체는 46.5%의 G + C 함량을 나타내며 3,004개의 단백질 코딩 유전자, 30개의 rRNA 유전자, 91개의 tRNA 유전자, 5개의 ncRNA 유전자를 포함한다. 또한, NCBI Prokaryotic Genome Annotation Pipeline을 통해 우레아제 서브유닛 암호화 유전자, 인돌-3-아세트산(IAA) 생성 관련 유전자, 질산염 환원효소 관련 유전자 등 잠재적인 식물 성장 촉진 유전자의 존재를 예측하였다.

Acknowledgments

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Crop Viruses and Pests Response Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (321097-3), Korea Basic Science Institute (National research Facilities and Equipment center) grant funded by the Ministry of Education (2021R1A6C101A416) and “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ017033)” Rural Development Administration, Republic of Korea.

Conflict of Interest

The authors have no conflict of interest to report.

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