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Complete genome sequence of Bacillus inaquosorum 1HC-NA assembled from the Oxford Nanopore sequencing data
Korean J. Microbiol. 2022;58(3):194-196
Published online September 30, 2022
© 2022 The Microbiological Society of Korea.

Min-Ji Kim1, TaeHyung Park2, Da-Ryung Jung1, GyuDae Lee1, and Jae-Ho Shin1,2*

1Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
2Department of Integrative Biology, 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 22, 2022; Revised August 19, 2022; Accepted August 22, 2022.
Abstract
Bacillus inaquosorum 1HC-NA is isolated from the soybean rhizosphere soil in Republic of Korea. The complete genome sequence of B. inaquosorum 1HC-NA was obtained through Oxford Nanopore sequencing. The total genome length of this strain is 4,240,317 bp with 44.0% of GC content. This strain contains 3,293 protein-coding genes, 30 rRNA genes, 86 tRNA genes and 5 ncRNA genes. Furthermore, Rapid Annotation using Subsystem Technology (RAST) predicted a total of 332 subsystems of the strain, including amino acids and derivatives and carbohydrates subsystem.
Keywords : Bacillus inaquosorum 1HC-NA, complete genome, long-read sequencing, Oxford Nanopore sequencing
Body

Ubiquitous in nature, Bacillus genera interact symbiotically with various microbes and hosts (Jo et al., 2020; Wilkes et al., 2020; Torres-Sánchez et al., 2021). Recently, Bacillus spp. have been suggested as the biocontrol agents to promote the eubiosis of the soil microbiome in the agricultural industry (Jo et al., 2020; Wilkes et al., 2020). Therefore, we isolated Bacillus inaquosorum 1HC-NA from the soybean rhizosphere soil and identified its genome sequence. The soybean rhizosphere soil was collected from Sangju-si, Gyeongsangbuk-do, Republic of Korea. From this, the bacterial isolation was conducted with the following methods: 1 g of soil attached to the soybean roots was taken, suspended in saline, 100 μl of suspension plated on nutrient agar (NA; Difco). Bacillus inaquosorum 1HC-NA is available in Korean Collection for Type Cultures under the accession number KCTC 13372BP.

The genomic DNA extraction was performed using Wizard® Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol. Qubit 3.0 fluorometer (Thermo Fisher Scientific) and Nanodrop One Spectrophotometer (Thermo Fisher Scientific) were used for DNA quantification and qualification, respectively. In order to prepare the Nanopore sequencing library, Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies [ONT]) and NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing Kit (NEB) were used. The prepared sequencing library was loaded into the FLO-MIN111 (R10.3, ONT). The sequencing was performed during 72 h and Guppy (v.4.4.1) was used for basecalling (Wick et al., 2019). A total of 501,492,938 reads with Phred score higher than 7 was generated with 20,700 bp of N50. De novo assembly of sequences was performed using Flye (v.2.9) with default parameters, excepting size option (option: –nano-raw –genome-size 4.0) (Kolmogorov et al., 2019).

The complete genome of B. inaquosorum 1HC-NA comprises one chromosome with 4,240,317 bp of genome length and 44.0% of GC content. Furthermore, the genome was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016) and the Rapid Annotations using Subsystem Technology (RAST) server (Aziz et al., 2008). Bacillus inaquosorum 1HC-NA contains 4,359 protein-coding genes, 30 rRNAs, 86 tRNAs, 5 ncRNAs, and 945 pseudo genes (Table 1).

Genome features of <italic>B. inaquosorum</italic> 1HC-NA genome sequence
Genome feature Value
Genome length (bp) 4,240,317
GC content (%) 44.0
Total number of genes 4,359
Number of protein-coding genes 3,293
Total number of RNA genes 121
rRNA genes (5S, 16S, 23S) 10, 10, 10
tRNA genes 86
ncRNA genes 5
Pseudo genes 945


The RAST server predicted 332 of classified subsystems with 30% of coverage. Amino acids and derivatives related features (372 genes), carbohydrates (324 genes), protein metabolism (248 genes), cofactors, vitamins, prosthetic groups, pigments subsystem (181 genes), cell wall and capsule (112 genes) and nucleosides and nucleotides (112 genes) were the predominant subsystems in the strain. Furthermore, the strain 1HC-NA has the ability to antimicrobial activities, such as bacilysin and antimicrobial peptide biosynthesis. The genome contains bacA, bacB, bacC, and bacG, which encode the bacilysin biosynthesis protein (Table 2). Additionally, antimicrobial peptides, including sublancin and plipastatin, can be produced through the sunA, sunS, and ppsA in the strain 1HC-NA. These results suggest that B. inaquosorum 1HC-NA is a potent biocontrol bacterium for agricultural purposes.

The list of biocontrol related genes in the genome of <italic>B. inaquosorum</italic> 1HC-NA
Gene Size (bp) Protein Locus-tag
bacA 615 Biosynthesis of the antibiotic bacilysin NM058_02385
bacB 708 Biosynthesis of the antibiotic bacilysin NM058_02390
bacC 762 Biosynthesis of the antibiotic bacilysin NM058_02395
bacE 1185 Self-protection to bacilysin NM058_02405
bacG 780 Biosynthesis of the antibiotic bacilysin NM058_02415
albG 702 Antilisterial bacteriocin (subtilosin) production NM058_02550
albD 1,311 Export of antilisterial bacteriocin (subtilosin) NM058_02565
albB 162 Antilisterial bacteriocin (subtilosin) production NM058_02575
albA 1,347 Antilisterial bacteriocin (subtilosin) production NM058_02580
sboA 132 Antimicrobial peptide NM058_02590
bpsB 507 Polyketide synthesis NM058_10865
sunA 171 Exported antimicrobial peptide NM058_11590
sunS 1,269 Biosynthesis of the antimicrobial peptide sublancin NM058_11605
ppsA 2,613 Production of the antibacterial compound plipastatin NM058_11755


Nucleotide sequence accession numbers

The complete genome sequence data of B. inaquosorum 1HC-NA was deposited in GenBank with accession number (CP101611.1).

적 요

한국의 콩 근권 토양으로부터 Bacillus inaquosorum 1HC-NA를 분리하였다. Oxford Nanopore sequencing을 통해 B. inaquosorum 1HC-NA의 전장 유전체가 확보되었다. 전장 유전체는 총 44.0%의 GC 함량을 포함하며 4,240,317 bp의 길이를 가졌다. 이 균주는 3,293개의 단백질 코딩 유전자와 30개의 rRNA, 86개의 tRNA, 5개의 ncRNA를 보유하고 있다. 또한, Rapid Annotation using Subsystem Technology (RAST)는 아미노산 및 탄수화물 subsystem을 포함하여 균주의 총 332개의 subsystem을 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 a project to train professional personnel in biological materials by the Ministry of Environment.

We especially thank the KNU NGS Core facility for their support and cooperation in the whole genome sequencing using Oxford Nanopore MinION.

Conflict of Interest

The authors have no conflict of interest to report.

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September 2022, 58 (3)