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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2023; 51(4): 535-537

https://doi.org/10.48022/mbl.2310.10001

Received: October 5, 2023; Accepted: November 7, 2023

Draft Genome Sequence of Bacillus thuringiensis serovar aizawai AS23, Isolated from the Rhizosphere of Korean Melon (Cucumis melo L.)

Da-Ryung Jung1, GyuDae Lee1, Kyeongmo Lim1, Yeonkyeong Lee2, Ga-Yeon Nam1, Do-Yeun Won3, Na-Yun Park3, Young-Jin Seo3, and Jae-Ho Shin1,2,4*

1Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
2Department of Integrative Biology, Kyungpook National University, Daegu 41566, Republic of Korea
3Seongju Korean Melon Fruit and Vegetable Research Institute, Seongju 40054, Republic of Korea
4NGS Core Facility, Kyungpook National University, Daegu 41566, Republic of Korea

Correspondence to :
Jae-Ho Shin,        jhshin@knu.ac.kr

Abstract

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We report the draft genome sequence of Bacillus thuringiensis serovar aizawai AS23, an insecticidal strain targeting lepidopteran pests, which was isolated from the rhizosphere of Korean melon (Cucumis melo L.). The genome of strain AS23 comprising 6,846,584 bp with a G + C content of 34.83% was assembled to 11 contigs obtained using hybrid assembly. Additionally, we mined the genome for pesticidal genes, identifying several insecticidal genes, including Cry1Aa3, Cry1Ca9, Cry1Da2, Cry1Ia44, Cry2Ab41, Cry9Ea9, Spp1Aa1, and Vip3Aa86.

Keywords: Bacillus thuringiensis serovar aizawai, draft genome, Korean melon, biopesticide

Body

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Bacillus thuringiensis is a Gram-positive, sporeforming bacterium that belongs to the Bacillus genus. It is widely recognized for its insecticidal properties, making it valuable in agricultural and biological pest control applications [1, 2]. B. thuringiensis produces insecticidal proteins, known as Cry and Cyt toxins (also called delta-endotoxins), which are toxic to a variety of insect pests, certain orders like Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), and Coleoptera (beetles) [3−5]. In particular, B. thuringiensis serovar aizawai AS23 is a subspecies that has an insecticidal effect on lepidopteran pests.

In this study, B. thuringiensis serovar aizawai AS23 was isolated from the rhizosphere soil of Korean melon (Cucumis melo L.), which was sampled from the greenhouse at the Seongju Korean Melon Fruit and Vegetable Research Institute (Seongju, Republic of Korea). The draft genome sequence of the strain is reported.

The genomic DNA of strain AS23 was extracted using the Wizard genomic DNA purification kit (Promega, USA) following the manufacturer’s instructions. The quality and quantity of the extracted DNA were assessed using the Qubit Flex Fluorometer (Thermo Fisher Scientific, USA) and NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific). Sequencing was performed on two platforms: MinION (Oxford Nanopore Technologies [ONT], UK) and DNBSEQ-G400RS (MGI Tech, China), at the NGS Core Facility (Kyungpook National University, Republic of Korea).

Long-read sequencing was carried out using the ONT MinION platform with the sequencing library prepared using a ligation sequencing kit SQK-LSK109 (ONT) and the NEBNext companion module (New England Biolabs, USA). The library was sequenced for 48 h on a FLOMIN111 flow cell R10.4.1 (ONT, USA). Guppy v4.4.1 software was employed in high-accuracy mode to perform base calling and generate FASTQ files. For quality trimming, sequences with Phred scores below 7 were excluded from further analyses.

For short-read sequencing, the sequencing library was prepared using the MGIEasy FS DNA Library Prep Kit and DNBSEQ-G400RS High-throughout Sequencing Kit PE100 (MGI Tech., China) following the manufacturer’s instructions. The same batched genomic DNA was sheared to approximately 100 bp using the Frag enzyme II and then end-repaired using the ERAT enzyme provided by the manufacturer. DNA Nano Ball (DNB) was constructed after circularization of the single-stranded DNA. The final DNB library was loaded into the flow cell and 2 ×100-bp pair-end sequenced for 72 h using a DNBSEQ-G400RS sequencer (MGI Tech.).

The hybrid assembly of long- and short-read sequences was performed using MaSuRCA (Maryland Super Read Cabog Assembler) version 4.0.9 with default settings [6]. Scaffolding was carried out using the assembled contigs as input in CSAR version 1.1.1 [7] with reference genome, i.e., B. thuringiensis serovar berliner ATCC 10792 (GenBank accession no. CM000753.1). The scaffolds generated by CSAR were subsequently polished using Polypolish version 0.5.0 [8]. The final assemblies were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and summarized results are presented in Table 1.

Table 1 . Genome feature of B. thuringiensis serovar aizawai AS23.

FeatureValue
Genome size (bp)6,846,584
Number of contigs11
G + C ratio (%)34.83
Total number of genes7,120
Number of protein-coding genes6,639
Total number of RNA genes155
rRNA genes (5S, 16S, 23S)14, 14, 14
tRNA genes108
ncRNA genes5
Pseudo genes326


For mining pesticidal genes, protein-coding sequences (CDS) were predicted using Prodigal version 2.6.3 with the “-p meta” option [9]. Insecticidal toxic gene database was downloaded from the Bacterial Pesticidal Protein Resource Center (https://www.bpprc-db.org [10]), and predicted CDSs were annotated using blastp with a minimum of 80% identity and only the top alignment score. In total, the following insecticidal genes were identified: Cry1Aa3, Cry1Ca9, Cry1Da2, Cry1Ia44, Cry2Ab41, Cry9Ea9, Spp1Aa1, and Vip3Aa86.

Acknowledgments

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This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ017033)” Rural Development Administration, Republic of Korea.

Data Availability

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The draft genome sequence of strain AS23 has been deposited in GeneBank under accession number PRJNA399840 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA399840).

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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