The genus Bacillus, first proposed by Cohn (1872), belongs to the family Bacillaceae within the phylum Bacillota. Bacillus species are typically Gram-stain-positive, rod-shaped bacteria, often exhibiting resistance to antibiotics or toxic compounds (Mohammed et al., 2002; Stein, 2005; Fiedler et al., 2019; Shleeva et al., 2023).

Strain AYGS-17 was isolated from sediment collected from the Nakdong River (36°31'17.7" N, 128°15'52.1" E) during a screening for bacteria with antimicrobial activity. In order to select microorganisms exhibiting antibacterial activity against antibiotic-resistant strains, both Staphylococcus aureus subsp. aureus CCARM 3090 and Escherichia coli CCARM 1G440, which are multi-drug resistant, were utilized as target microorganisms. The selection process involved streaking methods, with strains displaying inhibition zones after 24 h of cultivation at 25°C being initially chosen. Following this initial selection, microorganisms were further confirmed for antibacterial activity using the paper disc method. Here, we present the complete genome sequence of strain AYGS-17 and discuss its metabolic features based on genome analyses. A single colony of Bacillus velezensis AYGS-17 was grown for 3 days in R2A agar (BD Difco) at 30°C. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s protocols. Sanger sequencing of the 16S rRNA gene confirmed its affiliation with the Bacillus genus, sharing 99.9% similarity with Bacillus velezensis CR-502^T.

The whole genome of strain AYGS-17 was sequenced at Macrogen Inc. using a Pacific Biosciences (PacBio) Sequel platform. High-quality de novo assembly was performed using Canu v1.7 (Koren et al., 2017). In total, 114,317 subreads (1.0 Gb; N50 10,301 bp; mean length 9,032) were generated before undergoing quality filtering and adapter trimming using the Hierarchical Genome Assembly Process 3 (HGAP3) within PacBio SMRT analysis 2.3.0 (Chin et al., 2013). The completeness of the genome assembly was assessed as 100% by CheckM software (Parks et al., 2015). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) after being submitted to GenBank (Tatusova et al., 2016). The resulting whole genome of strain AYGS-17 was determined to be a circular chromosome of 4,064,612 bp, with a DNA G + C content of 46.1% and a depth of 121×. Annotation of the genome revealed 4,000 protein-coding sequences, 86 tRNA, and 27 rRNA genes (Table 1). Comparison of AYGS-17 with Bacillus velezensis type strain CR-502^T using OrthoANI indicated a close relationship (98.7% OrthoANI). This similarity exceeded the generally accepted threshold of 95 to 96% (Lee et al., 2016), confirming AYGS-17 as belonging to the species Bacillus velezensis (Jain et al., 2018).

Table 1

General genomic features of the strain AYGS-17

Features	Value
enome size (bp)	4,064,612
GC content (mol%)	46.1
No. of contigs	1
Genes (total)	4,118
CDSs (total)	4,000
Genes (coding)	3,909
No. of rRNA genes (5S, 16S, 23S)	27 (9, 9, 9)
No. of tRNA genes	86
No. of ncNA genes	5
No. of pseudo genes	91

In a previous study by Jeong et al. (2023), both strain AYGS-17 and its extract demonstrated antibacterial activity against periodontitis-related bacteria, including Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans KCTC 2581, as well as cavity-related bacteria such as Streptococcus mutans KCCM 40105. The study also investigated various antibacterial genes in the genome of B. velezensis AYGS-17. AntiSMASH shell analysis (Medema et al., 2011) revealed that the genome harbors 12 gene clusters related to ladderane, terpene, Polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS) biosynthesis. Bacillus velezensis is known for its ability to produce a diverse array of enzymes through biotechnological applications and the synthesis of physiologically active secondary metabolites (Zhao et al., 2017). These genes may play an important role in regulating bacterial physiological and biochemical activities, thus indirectly affecting antibacterial activity. Antimicrobial resistance genes were identified using ResFinder v4.1 (https://cge.food.dtu.dk/services/ResFinder) with default settings (> 90% threshold, > 60% minimum length) (Bortolaia et al., 2020). The genome of this strain exhibited 92 antibiotic resistance genes (ARGs) conferring resistance to diverse antimicrobials, including gentamicin, fluoroquinolone, amoxicillin, sulfamethoxazole, and tetracycline. These ARGs may play an important role in bacteria developing resistance to antibiotics.

This comprehensive genome analysis of Bacillus velezensis AYGS-17 provides valuable insights into its antimicrobial capabilities, contributing to our understanding of its potential applications in developing natural microbial extract antibiotics across diverse environments.

Strain and nucleotide sequence accession numbers

Strain AYGS-17 has been deposited in the Freshwater Bioresources Culture Collection (FBCC) under the number FBCC-B13530. The GenBank/EMBL/DDBJ accession number for the complete genome sequence of strain AYGS-17 is CP138627. The associated BioProject and BioSample accession numbers are PRJNA1037351 and SAMN38171755, respectively.

이 연구에서는 낙동강 퇴적물에서 분리한 항균활성을 갖는 AYGS-17 균주의 유전체 분석하였다. PacBio Sequel 플랫폼을 사용하여 시퀀싱된 전체 게놈은 46.1%의 GC 함량과 4.1 Mb의 원형 염색체 나타낸다. 게놈에는 4,000개의 코딩 서열(CDS), 86개의 tRNA, 27개의 rRNA 및 5개의 ncRNA를 포함하여 4,118개의 유전자가 포함되어 있다. 이 유전체 정보는 균주의 항균 활성에 대한 유전적 기반의 이해를 제공한다.

This work was supported by the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR202401103).

The authors have no conflict of interest to report.