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Complete genome sequence of Bacillus halotolerans F41-3 isolated from wild flower in Korea
Korean J. Microbiol 2019;55(3):306-308
Published online September 30, 2019
© 2019 The Microbiological Society of Korea.

Jun Heo, Soo-Jin Kim, Jeong-Seon Kim, Seung-Beom Hong, and Soon-Wo Kwon*

Agricultural Microbiology Division, National Institute of Agricultural Sciences, Wanju 55365, Republic of Korea
Correspondence to: *E-mail: swkwon1203@korea.kr; Tel.: +82-63-238-3024; Fax: +82-63-238-3845
Received September 9, 2019; Revised September 16, 2019; Accepted September 16, 2019.
Abstract
A number of Bacillus strains are known to have antimicrobial activity useful in various fields. In order to prevent Propionibacterium acnes, which is one of the factors of acne, we selected Bacillus halotolerans F41-3 which have high antimicrobial activities against P. acnes. We conducted complete genome sequencing of B. halotolerans F41-3 and analyzed genomic characteristics. This genome size is 4,144,458 bp with a G + C content of 43.76%, 4,145 total genes and 3,686 protein coding genes. Among the genes, we found that gene clulster of subtilosin, a kind of bacteriocin, synthesis and gene cluster of nickel transportation. Both of them may influence inhibition of P. acnes.
Keywords : Bacillus halotolerans, genome sequence, inhibition of Propionibacterium acnes, subtilosin
Body

Antibacterial activity is important especially for preventing pathogens. Some microbes and their antimicrobial substances were used for acne treatment as a natural antibiotic against Propionibacterium acnes which is a Gram-positive bacterium and one of the causative agents of acne (Lee and Song, 2018). A number of Bacillus strains were reported as antimicrobial bacteria against Gram-positive bacteria (Sumi et al., 2015). In this study, several Bacillus strains were isolated from a variety of flowers. The flower samples were suspended in distilled water and then spread and cultivated on R2A agar at 28°C for two days. Among them, B. halotolerans F41-3 which was isolated from a flower of Chinese redbud (Cercis chinensis) had higher antibacterial activity against P. acnes than other Bacillus strains. To investigate antibacterial factors in respect of genomics, we conducted whole genome sequencing of the B. halotolerans strain F41-3.

The genomic DNA was extracted using a Wizard genomic DNA isolation kit (Promega). Single Molecule, Real-Time (SMRT) library was prepared to sequence long fragments and sequencing was performed by de novo sequencing using PacBio RS II (Pacific Biosciences) at the Macrogen. De novo assembly was performed by using RS HGAP assembly version 3.0 (Chin et al., 2013). The genome was annotated by NCBI prokaryotic genome annotation pipeline (Tatusova et al., 2016), and submitted to the GenBank (http://www.ncbi.nlm.nih.gov/) database (CP041357).

The complete genome sequence of strain F41-3 was composed of a single circular chromosome of which genome coverage was 198.0 x and did not contain plasmid. The chromosome genome was 4,144,458 bp with a G + C content of 43.76% and contained 4,145 coding genes (CDS), 459 pseudo genes (404 genes are frameshifted), and 121 RNA genes. The genomic feature of strain F41-3 was summarized in Table 1.

Genome feature of Bacillus halotolerans F41-3

Attribute Value
Genome size (bp) 4,144,458
GC content (%) 43.96
No. of contigs 1 (CP041357)
Total genes (CDS) 4,145
Protein-coding genes 3,686
tRNAs 86
rRNAs 30
ncRNAs 5
Pseudogenes 459
Plasmid 0


We compared the ANI value between strain F41-3, and B. halotolerans ATCC 25096T (LPVF01000000) and B. mojavensis KCTC 3706T (AYTL01000000) which were the most closely related species on the basis of high 16S rRNA similarities (99.93% and 99.86%, respectively). The OrthoANI value was calculated using EZbiocloud (http://www.ezbiocloud.net/tools/ani) (Yoon et al., 2017). Strain F41-3 was the highest OrthoANI value of 98.03% with B. halotolerans ATCC 25096T and a value of 96.00% with B. mojavensis KCTC 3706T, which meant the strain should be classified into B. halotolerans (Chun et al., 2018).

Characteristically, strain F41-3 contained two gene clusters which were absent in the genome of type strain ATCC 25096T; subtilosin (FLQ13_05815) and associated genes (FLQ13_05820 to FLQ13_05855), and nickel transporter system genes (FLQ13_00230 to FLQ13_00255). Subtilosin which was first reported to be produced by Bacillus subtilis strain 168 inhibited the growth of some Gram-positive bacteria (Babasaki et al., 1985). The nickel transport system present in diverse microorganisms was reported that nickel into cell influenced formation of subtilosin (Kawulka et al., 2004). Therefore, we assume that this may be the cause of high antibacterial activity.

Availability of the sequence data and strain

The complete genome sequence of Bacillus halotolerans F41-3 has been deposited to GenBank under accession number CP041357. The strain is available at the Korean Agricultural Culture Collection (accession number KACC 21447).

적 요

많은 Bacillus 속 균주들이 다양한 분야에서 이용할 수 있는 항균 활성을 가지고 있는 것으로 알려져 있다. 여드름을 유발하는 인자 중 하나인 Propionibacterium acne를 억제하기 위해 P. acnes에 대해 높은 항균활성을 가지는 Bacillus halotolerans F41-3 균주를 선발하였다. 우리는 B. halotolerans F41-3 균주에 대한 유전체 분석을 하였고 유전적 특성을 분석하였다. 이 유전체는 4,144,458 bp의 크기로 G + C 함량은 43.76%, 유전자의 수는 4,145개, 단백질 암호화 유전자는 3,686개로 구성되어 있다. 유전체 가운데 bacteriocin 중 하나인 subtilosin에 연관된 유전자 클러스터와 subtilosin 합성에 관여하는 nickel 수송에 연관된 유전자 클러스터가 포함되어 있는 것을 확인하였다. 두 클러스터가 P. acnes를 저해하는데 영향을 줄 것으로 보인다.

Acknowledgements

This study was carried out with the support (PJ013549) of National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea.

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