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Draft genome sequence of Bacillus sp. strain B1-b2 isolated from meconium
Korean J. Microbiol. 2021;57(1):55-57
Published online March 31, 2021
© 2021 The Microbiological Society of Korea.

Young Mi Yoon1, Minji Kim2, Eui Tae Kim3*, and Soo-Je Park2*

1Department of Pediatrics, Jeju National University Hospital, Jeju 63241, Republic of Korea
2Department of Biology, Jeju National University, Jeju 63243, Republic of Korea
3Department of Microbiology and Immunology, Jeju National University School of Medicine, Jeju 63241, Republic of Korea
Correspondence to: (E.T. Kim) E-mail: tae@jejunu.ac.kr;
Tel.: +82-64-754-8111; Fax: +82-64-725-3593 /
(S.J. Park) E-mail: sjpark@jejunu.ac.kr;
Tel.: +82-64-754-3524; Fax: +82-64-756-3541
Received December 23, 2020; Revised January 27, 2021; Accepted January 27, 2021.
Abstract
The genus Bacillus belonging to Firmicutes division has been recognized as a phenotypically diverse and Gram-positive or Gram-variable group. Also, the genus Bacillus has a wide range of physiological characteristics and its abilities are used in medical, pharmaceutical, and agricultural fields. In the present study, strain B1-b2 was isolated from the meconium sample and we analyzed its whole-genome sequence. The draft genome consisted of 80 contigs, and their predicted genome size and a mean G + C content were about 5.41 Mbp and 34.9%, respectively. The draft genome sequence was predicted to have 4,706 coding sequences with protein, 26 ribosomal RNAs and 90 transfer RNA genes. Strain B1-b2 genome encoded proteins for biosynthesis for polyamine and glycogen biosynthesis and degradation. Also, the draft genome of the strain B1-b2 encoded transporters for osmotic stress.
Keywords : Bacillus, draft genome, meconium, newborn
Body

Probiotics use in man has been focused as various live microorganisms on expecting to maintain the health and the well-being. Among the many microorganisms, Bacillus spp. have been known for sporulation capacity that makes them survive extreme conditions (e.g., stomach) in the gastrointestinal tract, as non-pathogenic and non-toxic. They can also produce probiotic supplements that make them more suitable for humans (Mingmongkolchai and Panbangred, 2018; Lee et al., 2019). This study attempted to isolate a microbial candidate as a potential possibility for human dietary supplements and analyze its genomic traits. We received the Institutional Review Board (IRB) approval (IRB No. 2019-05-018) from Jeju National University Hospital.

For bacterial isolation, the meconium sample was harvested within the first 24 h after birth using a sterilized spatula and quickly mixed with phosphate-buffered saline (PBS) solution (Sigma-Aldrich). The mixture was serially diluted by ten-fold to 10-6 with PBS solution. Then 100 µl of the aliquot from the diluted sample was spread on Brain Heart Infusion (BHI) agar (BD DifcoTM) plates and incubated at 37°C for two weeks under aerobic conditions. We selected several strains based on colony morphology and successively transferred them to new BHI agar plates to obtain the purified colony. Finally, one strain was isolated and designed into strain B1-b2. The isolated strain B1-b2 has been deposited at the Korean Culture Center for Microorganisms (KCCM) as KCCM 43328. For taxonomic and phylogenetic position, the genomic DNA (gDNA) of the strain B1-b2 was extracted using a commercial DNA extraction kit (GeneAll Biotechnology. Co. Ltd.) and used to sequence the 16S ribosomal RNA gene, according to the previous study (Koh et al., 2015). Then, we found that the strain B1-b2 was most closely related to Bacillus circulans ATCC 4513T (AY724690, 99.2% 16S rRNA gene sequence similarity) using EzBioCloud server (https://www.ezbiocloud.net/). Recently, based on the phylogenomics and comparative genomic analyses, Bacillus circulans was proposed to transfer to Niallia circulans (Gupta et al., 2020). For genome sequencing, gDNA of strain B1-b2 was extracted using a commercial DNA extraction kit (GeneAll Biotechnology Co., Ltd.) according to the manufacturer’s instructions. Whole-genome sequencing was performed at Macrogen Inc. using the HiSeq 2500 sequencing platform (Illumina Inc.). Total read bases about 2.4 Gb (after passed filtering) were used into contig assembly. The trimming of the resulting nucleotide sequences and assembling de novo were accomplished by SPAdes (v.3.13). Finally, 80 contigs (436 X depth, N50 199,897 bp) were obtained after assembly. To estimate genome completeness and quality, we used CheckM v1.0.12 (Parks et al., 2015). The resulting assembled contigs were annotated by NCBI Prokaryotic Genome Annotation Pipeline with GeneMarkS-2+ version 4.9, using the best-placed reference protein method (Angiuoli et al., 2008). In addition, protein sequences were annotated using BLAST hit against the NCBI NR database and performed using Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database references, as described previously (Kim et al., 2020). Finally, the assembled sequence length of the strain B1-b2 is 5,133,037 bp with 34.9% mean G + C content. The result of CheckM estimation indicated that genome completeness at 93.3% with 1.6% contamination and 0.0% strain heterogeneity. The putative genome size was expected to about 5.41 Mbp as previously described (Ghai et al., 2013). To construct a phylogenomic tree, single-copy marker genes (n = 161) for strain B1-b2 and more closely related type strain genome sequences were used to infer concatenated-protein phylogeny as previously described (Park et al., 2020).

The draft genome of the strain B1-b2, estimated by PGAP, included 4,956 predicted genes, 26 ribosomal RNA and 80 transfer RNA genes (Table 1). Despite the similarity for 16S rRNA gene sequence was high (99.2%), the result (21.1%) of digital DDH estimated by GGDC (Meier-Kolthoff et al., 2013) indicated that strain B1-b2 is a potential new species of the genus Bacillus (or newly proposed genus Niallia), against Bacillus circulans (or proposed Niallia circulans) NBRC 13626T (GCF_001591585). This position of strain B1-b2 within the genus Niallia was also strongly supported by the result of phylogenomics tree (Fig. 1).

Bacillus sp. strain B1-b2 genome assembly and its general features

Item Description
Genome assembly data
Assembly method SPAdes v. 3.13
Genome coverage 436X
Sequencing technology Illumina HiSeq
No. of contigs 80
Genome features
Assembled length (Mbp) 5.13
Estimated size (Mbp) 5.41
GC content (%) 34.9
No. of total predicted genes* 4,956
No. of total coding sequences* 4,836
No. of coding sequences with protein* 4,706
rRNA (23S, 16S, 5S)* 26 (11, 9, 6)
tRNA* 80

* Predicted by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).



Fig. 1. Phylogenomic tree of strain B1-b2 and relative type strains. Whole-genome phylogenies based on a maximum likelihood tree inferred from 13 genomes. The draft genome of the strain B1-b2 was highlighted in boldface. The strength of support for internal nodes was assessed by performing bootstrap replicates and was shown > 50%. The genome sequence of Staphylococcus warneri 22.1 (GCF 003571725) was used as the outgroup. A midpoint root was generated when selecting outgroup sequences. Bar, 0.1 substitutions per nucleotide position.

KEGG analysis revealed that the draft genome of the strain B1-b2 encoded proteins for glycogen degradation and biosynthesis with carbon storage regulator (CsrA as a two-component system), polyamine biosynthesis, and sporulation. Unexpectedly, transporters for compatible solute substrates (e.g., glycine betaine, osmoprotectant) for osmotic regulation and resistance were identified in the draft genome of the strain B1-b2. Also, the draft genome harbored genes encoding for enzymes involved in two-component regulatory systems for phosphate limitation (PhoRPA), salt stress (DegSU), low temperature (DesKR), cell wall-active-antibiotics (LiaSRIHFS), bacitracin (BceSRAB), efflux (VraDE), and cationic antimicrobial peptide (YxdKJLM).

Neucleotide sequence accession number

This whole genome shotgun project of the strain B1-b2 (= KCCM 43328) has been deposited at DDBJ/ENA/GenBank under the accession WEHU00000000. The version described in this paper is version WEHU01000000 and consists of sequences WEHU01000001-WEHU01000080.

적 요

Firmicutes에 속하는 Bacillus 속은 다양한 표현형과 함께 그람 양성 혹은 그람 다양성 세균으로 알려져 있다. Bacillus 속은 다양한 생리적 특성을 지니고 있으며, 의료, 제약 농업 분야 등에서 사용된다. 본 연구에서는 태변 샘플로 부터 B1-b2 균주를 분리하고 그 전장유전체를 분석하였다. 80개 콘티그로 구성된, 불완전한 전장유전체로, 약 5.41 Mb의 크기와 34.9%의 평균 G + C 함량을 가진 것으로 확인되었다. 불완전한 전장유전체는 4,706개의 단백질 코딩 서열, 26개 리보좀 RNA와 90개의 전사 RNA 유전자를 포함하였다. B1-b2 균주의 유전체는 폴리아민 생합성과 글리코겐 생합성 및 분해에 관련된 유전자와 삼투 조절과 관련된 수송체를 암호화하는 것이 확인되었다.

Acknowledgments

This work was supported by the research grant of Jeju National University in 2019.

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