Draft Genome Sequence of Bacillus Sp. Strain B1-B2 Isolated from Meconium

Draft genome sequence of Bacillus sp. strain B1-b2 isolated from meconium

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

태변으로부터 분리된 Bacillus 속 B1-b2 균주의 유전체 분석

윤영미1 ･ 김민지2 ･ 김의태3* ･ 박수제2* 1제주대학교병원, 2제주대학교 생물학과, 3제주대학교 의과대학

(Received December 23, 2020; Revised January 27, 2021; Accepted January 27, 2021)

The genus Bacillus belonging to Firmicutes division has been tract, as non-pathogenic and non-toxic. They can also produce recognized as a phenotypically diverse and Gram-positive or probiotic supplements that make them more suitable for humans Gram-variable group. Also, the genus Bacillus has a wide (Mingmongkolchai and Panbangred, 2018; Lee et al., 2019). range of physiological characteristics and its abilities are used This study attempted to isolate a microbial candidate as a in medical, pharmaceutical, and agricultural fields. In the potential possibility for human dietary supplements and analyze present study, strain B1-b2 was isolated from the meconium sample and we analyzed its whole-genome sequence. The draft its genomic traits. We received the Institutional Review Board genome consisted of 80 contigs, and their predicted genome (IRB) approval (IRB No. 2019-05-018) from Jeju National size and a mean G + C content were about 5.41 Mbp and 34.9%, University Hospital. respectively. The draft genome sequence was predicted to have For bacterial isolation, the meconium sample was harvested 4,706 coding sequences with protein, 26 ribosomal RNAs and within the first 24 h after birth using a sterilized spatula and 90 transfer RNA genes. Strain B1-b2 genome encoded proteins quickly mixed with phosphate-buffered saline (PBS) solution for biosynthesis for polyamine and glycogen biosynthesis and (Sigma-Aldrich). The mixture was serially diluted by ten-fold degradation. Also, the draft genome of the strain B1-b2 encoded -6 transporters for osmotic stress. to 10 with PBS solution. Then 100 µl of the aliquot from the diluted sample was spread on Brain Heart Infusion (BHI) agar Keywords: Bacillus, draft genome, meconium, newborn (BD DifcoTM) plates and incubated at 37°C for two weeks under aerobic conditions. We selected several strains based on Probiotics use in man has been focused as various live colony morphology and successively transferred them to new microorganisms on expecting to maintain the health and the BHI agar plates to obtain the purified colony. Finally, one well-being. Among the many microorganisms, Bacillus spp. strain was isolated and designed into strain B1-b2. The isolated have been known for sporulation capacity that makes them strain B1-b2 has been deposited at the Korean Culture Center survive extreme conditions (e.g., stomach) in the gastrointestinal for Microorganisms (KCCM) as KCCM 43328. For taxonomic and phylogenetic position, the genomic DNA (gDNA) of the *For correspondence. (E.T. Kim) E-mail: [email protected]; Tel.: +82-64-754-8111; Fax: +82-64-725-3593 / strain B1-b2 was extracted using a commercial DNA extraction (S.J. Park) E-mail: [email protected]; kit (GeneAll Biotechnology. Co. Ltd.) and used to sequence the Tel.: +82-64-754-3524; Fax: +82-64-756-3541 56 ∙ Yoon et al.

16S ribosomal RNA gene, according to the previous study (Koh B1-b2 is 5,133,037 bp with 34.9% mean G + C content. The et al., 2015). Then, we found that the strain B1-b2 was most result of CheckM estimation indicated that genome completeness closely related to Bacillus circulans ATCC 4513T (AY724690, at 93.3% with 1.6% contamination and 0.0% strain heterogeneity. 99.2% 16S rRNA gene sequence similarity) using EzBioCloud The putative genome size was expected to about 5.41 Mbp as server (https://www.ezbiocloud.net/). Recently, based on the previously described (Ghai et al., 2013). To construct a phylo- phylogenomics and comparative genomic analyses, Bacillus genomic tree, single-copy marker genes (n = 161) for strain circulans was proposed to transfer to Niallia circulans (Gupta B1-b2 and more closely related type strain genome sequences et al., 2020). For genome sequencing, gDNA of strain B1-b2 were used to infer concatenated-protein phylogeny as previously was extracted using a commercial DNA extraction kit (GeneAll described (Park et al., 2020). Biotechnology Co., Ltd.) according to the manufacturer’s The draft genome of the strain B1-b2, estimated by PGAP, instructions. Whole-genome sequencing was performed at included 4,956 predicted genes, 26 ribosomal RNA and 80 Macrogen Inc. using the HiSeq 2500 sequencing platform transfer RNA genes (Table 1). Despite the similarity for 16S (Illumina Inc.). Total read bases about 2.4 Gb (after passed rRNA gene sequence was high (99.2%), the result (21.1%) of filtering) were used into contig assembly. The trimming of the digital DDH estimated by GGDC (Meier-Kolthoff et al., 2013) resulting nucleotide sequences and assembling de novo were indicated that strain B1-b2 is a potential new species of the accomplished by SPAdes (v.3.13). Finally, 80 contigs (436 X genus Bacillus (or newly proposed genus Niallia), against depth, N50 199,897 bp) were obtained after assembly. To Bacillus circulans (or proposed Niallia circulans) NBRC 13626T estimate genome completeness and quality, we used CheckM (GCF_001591585). This position of strain B1-b2 within the v1.0.12 (Parks et al., 2015). The resulting assembled contigs genus Niallia was also strongly supported by the result of were annotated by NCBI Prokaryotic Genome Annotation phylogenomics tree (Fig. 1). Pipeline with GeneMarkS-2+ version 4.9, using the best-placed KEGG analysis revealed that the draft genome of the strain reference protein method (Angiuoli et al., 2008). In addition, B1-b2 encoded proteins for glycogen degradation and bio- protein sequences were annotated using BLAST hit against the synthesis with carbon storage regulator (CsrA as a two-component NCBI NR database and performed using Clusters of Orthologous system), polyamine biosynthesis, and sporulation. Unexpectedly, Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes transporters for compatible solute substrates (e.g., glycine (KEGG) database references, as described previously (Kim et betaine, osmoprotectant) for osmotic regulation and resistance al., 2020). Finally, the assembled sequence length of the strain were identified in the draft genome of the strain B1-b2. Also,

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.

미생물학회지 제57권 제1호 Bacillus sp. strain B1-b2 genome ∙ 57

Table 1. Bacillus sp. strain B1-b2 genome assembly and its general features 삼투 조절과 관련된 수송체를 암호화하는 것이 확인되었다.

Item Description Genome assembly data Assembly method SPAdes v. 3.13 Acknowledgments Genome coverage 436X Sequencing technology Illumina HiSeq This work was supported by the research grant of Jeju No. of contigs 80 National University in 2019. Genome features Assembled length (Mbp) 5.13 Estimated size (Mbp) 5.41 References GC content (%) 34.9

No. of total predicted genes* 4,956 Angiuoli SV, Gussman A, Klimke W, Cochrane G, Field D, Garrity G, No. of total coding sequences* 4,836 Kodira CD, Kyrpides N, Madupu R, Markowitz V, et al. 2008. No. of coding sequences with protein* 4,706 Toward an online repository of Standard Operating Procedures rRNA (23S, 16S, 5S)* 26 (11, 9, 6) (SOPs) for (meta)genomic annotation. OMICS 12, 137–141. tRNA* 80 Ghai R, Mizuno CM, Picazo A, Camacho A, and Rodriguez-Valera F. * Predicted by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). 2013. Metagenomics uncovers a new group of low GC and ultra-small marine Actinobacteria. Sci. Rep. 3, 2471. Gupta RS, Patel S, Saini N, and Chen S. 2020. Robust demarcation of the draft genome harbored genes encoding for enzymes involved 17 distinct Bacillus species clades, proposed as novel Bacillaceae in two-component regulatory systems for phosphate limitation genera, by phylogenomics and comparative genomic analyses: (PhoRPA), salt stress (DegSU), low temperature (DesKR), cell description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of wall-active-antibiotics (LiaSRIHFS), bacitracin (BceSRAB), the Subtilis and Cereus clades of species. Int. J. Syst. Evol. efflux (VraDE), and cationic antimicrobial peptide (YxdKJLM). Microbiol. 70, 5753–5798. Kim M, Cha IT, Lee KE, Lee EY, and Park SJ. 2020. Genomics reveals Neucleotide sequence accession number the metabolic potential and functions in the redistribution of dissolved organic matter in marine environments of the genus This whole genome shotgun project of the strain B1-b2 (= Thalassotalea. Microorganisms 8, 1412. KCCM 43328) has been deposited at DDBJ/ENA/GenBank Koh HW, Hong H, Min UG, Kang MS, Kim SG, Na JG, Rhee SK, under the accession WEHU00000000. The version described in and Park SJ. 2015. Rhodanobacter aciditrophus sp. nov., an this paper is version WEHU01000000 and consists of sequences acidophilic bacterium isolated from mine wastewater. Int. J. Syst. Evol. Microbiol. 65, 4574–4579. WEHU01000001-WEHU01000080. Lee NK, Kim WS, and Paik HD. 2019. Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier. Food Sci. Biotechnol. 28, 1297–1305. 적 요 Meier-Kolthoff JP, Auch AF, Klenk HP, and Göker M. 2013. Genome sequence-based species delimitation with confidence intervals Firmicutes에 속하는 Bacillus 속은 다양한 표현형과 함께 and improved distance functions. BMC Bioinformatics 14, 60. 그람 양성 혹은 그람 다양성 세균으로 알려져 있다. Bacillus 속 Mingmongkolchai S and Panbangred W. 2018. Bacillus probiotics: an alternative to antibiotics for livestock production. J. Appl. 은 다양한 생리적 특성을 지니고 있으며, 의료, 제약 농업 분야 Microbiol. 124, 1334–1346. 등에서 사용된다. 본 연구에서는 태변 샘플로 부터 B1-b2 균주 Park SJ, Andrei AŞ, Bulzu PA, Kavagutti VS, Ghai R, and Mosier AC. 를 분리하고 그 전장유전체를 분석하였다. 80개 콘티그로 구 2020. Expanded diversity and metabolic versatility of marine 성된, 불완전한 전장유전체로, 약 5.41 Mb의 크기와 34.9%의 nitrite-oxidizing bacteria revealed by cultivation- and genomics- based approaches. Appl. Environ. Microbiol. 86, e01667-20. 평균 G + C 함량을 가진 것으로 확인되었다. 불완전한 전장유 Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, and Tyson GW. 전체는 4,706개의 단백질 코딩 서열, 26개 리보좀 RNA와 90개 2015. CheckM: assessing the quality of microbial genomes 의 전사 RNA 유전자를 포함하였다. B1-b2 균주의 유전체는 폴 recovered from isolates, single cells, and metagenomes. Genome 리아민 생합성과 글리코겐 생합성 및 분해에 관련된 유전자와 Res. 25, 1043–1055.

Korean Journal of Microbiology, Vol. 57, No. 1