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Draft genome sequence of bacterial strain BA299 in the genus Alkalihalobacillus, isolated from soil of a solar saltern in Korea
Korean J. Microbiol. 2021;57(2):125-128
Published online June 30, 2021
© 2021 The Microbiological Society of Korea.

Ki-Hong Yoon*

Food Science & Biotechnology Major, Woosong University, Daejeon 34606, Republic of Korea
Correspondence to: E-mail: ykh@wsu.ac.kr; Tel.: +82-42-630-9742; Fax: +82-42-630-9389
Received April 26, 2021; Revised June 15, 2021; Accepted June 22, 2021.
Abstract
A bacterial strain BA299, belonging to the genus Alkalihalobacillus, was isolated from a saltern soil sampled at Anmyeon Island located in Taean, South Korea. The draft genome sequence of BA299 was determined using Illumina MiSeq platform. The genome comprises 69 contigs with genome size of 5,137,563 bp long and GC content of 36.7%. The draft genome contains 4,844 protein-coding genes, 106 pseudogenes and 160 RNA genes including 34 ribosomal RNA genes, 119 transfer RNA (tRNA) genes and 7 non-coding RNA (ncRNA) genes. The phylogenomic analysis based on the whole genome sequence demonstrated that strain BA299 formed a distinct phyletic line from Alkalihalobacillus alkalinitrilicus DSM 22532T showing average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values of 80.5 and 25.8%, respectively. The strain BA299 contains exclusively a hydantoin racemase gene from other members of the genus Alkalihalobacillus, which is involved in the production of optically pure amino acids.
Keywords : Alkalihalobacillus, BA299, draft genome sequence, hydantoin racemase, Illumina MiSeq
Body

The genus Alkalihalobacillus was proposed by Patel and Gupta (2020) with the reclassification of 34 species in the genus Bacillus within the phylum Firmicutes. The genus Alkalihalobacillus comprised 39 recognized species with validly published and correct names (lpsn.dsmz.de/genus/alkalihalo bacillus) including additional reclassification of 7 species in the genus Bacillus by Gupta et al. (2020), and Alkalihalobacillus hunanensis (Patel and Gupta, 2020). Members of the genus are aerobic or facultatively anaerobic, Gram-positive or Gram-variable, rod shaped, endospore-forming bacteria, and motile through peritrichous flagella. Many alkaliphilic and halophilic or halotolerant Alkalihalobacillus species have been isolated from a wide range of hypersaline or alkaline habitats, such as salterns, soda lakes, seawater, sponges, and rhizosphere soils (Patel and Gupta, 2020). These bacteria have great potential in biotechnology and industrial applications due to their production of alkaline active enzymes capable of function at salt concentration above 10% (Aharon, 2010). In this study, I present the draft genome sequence and annotation of a Alkalihalobacillus sp. strain BA299 isolated from a saltern soil in South Korea.

The strain BA299 was incubated on Reasoner’s 2A agar (BD Difco) supplemented with 5% (w/v) NaCl and adjusted to pH 8.0 at 35°C for 2 days. Genomic DNA of strain BA299 was extracted using Genomic DNA Prep Kit (Biofact). The comparison of the 16S rRNA sequence showed the highest similarity to Alkalihalobacillus alkalinitrilicus DSM 22532T (98.1%). The whole-genome sequencing and analysis of strain BA299 was performed using the paired-end sequencing method on the MiSeq platform (Illumina) at ChunLab. The estimation of genome completeness and quality was verified with CheckM (Version 1.0.12) (Parks et al., 2015). The resulting short sequencing reads were assembled in contigs using the SPAdes 3.13.0 program (Bankevich et al., 2012). The genome sequence was annotated with the Rapid Annotation of microbial genomes using Subsystems Technology (RAST) (Aziz et al., 2008), and the protein-coding sequences were determined using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016). The average nucleotide identity (ANI) values between strain BA299 and available type strains of species in the genus Alkalihalobacillus were calculated using the ANI Calculator (www.ezbiocloud.net/tools/ani) (Yoon et al., 2017). To determine genomic relatedness, digital DDH (dDDH) values were computed using Genome-to-Genome Distance Calculator (GGDC) version 2.1 (Meier-Kolthoff et al., 2013).

The draft genome of the strain BA299 composed of 69 contigs with a chromosome length of 5,137,563 bp (N50 value 223,035 bp) with 350× sequencing coverage, and 36.7% GC content. The result of CheckM estimation indicated that genome completeness was 98.01% with 1.21% contamination and 0.0% strain heterogeneity. The NCBI PGAP annotation revealed that the genome contains 4,844 protein-coding genes, 106 pseudogenes and 160 RNA genes including 34 rRNA genes (twelve 5S ribosomal RNA, twelve 16S ribosomal RNA, ten 23S ribosomal RNA), 119 transfer RNA (tRNA) genes and 7 non-coding RNA (ncRNA) genes (Table 1). Ten conserved signature indels (CSIs) that are shared exclusively by the members of the genus Alkalihalobacillus (Patel and Gupta, 2020) were found in the genome of strain BA299; RNase adapter protein (RapZ) [J0N78_RS20410], flagellar M-ring protein (FliF) [J0N78_RS07380], 7-carboxy-7-deazaguanine synthase (QueE) [J0N78_RS19040], peptide chain release factor 3 [J0N78_RS21300], transcription-repair coupling factor (Mfd) [J0N78_RS23900], tRNA-5-carboxymethyl-aminomethyl-2-thiouridine (34) synthesis protein (MnmG) [J0N78_RS21735], ribosomal protein L11 methyltransferase (PrmA) [J0N78_RS 10370], homoserine kinase [J0N78_RS24770], type I glutamate-ammonia ligase (GlnA) [J0N78_RS06890], and tRNA threonylcarbamoyladenosine dehydratase [J0N78_RS09630]. The average nucleotide identity (ANI) and dDDH value between genome of strain BA299 and closest type strain, Alkalihalobacillus alkalinitrilicus DSM 22532T was 80.49% and 25.8%, respectively, far below the cut-off points for the novel species (< 95% ANI, < 70% dDDH) (Meier-Kolthoff et al., 2013). The phylogenomic analyses based on whole genome sequences showed strain BA299 was located in the genus Alkalihalobacillus, forming a distinct phyletic lineage from Alkalihalobacillus alkalinitrilicus DSM 22532T indicating a novel line of species in the genus Alkalihalobacillus (Fig. 1).

General genomic features of Alkalihalobacillus sp. BA299

Features Alkalihalobacillus sp. BA299
Genome length (bp) 5,137,563
GC-content (%) 36.7
Contigs 69
Total genes 4,950
Protein-coding genes 4,844
Pseudogenes 106
rRNA genes (5S, 16S, 23S) 34 (12,12,10)a
tRNA genes 119
ncRNA genes 7

a rRNA genes including 10 (5S) complete rRNAs, and 2, 12, 10 (5S, 16S, 23S) partial rRNAs.



Fig. 1. Phylogenomic tree based on genome sequences of the strain BA299 and type species of closely related taxa, created with the TYGS platform (https://tygs.dsmz.de/). Tree inferred from Genome BLAST Distance Phylogeny approach (GBDP) distances calculated from genome sequences. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 28.2%. The tree was rooted at the midpoint. Genome accession numbers are indicated in parenthesis.

In the PGAP analysis, I also found that the strain BA299 contains a gene encoding hydantoin racemase (WP_209123644.1) exclusively unlike other 32 strains of the genus Alkalihalobacillus, which is used in the production of optically pure amino acid as valuable intermediates in the production of various sweeteners, antibiotics, pesticides and pharmaceuticals (Suzuki et al., 2005).

Nucleotide sequence and strain accession numbers

The draft genome sequence of the strain BA299 have been deposited at GenBank under the accession JAFLJM010000000. The strain BA299 has been deposited at Korean Collection for Type Cultures (KCTC 3948).

적 요

Alkalihalobacillus 속에 속하는 신종 균주 BA299를 충남 태안군 안면도 소재 천일염전 토양으로부터 분리하였다. BA299 균주의 전장 유전체는 5,137,563 bp 크기로 69개의 contig로 구성되어 있으며 GC 함량은 36.7%이다. 유전체는 4,844개의 단백질 코딩 유전자로 되어 있으며, 106개의 유사 유전자 및 34개의 리보솜 RNA 유전자, 119개의 운반 RNA (tRNA) 유전자 및 7개의 비 코딩 RNA 유전자(ncRNA) 등 160개의 RNA 유전자를 포함하고 있다. 전장 유전체 서열을 이용하여 계통분석한 결과 BA299 균주는 Alkalihalobacillus alkalinitrilicus DSM 22532T 균주로부터 가장 가까운 계통군을 형성하였으며, 유사도는 80.5%의 ANI값과 25.8%의 dDDH 값을 나타냈다. Alkalihalobacillus 속의 32개 균주의 유전체와 비교한 결과, BA299 균주가 광학적으로 순수한 아미노산을 생산하는 핵심효소인 hydantoin racemase를 유일하게 가지고 있는 것으로 확인되었다.

Acknowledgments

This research is based on the support of 2020 Woosong University Academic Research Funding.

Conflict of Interest

The author declares that there are no conflicts of interest.

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June 2021, 57 (2)