Open access
Environmental Microbiology
Announcement
7 March 2022

Complete Genome Sequence of Bacillus cereus Strain HT18, Isolated from Forest Soil

ABSTRACT

The genome sequence of Bacillus cereus strain HT18, isolated from forest soil, was 5,333,415 bp long. The genome included 5,825 putative coding sequences and 35.2% GC content; the strain had 5 plasmids. Average nucleotide identity based on BLAST+ (ANIb) and digital DNA-DNA hybridization (dDDH) results showed that HT18 was 98.78% and 90.70% homologous, respectively, to B. cereus ATCC 14579T.

ANNOUNCEMENT

The Bacillus cereus group (phylum Firmicutes) comprises Gram-positive, spore-forming, facultative, anaerobic, rod-shaped bacteria with low-GC-content genomes (1). It includes eight closely related species with high genomic homology and 16S rRNA gene sequence similarity—B. anthracis, B. cereus, B. cytotoxicus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. toyonensis, and B. weihenstephanensis (2). Phenotypic features, such as motility and hemolysis, used to classify species within this group can differ within and among species, leading to the use of the average nucleotide identity based on BLAST+ (ANIb) and digital DNA-DNA hybridization (dDDH) as classification indices (3, 4).
Strain HT18 was isolated from forest soil in Hashimoto, Wakayama, Japan. The soil samples were flooded, filtered, and incubated overnight at 37°C on LB agar (Nacalai Tesque). After colony isolation, the cells were cultured in LB broth at 37°C for 24 h. Then, genomic DNA was isolated using Marmur’s method (5).
Sequencing was a combination of short and long reads. Short-read sequencing libraries were constructed using the NEBNext Ultra II FS DNA library prep kit (New England Biolabs [NEB]) and decoded on a MiSeq instrument (Illumina). A total of 1,488,012 reads with 431,000,004 bases were decoded with an average insert size of 641 bp and spot length of 602 bp with 2 × 300-bp paired ends. Low-quality bases (Q scores of <15) were trimmed, and short reads (<25 bp) were removed using Platanus trim version 1.0.7 (http://platanus.bio.titech.ac.jp/pltanus_trim) (6). Long-read sequence libraries were constructed using the rapid barcoding kit (SQK-RBK004), and sequencing was performed on a MinION device (Oxford Nanopore Technology [ONT]) using an R9.4.1 flow cell. No size selection was performed before library preparation. A total of 48,894 reads with 455,539,372 bases were decoded using Guppy version 4.0.15. The recovered data with NanoFilter technology (quality, ≥10; length, ≥2 kb; head crop, 100 bp) revealed 23,033 ONT reads with a mean read length of 17,050 bp and an N50 value of 27,780 bp. De novo assembly was performed using Unicycler version 0.4.8 (https://github.com/rrwick/Unicycler) (7). The genome annotation and rotation of the chromosome to bring dnaA first was performed with DFAST version 1.4.0 (https://dfast.ddbj.nig.ac.jp) (8). ANIb and dDDH values were calculated from the JSpeciesWS online service version 3.8.5 (http://jspecies.ribohost.com/jspeciesws/) and the Genome-to-Genome Distance Calculator version 3.0 (http://ggdc.dsmz.de/ggdc.php) (9) using the reported genome sequences of type strains of the B. cereus group. Default parameters were used for all software, unless specified.
One contig of the assembled genome sequence was 5,333,415 bp long (35.2% GC content) with a sequence depth of approximately 80. A total of 5,825 coding regions, 107 tRNAs, 42 rRNAs, and 3 CRISPR regions were annotated. Five plasmid DNA sequences were also assembled (Table 1).
TABLE 1
TABLE 1 Characteristics of the genome of Bacillus cereus strain HT18
Genetic elementAssembly size (bp)G+C content (%)No. of CDSsaNo. of rRNAsNo. of tRNAsAvg read depth (×)Accession no.
Chromosome5,333,41535.25,4884210775.8AP024504
Plasmids      
 pHT1358,13933.435500130.4AP024505
 pHT29,51831.61200351.7AP024506
 pHT39,19031.41000537.4AP024507
 pHT47,21532.1700687.5AP024508
 pHT53,16633.1300476.0AP024509
a
CDS, coding DNA sequence.
Based on the ANIb (%) and dDDH (%) results, strain HT18 was 99.9% and 100% homologous to both B. cereus strains WPySW2 and AFA01, and 99.9% and 99.9% homologous to the foodborne pathogen FORC021 (10) (Table S1, posted at https://figshare.com/articles/dataset/suppl_Table_pdf/17111096). Although strain HT18 showed high homology to B. thuringiensis serovar Berliner ATCC 10792T, it was closely related to B. cereus ATCC 14579T when it was compared to reference strains of the B. cereus group. Therefore, strain HT18 was identified as a B. cereus strain.

Data availability.

The genome sequence and annotation data for strain HT18 were deposited in DDBJ/GenBank under BioProject number PRJDB11181, BioSample number SAMD00278679, DRA number DRA011610, accession numbers AP024504 to AP024509, and SRA numbers DRX266213 (Illumina) and DRX266214 (ONT).

ACKNOWLEDGMENTS

A part of this work was supported by JSPS KAKENHI grant numbers JP20H02903 and 16H06279 (PAGS).
We thank Editage for English language editing.

REFERENCES

1.
Whitman W. 2015. Bergey’s manual of determinative bacteriology, Wiley, Hoboken, NJ.
2.
Ehling-Schulz M, Lereclus D, Koehler TM. 2019. The Bacillus cereus group: Bacillus species with pathogenic potential. Microbiol Spectr 7:7.3.6.
3.
Liu Y, Du J, Lai Q, Zeng R, Ye D, Xu J, Shao Z. 2017. Proposal of nine novel species of the Bacillus cereus group. Int J Syst Evol Microbiol 67:2499–2508.
4.
Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131.
5.
Marmur J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218.
6.
Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T. 2014. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res 24:1384–1395.
7.
Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595.
8.
Tanizawa Y, Fujisawa T, Nakamura Y. 2018. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics 34:1037–1039.
9.
Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60.
10.
Chung HY, Lee KH, Ryu S, Yoon H, Lee JH, Kim HB, Kim H, Jeong HG, Choi SH, Kim BS. 2016. Genome sequence of Bacillus cereus FORC_021, a food-borne pathogen isolated from a knife at a sashimi restaurant. J Microbiol Biotechnol 26:2030–2035.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 11Number 317 March 2022
eLocator: e01106-21
Editor: Steven R. Gill, University of Rochester School of Medicine and Dentistry
PubMed: 35254113

History

Received: 8 December 2021
Accepted: 5 February 2022
Published online: 7 March 2022

Contributors

Authors

Minami Matsunaka
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Nguyen Cong Thanh
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Plant Protection Research Institute, Hanoi, Vietnam
Tatsuya Uedoi
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Takashi Iida
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Osaka, Japan
Yasuhiro Fujino
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Taketo Ohmori
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Osaka, Japan
Yasuaki Hiromasa
Attached Promotive Center for International Education and Research of Agriculture, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Toshihisa Ohshima
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Osaka, Japan
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

Editor

Steven R. Gill
Editor
University of Rochester School of Medicine and Dentistry

Notes

The authors declare no conflict of interest.

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