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Microbiol Resour Announc
Microbiol Resour Announc
mra
Microbiology Resource Announcements
2576-098X
American Society for Microbiology 1752 N St., N.W., Washington, DC

39162442
mra00573-24
10.1128/mra.00573-24
mra.00573-24
Genome Sequences
bacteriologyBacteriologyComplete genome sequences of Geobacillus stearothermophilus strains EF60045 and SJEF4-2 from Korean hot springs
https://orcid.org/0000-0002-2475-8743
Sung Jae-Yoon 1
https://orcid.org/0000-0003-4244-5465
Ganbat Dariimaa 2
https://orcid.org/0000-0003-4278-9313
Kim Seong Bo 3
https://orcid.org/0000-0002-9516-3165
Lee Sang-Jae 2 sans76@silla.ac.kr

https://orcid.org/0000-0002-2272-8321
Lee Dong-Woo 1 leehicam@yonsei.ac.kr

1 Department of Biotechnology, Yonsei University , Seoul, South Korea
2 Department of Bioscience and Research Center for Extremophiles and Marine Microbiology, Silla University , Busan, South Korea
3 Bio-Living Engineering Major, Global Leaders College, Yonsei University , Seoul, South Korea
Editor Maresca Julia A. SUNY College of Environmental Science and Forestry , Syracuse, New York, USA

Address correspondence to Sang-Jae Lee, sans76@silla.ac.kr
Address correspondence to Dong-Woo Lee, leehicam@yonsei.ac.kr
Jae-Yoon Sung and Dariimaa Ganbat contributed equally to this article. Author order was determined by drawing straws.

The authors declare no conflict of interest.

9 2024
20 8 2024
20 8 2024
13 9 e00573-2429 5 2024
25 7 2024
Copyright © 2024 Sung et al.
2024
Sung et al.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

We report the complete genomes of Geobacillus stearothermophilus strains EF60045 and SJEF4-2 from Korean hot springs, with 3,769 and 3,625 thermophilic genes, respectively. G. stearothermophilus EF60045 shows four methylation patterns. G. stearothermophilus SJEF4-2 harbors three plasmids. These findings enhance understanding of Geobacillus strains, aiding in their development as microbial platform hosts.

KEYWORDS

Geobacillus stearothermophilus
thermophile
genome
plasmid
hot springs
cover-dateSeptember 2024
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pmcANNOUNCEMENT

Geobacillus spp., sourced from Korean hot springs (1), are recognized for their potent enzyme activity and thermostability, making them ideal for industrial applications (2–5). We sequenced the genomes of Geobacillus stearothermophilus strains EF60045 and SJEF4-2, isolated from Neungam (37°05'36.5"N 127°48'06.6"E) and Deokgu (37°04'43.8"N 129°16'59.9"E) hot springs. Samples were cultured on marine agar plates (Difco 2216) at 60°C for 5 days to assess their genomic capabilities and biotechnological potential (6).

The Geobacillus strains were aerobically cultured in modified Luria-Bertani (LB) medium at 60°C overnight (7). Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). For EF60045, DNA was sheared using a g-TUBE (Covaris Inc.) and purified for fragments under 17 kb using AMPurePB magnetic beads (Beckman Coulter Inc.). Sequencing libraries were prepared using the PacBio DNA Template Prep Kit 1.0. SMRTbell (Pacific Biosciences) and sequenced on the PacBio Sequel II platform with Sequel Sequencing Kit 3.0, yielding 78,931 raw reads (651,140,287 bases), which produced a circular consensus sequence averaging 8,249 bp (N50 length, 10,526 bp) and compiled a 3,650,785 bp chromosome with 178.0× coverage (Table 1). Further quality enhancement for EF60045 involved aligning short reads generated by the TruSeq Nano DNA Sample Prep Kit (Illumina) using the same genomic DNA. Short-read paired-end libraries (2 × 150 bp) were sequenced on an NovaSeq 6000 instrument (Illumina), yielding 14,759,206 reads. Adapter trimming and quality control were performed using Trimmomatic v0.39 (8) and FastQC v0.11.9 (9), with variant calling via bcftools (“--minDP 5—minQ 30”) (10). Finally, the bcftools consensus function was applied against the pre-assembled contig of Geobacillus.

TABLE 1 Genomic features of G. stearothermophilus strains EF60045 and SJEF4-2

Feature	EF60045	SJEF4-2	
Genome size (bp)	3,650,785	3,471,328	
No. of contigs	1	1	
GC content (%)	52.0	52.5	
Total number of genes	3,769	3,625	
Protein coding genes (CDS)	3,459	3,310	
rRNA genes (5S, 16S, and 23S)	29 (9, 10, and 10)	29 (9, 10, and 10)	
tRNA genes	89	88	
ncRNA	5	5	
Pseudogenes	190	220	
CRISPR arrays	5	5	
GenBank Accession	CP1298453	CP128449	
a ncRNA, Non-coding RNA.

SJEF4-2 was sequenced using the Oxford Nanopore MinION platform with the Ligation Sequencing kit SQK-LSK112.24 (Oxford Nanopore Technologies) without DNA shearing or size selection. The sequencing library loaded into the FLO-MIN112 flow cell (R10.4, Oxford Nanopore Technologies) yielded 309,178 reads totaling 1,999,383,409 bp, with a read length N50 of 14,234 bp. Adapter trimming and quality control were conducted using Trimmomatic v0.3.2 (8), Nanostat v1.4.0 (11), and FastQC v0.12.0 (9). Flye v2.8.2 (12) with the iteration parameter set to five assembled four circular contigs, with error correction achieved using 25,729,549 Illumina reads for a 519× coverage of the 3,471,328 bp chromosome (Table 1). Additionally, three plasmids—pSJEF4-2-1 (55,393 bp with 727× coverage), pSJEF4-2-2 (13,067 bp with 472× coverage), and pSJEF4-2-3 (5,886 bp with 266× coverage)—were identified. The dnaA gene was relocated to the 0 position of the circular genome using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) to standardize the starting point. The coding sequences of both strains were predicted by the PGAP v6.5 with default parameters (13).

Both strains exhibited larger genomes than the 2.8 Mb genome of ATCC 12980T (GCA_030369615.1), suggesting enhanced carbohydrate-degrading capabilities potentially acquired through horizontal gene transfer (14). Key genes like mtl, abf, and xyn, important for carbohydrate degradation (15, 16), alongside metabolic pathway genes such as pgi, gntK, and acsA, highlight their adaptability and industrial potential (17). This analysis provides a solid foundation for future genetic engineering and utilization of these strains as thermophilic platform hosts (17).

ACKNOWLEDGMENTS

This work was partly supported by the National Research Foundation (NRF) of Korea through grant (2023R1A2C100758711 to DWL) and the Bio and Medical Technology Development Program grant (2021M3A9I4021431 to DWL), funded by the Ministry of Science and ICT (MSIT), Republic of Korea. Additional fundings were provided by the Technology Innovation Program (grant number 20015807 to SBK and SJL), supported by the Ministry of Trade, Industry and Energy (MOTIE, Korea) and the high seas bioresources programme of Korea Insitute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (grant number KIMST-20210646). Sequencing with the Oxford Nanopore MinION platform was conducted at the next-generation sequencing (NGS) core facility, Kyungpook National University, South Korea.

DATA AVAILABILITY

The whole-genome sequences of G. stearothermophilus strains EF60045 and SJEF4-2 have been deposited in the NCBI database under GenBank (BioProject: PRJNA983507) accession numbers CP128453 and CP128449, respectively. Raw sequencing data are available under SRA accession numbers SRR29080532 and SRR29120474 for BioSample SAMN35768001 and SRR29081530 and SRR29081531 for SAMN35766928. Three plasmids from SJEF4-2 are listed under CP128450, CP128451, and CP128452. Methylation motifs for EF60045 are available under SUPPF_0000005608.
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REFERENCES

1 Lee YJ, Ganbat D, Oh D, Kim H, Jeong GE, Cha IT, Kim SB, Nam G, Jung YJ, Lee SJ. 2022. Isolation and characterization of thermophilic bacteria from hot springs in Republic of Korea. Microorganisms 10 :2375. doi:10.3390/microorganisms10122375 36557626
2 Feng L, Wang W, Cheng J, Ren Y, Zhao G, Gao C, Tang Y, Liu X, Han W, Peng X, Liu R, Wang L. 2007. Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc Natl Acad Sci U S A 104 :5602–5607. doi:10.1073/pnas.0609650104 17372208
3 Abd Rahman RNZR, Leow TC, Salleh AB, Basri M. 2007. Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia. BMC Microbiol 7 :77. doi:10.1186/1471-2180-7-77 17692114
4 Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, Lysenko AM, Petrunyaka VV, Osipov GA, Belyaev SS, Ivanov MV. 2001. Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th. Int J Syst Evol Microbiol 51 :433–446. doi:10.1099/00207713-51-2-433 11321089
5 Lee D, Koh Y, Kim K, Kim B, Choi H, Kim D, Suhartono MT, Pyun Y. 1999. Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol Lett 179 :393–400. doi:10.1111/j.1574-6968.1999.tb08754.x 10518742
6 McMullan G, Christie JM, Rahman TJ, Banat IM, Ternan NG, Marchant R. 2004. Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus. Biochem Soc Trans 32 :214–217. doi:10.1042/bst0320214 15046574
7 Zeigler D. 2001. The genus Geobacillus - introduction and strain catalog. In Bacillus genetic stock center. Vol. 3 .
8 Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 :2114–2120. doi:10.1093/bioinformatics/btu170 24695404
9 Simon A. 2010. FastQC: a quality control tool for high throughput sequence data. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc
10 Li H. 2011. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27 :2987–2993. doi:10.1093/bioinformatics/btr509 21903627
11 De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34 :2666–2669. doi:10.1093/bioinformatics/bty149 29547981
12 Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37 :540–546. doi:10.1038/s41587-019-0072-8 30936562
13 Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44 :6614–6624. doi:10.1093/nar/gkw569 27342282
14 Burgess SA, Flint SH, Lindsay D, Cox MP, Biggs PJ. 2017. Insights into the Geobacillus stearothermophilus species based on phylogenomic principles. BMC Microbiol 17 :140. doi:10.1186/s12866-017-1047-x 28651524
15 Shulami S, Shenker O, Langut Y, Lavid N, Gat O, Zaide G, Zehavi A, Sonenshein AL, Shoham Y. 2014. Multiple regulatory mechanisms control the expression of the Geobacillus stearothermophilus gene for extracellular xylanase. J Biol Chem 289 :25957–25975. doi:10.1074/jbc.M114.592873 25070894
16 Shulami S, Zehavi A, Belakhov V, Salama R, Lansky S, Baasov T, Shoham G, Shoham Y. 2020. Cross-utilization of β-galactosides and cellobiose in Geobacillus stearothermophilus. J Biol Chem 295 :10766–10780. doi:10.1074/jbc.RA120.014029 32493770
17 Sarmiento F, Peralta R, Blamey JM. 2015. Cold and hot extremozymes: industrial relevance and current trends. Front Bioeng Biotechnol 3 :148. doi:10.3389/fbioe.2015.00148 26539430
