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. 2026 Jun 29;15(8):e00206-26. doi: 10.1128/mra.00206-26

Whole-genome sequences of Okibacterium fritillariae MBRM-U4 and Bacillus subtilis MBRM-U5, isolated as recurring laboratory contaminants in high-salt media

Maryssa A Beasley 1, Amanda N Scholes 1, Rebecca L Mickol 1,✉
Editor: Frank J Stewart2
PMCID: PMC13460149  PMID: 42370837

ABSTRACT

Here, we report the whole-genome sequences of Okibacterium fritillariae MBRM-U4 and Bacillus subtilis MBRM-U5, gram-positive bacteria isolated as recurring contaminants in marine medium. Genomes were sequenced using Oxford Nanopore Technology, generating a 3.2 Mbp genome for O. fritillariae MBRM-U4 and a 4.4 Mbp genome for B. subtilis MBRM-U5.

KEYWORDS: biotechnology, bioremediation, environmental, biodegradation

ANNOUNCEMENT

While both organisms are representative of soil bacteria, Okibacterium spp. and Bacillus spp. are classified within the phylum Actinomycetota (1) and the phylum Bacillota (2), respectively, indicating distinct evolutionary histories. Okibacterium are gram-positive, aerobic bacteria often associated with plants (e.g., Fritillaria spp., Clematis spp., and Salsola spp.) with only two named species (1, 3, 4). A third isolate, Okibacterium sp., AP_21104-1, has been shown to degrade petroleum hydrocarbons (5). Bacillus spp. are gram-positive, aerobic, spore-forming bacteria commonly isolated from soil (2). Bacillus subtilis, in particular, has been shown to degrade polyethylene (6, 7), polyurethane (8, 9), and other environmental contaminants (10–17), indicating usefulness in bioremediation strategies (18–21).

Here, we present the whole-genome sequences for two novel strains, Okibacterium fritillariae MBRM-U4 and B. subtilis MBRM-U5, isolated as recurring contaminants during cultivation of marine bacteria in high-salt (ca. 18.85 g/L Cl−) media at the U.S. Naval Research Laboratory. Each contaminant was initially identified by its colony morphology (round, yellow [1], O. fritillariae MBRM-U4; white, fluffy [22–25], B. subtilis MBRM-U5) on BB (per L: 18.7 g Difco Marine Broth 2216, 5 g tryptone, 2.5 g yeast extract, and 5 g sodium chloride) (26) agar plates incubated at 30°C. Pure monocultures were obtained by selecting single colonies of each contaminant and re-streaking on BB agar plates twice. Genomic DNA was purified from cultures grown overnight in 5 mL BB medium at 30°C with shaking (180 rpm), using the Wizard Genomic DNA Purification Kit (Promega) with an additional RNase digestion step. Purified DNA was quantified using a double-stranded (ds)DNA High Sensitivity Assay Kit (Life Technologies) and a Qubit 3 fluorometer (Invitrogen).

Genome sequencing was performed by Eurofins Genomics, LLC. Libraries were created using the Oxford Nanopore Technologies (ONT) Rapid Barcoding Kit 96 (part# SQK-RBK114.96) with v14 chemistry followed by amplification-free long-read sequencing on a MinION flow cell (R10.4.1; ONT) with no size selection. Base-calling occurred in real time using Dorado (v0.5.3) (27) within MinKNOW (v22.10.5; ONT). This resulted in 34,782 reads for O. fritillariae MBRM-U4 and 101,051 reads for B. subtilis MBRM-U5. Raw read N50s were calculated using bioconda (28) n50 (v1.9.3) (29). All data are available in Table 1. Assembly for O. fritillariae MBRM-U4 was performed by Eurofins Genomics, LLC using Flye (v2.9.5) (30), which includes error correction and trimming of overlapping ends, with genomes not rotated to a specific base. Only reads with a quality score >7 were considered for assembly, according to the Eurofins Bacterial Genome Sequencing Analysis Pipeline (v1.0). Assembly for B. subtilis MBRM-U5 was performed in KBase (31) using Flye (v2.9.4) (30), after filtering out raw reads smaller than 1,000 bp using KBase (31) wrapper Filtlong (v0.2.1) (32, 33). Default parameters were used for all software unless otherwise specified. Assembly parameters including genome size, GC content, and N50 contig length were assessed using Quast (v4.4) (34). Assembly resulted in one single, circular chromosome with an overall genome length of 3,154,517 bp for O. fritillariae MBRM-U4 and seven linear contigs for B. subtilis MBRM-U5. Genome completeness was determined by CheckM (v1.0.18) (35). The pair-wise average nucleotide identity (ANI) was calculated using FastANI (36) within KBase (31) toolkit GTDB-Tk (V2.3.2) (37). Digital DNA-DNA hybridization (dDDH) scores were determined using the Type (Strain) Genome Server (TYGS) (38, 39). Genome annotation was conducted using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP, v6.10) (40–42), providing the number of protein-coding genes, transfer (t)RNAs, ribosomal (r)RNAs, and non-coding (nc)RNAs.

TABLE 1.

Genome information for O. fritillariae MBRM-U4 and B. subtilis MBRM-U5

O. fritillariae MBRM-U4 B. subtilis MBRM-U5
Topology Circular Linear
Size (bp) 3,154,517 4,396,146 [contig_11: 1,421,733; contig_12: 87,074; contig_13: 10,760; contig_14: 273,894; contig_3: 392,559; contig_4: 1,794,017; contig_9: 416,109]
GC Content (%) 67.75 43.49
Protein-coding genes 2,824 4,150
tRNA number 45 88
rRNA number 6 30
Non-coding RNA regions 3 5
Sequence-cleaned bases (bp) 188,847,982 268,567,779
Coverage Depth 56× 53×
Raw Reads 34,782 101,051
Raw Reads N50 24,302 5,802
Average read length 5,429.47 2,657.74
Completeness (%) 98.99 99.16
Contamination (%) 0.76 1.24
Number of contigs 1 7
Contig N50 3,154,517 1,421,733
ANI comparison organism O. fritillariae VKM Ac-2059 (GCF_900167575.1) B. subtilis subsp. subtilis strain 168 (GCA_000009045.1)
ANI 98.32% 98.52%
dDDHa 95.7% 93.0%
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Digital DNA-DNA hybridization (dDDH) scores were calculated using the Genome BLAST Distance Phylogeny (GBDP) (43) formula, d0 (length of all high-scoring segment pairs divided by the total genome length) (44).

ACKNOWLEDGMENTS

We thank the Office of the Under Secretary of Defense for Research and Engineering (OUSD(R&E)) Tri-Service Biotechnology for a Resilient Supply Chain (T-BRSC) program and the Office of Naval Research for their funding and support of this work.

Contributor Information

Rebecca L. Mickol, Email: rebecca.l.mickol.civ@us.navy.mil.

Frank J. Stewart, Montana State University, Bozeman, Montana, USA

DATA AVAILABILITY

These whole-genome sequences have been deposited in GenBank under accession numbers CP199917 (O. fritillariae MBRM-U4) and JBRATE000000000 (B. subtilis MBRM-U5). BioSample metadata are available in the NCBI BioSample database under accession numbers SAMN51297742 (O. fritillariae MBRM-U4) and SAMN51297842 (B. subtilis MBRM-U5). Raw data have been deposited in the Sequence Read Archive under the accession numbers SRR35368367 (O. fritillariae MBRM-U4) and SRR35368366 (B. subtilis MBRM-U5). All versions are version 1.0 except for the whole-genome sequence for B. subtilis MBRM-U5 (version 2.0)

REFERENCES

  • 1. Evtushenko LI. 2015. Okibacterium. In Bergey’s manual of systematics of archaea and bacteria:1-9 [Google Scholar]
  • 2. Logan NA, Vos PD. 2015. Bacillus. In Bergey’s manual of systematics of archaea and bacteria:1-163 [Google Scholar]
  • 3. Wang H-F, Zhang Y-G, Li L, Liu W-H, Hozzein WN, Chen J-Y, Guo J-W, Zhang Y-M, Li W-J. 2015. Okibacterium endophyticum sp. nov., a novel endophytic actinobacterium isolated from roots of Salsola affinis C. A. Mey. Antonie Van Leeuwenhoek 107:835–843. doi: 10.1007/s10482-014-0376-0 [DOI] [PubMed] [Google Scholar]
  • 4. Evtushenko LI, Dorofeeva LV, Krausova VI, Gavrish EY, Yashina SG, Takeuchi M. 2002. Okibacterium fritillariae gen. nov., sp. nov., a novel genus of the family Microbacteriaceae. Int J Syst Evol Microbiol 52:987–993. doi: 10.1099/00207713-52-3-987 [DOI] [PubMed] [Google Scholar]
  • 5. Bogatyrenko EA, Kim AV, Dunkai TI, Ponomareva AL, Es’kova AI, Sidorenko ML, Okulov AK. 2021. Taxonomic diversity of culturable hydrocarbon-oxidizing bacteria in the sea of Japan. Russ J Mar Biol 47:232–239. doi: 10.1134/S1063074021030032 [DOI] [Google Scholar]
  • 6. Vimala PP, Mathew L. 2016. Biodegradation of polyethylene using Bacillus subtilis. Procedia Technology 24:232–239. doi: 10.1016/j.protcy.2016.05.031 [DOI] [Google Scholar]
  • 7. Yao Z, Seong HJ, Jang Y-S. 2022. Degradation of low density polyethylene by Bacillus species. Appl Biol Chem 65:84. doi: 10.1186/s13765-022-00753-3 [DOI] [Google Scholar]
  • 8. Shah Z, Krumholz L, Aktas DF, Hasan F, Khattak M, Shah AA. 2013. Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75. Biodegradation 24:865–877. doi: 10.1007/s10532-013-9634-5 [DOI] [PubMed] [Google Scholar]
  • 9. Rowe L, Howard GT. 2002. Growth of Bacillus subtilis on polyurethane and the purification and characterization of a polyurethanase-lipase enzyme. International Biodeterioration & Biodegradation 50:33–40. doi: 10.1016/S0964-8305(02)00047-1 [DOI] [Google Scholar]
  • 10. Zissi U, Lyberatos G, Pavlou S. 1997. Biodegradation of p -aminoazobenzene by Bacillus subtilis under aerobic conditions. Journal of Industrial Microbiology and Biotechnology 19:49–55. doi: 10.1038/sj.jim.2900418 [DOI] [Google Scholar]
  • 11. Quan CS, Liu Q, Tian WJ, Kikuchi J, Fan SD. 2005. Biodegradation of an endocrine-disrupting chemical, di-2-ethylhexyl phthalate, by Bacillus subtilis No. 66. Appl Microbiol Biotechnol 66:702–710. doi: 10.1007/s00253-004-1683-6 [DOI] [PubMed] [Google Scholar]
  • 12. Vidhya R, Thatheyus A. 2013. Biodegradation of dimethylformamide using Bacillus subtilis. Am J Microbiol Res 1:10–15. doi: 10.12691/ajmr-1-1-3 [DOI] [Google Scholar]
  • 13. Darsa KV, Thatheyus AJ, Ramya D. 2014. Biodegradation of petroleum compound using the bacterium Bacillus subtilis. Science International 2:20–25. doi: 10.17311/sciintl.2014.20.25 [DOI] [Google Scholar]
  • 14. Montagnolli RN, Lopes PRM, Bidoia ED. 2015. Assessing Bacillus subtilis biosurfactant effects on the biodegradation of petroleum products. Environ Monit Assess 187:4116. doi: 10.1007/s10661-014-4116-8 [DOI] [PubMed] [Google Scholar]
  • 15. Wang D, Lin J, Lin J, Wang W, Li S. 2019. Biodegradation of petroleum hydrocarbons by Bacillus subtilis BL-27, a strain with weak hydrophobicity. Molecules 24:3021. doi: 10.3390/molecules24173021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Haq I, Kalamdhad AS, Malik A. 2024. Bioremediation of petroleum refinery wastewater using Bacillus subtilis IH-1 and assessment of its toxicity. Arch Microbiol 206:296. doi: 10.1007/s00203-024-04027-y [DOI] [PubMed] [Google Scholar]
  • 17. Ghorbannezhad H, Moghimi H, Dastgheib SMM. 2022. Biodegradation of high molecular weight hydrocarbons under saline condition by halotolerant Bacillus subtilis and its mixed cultures with Pseudomonas species. Sci Rep 12:13227. doi: 10.1038/s41598-022-17001-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Xiao Y, Chen S, Gao Y, Hu W, Hu M, Zhong G. 2015. Isolation of a novel beta-cypermethrin degrading strain Bacillus subtilis BSF01 and its biodegradation pathway. Appl Microbiol Biotechnol 99:2849–2859. doi: 10.1007/s00253-014-6164-y [DOI] [PubMed] [Google Scholar]
  • 19. Ganesh Kumar A, Manisha D, Nivedha Rajan N, Sujitha K, Magesh Peter D, Kirubagaran R, Dharani G. 2023. Biodegradation of phenanthrene by piezotolerant Bacillus subtilis EB1 and genomic insights for bioremediation. Mar Pollut Bull 194:115151. doi: 10.1016/j.marpolbul.2023.115151 [DOI] [PubMed] [Google Scholar]
  • 20. Wang X, Liu S, Ding X, Zhang L, Lv X, Li J, Song C, Zhang C, Wang S. 2025. Coexistence of diverse metabolic pathways promotes p-cresol biodegradation by Bacillus subtilis ZW. Int Biodeterior Biodegradation 196:105933. doi: 10.2139/ssrn.4920853 [DOI] [Google Scholar]
  • 21. Oyetibo GO, Chien M-F, Ikeda-Ohtsubo W, Suzuki H, Obayori OS, Adebusoye SA, Ilori MO, Amund OO, Endo G. 2017. Biodegradation of crude oil and phenanthrene by heavy metal resistant Bacillus subtilis isolated from a multi-polluted industrial wastewater creek. Int Biodeterior Biodegradation 120:143–151. doi: 10.1016/j.ibiod.2017.02.021 [DOI] [Google Scholar]
  • 22. Fujikawa H. 1992. Periodic growth of Bacillus subtilis colonies on agar plates. Physica A: Statistical Mechanics and its Applications 189:15–21. doi: 10.1016/0378-4371(92)90123-8 [DOI] [Google Scholar]
  • 23.Fujikawa H1994. Diversity of the growth patterns of Bacillus subtilis colonies on agar plates. FEMS Microbiol Ecol 13:159–168. doi: 10.1111/j.1574-6941.1994.tb00062.x [DOI] [Google Scholar]
  • 24. Shapiro JA. 1995. The significances of bacterial colony patterns. Bioessays 17:597–607. doi: 10.1002/bies.950170706 [DOI] [PubMed] [Google Scholar]
  • 25. Aguilar C, Vlamakis H, Losick R, Kolter R. 2007. Thinking about Bacillus subtilis as a multicellular organism. Curr Opin Microbiol 10:638–643. doi: 10.1016/j.mib.2007.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Bird LJ, Wang Z, Malanoski AP, Onderko EL, Johnson BJ, Moore MH, Phillips DA, Chu BJ, Doyle JF, Eddie BJ, Glaven SM. 2018. Development of a genetic system for Marinobacter atlanticus CP1 (sp. nov.), a wax ester producing strain isolated from an autotrophic biocathode. Front Microbiol 9:3176. doi: 10.3389/fmicb.2018.03176 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Nanopore O. 2025. Oxford nanopore’s basecaller. GitHub. https://github.com/nanoporetech/dorado. [Google Scholar]
  • 28. Grüning B, Dale R, Sjödin A, Chapman BA, Rowe J, Tomkins-Tinch CH, Valieris R, Köster J, Bioconda Team . 2018. Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods 15:475–476. doi: 10.1038/s41592-018-0046-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Telatin A, Fariselli P, Birolo G. 2021. SeqFu: a suite of utilities for the robust and reproducible manipulation of sequence files. Bioengineering (Basel) 8:59. doi: 10.3390/bioengineering8050059 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. 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 [DOI] [PubMed] [Google Scholar]
  • 31. Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, Dehal P, Ware D, Perez F, Canon S, et al. 2018. KBase: the United States department of energy systems biology knowledgebase. Nat Biotechnol 36:566–569. doi: 10.1038/nbt.4163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Wick R. 2021. Filtlong: quality filtering tool for long reads. GitHub. https://github.com/rrwick/Filtlong. [Google Scholar]
  • 33. Chandonia J-M. 2024. App to wrap filtlong in kbase. GitHub. https://github.com/kbaseapps/kb_filtlong. [Google Scholar]
  • 34. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055. doi: 10.1101/gr.186072.114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114. doi: 10.1038/s41467-018-07641-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. 2022. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics 38:5315–5316. doi: 10.1093/bioinformatics/btac672 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Meier-Kolthoff JP, Göker M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182. doi: 10.1038/s41467-019-10210-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Meier-Kolthoff JP, Carbasse JS, Peinado-Olarte RL, Göker M. 2022. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res 50:D801–D807. doi: 10.1093/nar/gkab902 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O’Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res 46:D851–D860. doi: 10.1093/nar/gkx1068 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Li W, O’Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ, Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res 49:D1020–D1028. doi: 10.1093/nar/gkaa1105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. 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 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Henz SR, Huson DH, Auch AF, Nieselt-Struwe K, Schuster SC. 2005. Whole-genome prokaryotic phylogeny. Bioinformatics 21:2329–2335. doi: 10.1093/bioinformatics/bth324 [DOI] [PubMed] [Google Scholar]
  • 44. Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60. doi: 10.1186/1471-2105-14-60 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

These whole-genome sequences have been deposited in GenBank under accession numbers CP199917 (O. fritillariae MBRM-U4) and JBRATE000000000 (B. subtilis MBRM-U5). BioSample metadata are available in the NCBI BioSample database under accession numbers SAMN51297742 (O. fritillariae MBRM-U4) and SAMN51297842 (B. subtilis MBRM-U5). Raw data have been deposited in the Sequence Read Archive under the accession numbers SRR35368367 (O. fritillariae MBRM-U4) and SRR35368366 (B. subtilis MBRM-U5). All versions are version 1.0 except for the whole-genome sequence for B. subtilis MBRM-U5 (version 2.0)

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