ABSTRACT
Knowledge about bacterial species in bee environments is essential for maintaining healthy honeybee colonies. Therefore, we performed whole-genome sequence analysis on bacteria isolated from honey harvested in Japan. This study reports the genomic sequences of the five bacterial strains identified.
ANNOUNCEMENT
Although honeybees are essential livestock for pollination and providing honey, research on infectious diseases affecting honeybees is not comprehensive. Previously, we isolated bacteria from honey in Japan (1) and performed whole-genome shotgun sequencing to investigate honeybee pathogens. However, obtaining complete genomic sequences is key to comprehensively understanding the toxicity and genetic profiles of the bacteria. In this study, we performed complete genomic analysis of five representative strains. Here, we report the complete genomic data for Bacillus paralicheniformis and Paenibacillus spp. found in honey.
Bacteria isolated from honey and stored as glycerol stocks were subsequently recovered and their genomic DNA purified (1). Bacteria were cultured on Columbia agar with 5% defibrinated sheep blood, harvested, and suspended in 550 μL Tris-EDTA buffer. The cells were treated with approximately 10 mg/mL lysozyme and 50 U/mL mutanolysin (Sigma-Aldrich, USA) at 37°C and lysed with 1% sodium dodecyl-sulfate. The lysates were mixed with once with phenol, thrice with phenol-chloroform-isoamyl alcohol (PCI), and once with chloroform to extract DNA and remove the proteins. After ethanol precipitation, 100 μg/mL RNase (Nippon Gene, Japan) treatment, PCI and chloroform extraction, ethanol precipitation, and a 70% ethanol rinse, the purified DNA was suspended in 10 mM Tris-HCl (pH 8.5) and stored at −20°C.
Whole-genome sequencing was performed using the PacBio RS II platform combined with a SMRT cell 8Pac ver. 3 and a DNA polymerase binding kit P6 (Pacific Biosciences, USA). For Illumina sequencing, the NovaSeq 6000 (Illumina, USA) system was used with 150-bp paired-end read settings. Samples were prepared using the TruSeq DNA PCR free kit (Illumina, USA) per the manufacturer’s instructions. Read quality control, raw read filtering, and de novo sequence assembly were performed using HGAP ver. 3 software (Pacific Biosciences) (2) and polished using Quiver (2) with default settings. The assembly accuracy was validated by mapping Illumina reads obtained in a previous study (1) using Pilon ver. 1.21 (3) with default settings, with 100.00% coverage (except for Paenibacillus azoreducens J34TS1; 99.97%). After assembly, a self-dot plot was created using UGENE ver. 35.0 (http://ugene.net) to verify the plasmid and chromosome circularization by checking the contig end overlap and the 5′ and 3′ end connections. Analysis was performed with BUSCO ver. 3.0 software (4), using the bacteria_odb9 database to validate the completeness of genome assembly; for all genomes, quality scores of >98.7% were obtained. The general features of each genome are listed in Table 1.
TABLE 1.
General features of the bacterial genomes presented in this studya
| Species | Isolate | Total no. of subreads | N50 (bp) | Total no. of subread bases | Size (bp) | GC content (%) | Coverage (×) | BioSample accession no. | SRA accession no. |
|---|---|---|---|---|---|---|---|---|---|
| Bacillus paralicheniformis | J25TS1 | 78,163 (P), 37,219,662 (I) | 16,453 | 708,855,100 (P), 5,620,168,962 (I) | 4,394,709 | 45.9 | 129 (P), 1,152 (I) | SAMD00425495 | DRX320385 (P), DRX321129 (I) |
| J36TS2 | 74,813 (P), 22,269,482 (I) | 15,585 | 651,721,606 (P), 3,362,691,782 (I) | 4,404,119 | 45.9 | 116 (P), 678 (I) | SAMD00425496 | DRX320386 (P), DRX321130 (I) | |
| J36TS2 plasmidb | 200,793 | 36.5 | 159 (P) | ||||||
| J41TS8 | 90,978 (P), 22,392,292 (I) | 15,975 | 914,167,224 (P), 3,381,236,092 (I) | 4,501,066 | 45.8 | 171 (P), 699 (I) | SAMD00425497 | DRX320387 (P), DRX321131 (I) | |
| Paenibacillus azoreducens | J34TS1 | 96,372 (P), 36,125,506 (I) | 13,745 | 840,311,025 (P), 5,454,951,406 (I) | 7,316,152 | 48.0 | 96 (P), 676 (I) | SAMD00425498 | DRX320388 (P), DRX321132 (I) |
| Paenibacillus dendritiformis | J27TS7 | 130,145 (P), 32,933,880 (I) | 15,307 | 1,359,398,548 (P), 4,973,015,880 (I) | 6,547,390 | 54.7 | 124 (P), 690 (I) | SAMD00425499 | DRX320389 (P), DRX321133 (I) |
The total number of subreads and subread bases and the coverage depth values were obtained by PacBio (P) and Illumina (I) sequencing. The N50, size, and GC content values were obtained by PacBio sequencing.
B. paralicheniformis J36TS2 has a single plasmid, pBP1.
Few genomes of P. azoreducens and Paenibacillus dendritiformis have been registered. Although NCBI contains genomic information on approximately 100 B. paralicheniformis strains, complete genome sequences are absent for honey-derived strains. The data obtained in this study are valuable for understanding bacteria in bee environments to promote future research.
Data availability.
The whole-genome sequences have been deposited at DDBJ/GenBank under BioProject accession number PRJDB12645 and BioSample accession numbers SAMD00425495 to SAMD00425499 (Table 1). The raw sequence reads have been deposited in the DDBJ DRA/NCBI SRA, and the accession numbers are listed in Table 1.
ACKNOWLEDGMENTS
This study was supported by a Grant-in-Aid for Scientific Research (A) (JP21H04757) from the Japan Society for Promotion of Science. We declare that we have no conflicts of interest.
All authors meet the MRA authorship criteria. K.O., M.O., and D.T. were responsible for the organization and coordination of the analysis. D.T. was the chief investigator and was responsible for the data analysis. All authors contributed to the writing of the final manuscript.
Contributor Information
Daisuke Takamatsu, Email: p1013dt@affrc.go.jp.
Steven R. Gill, University of Rochester School of Medicine and Dentistry
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The whole-genome sequences have been deposited at DDBJ/GenBank under BioProject accession number PRJDB12645 and BioSample accession numbers SAMD00425495 to SAMD00425499 (Table 1). The raw sequence reads have been deposited in the DDBJ DRA/NCBI SRA, and the accession numbers are listed in Table 1.