The Bacillus subtilis subsp. subtilis type strain DSM10 has been used as a reference in various studies. However, detailed information about the genome has not been available. Therefore, whole-genome sequencing was performed, and the sequence was compared with that of the related B. subtilis strain NCIB3610.
ABSTRACT
The Bacillus subtilis subsp. subtilis type strain DSM10 has been used as a reference in various studies. However, detailed information about the genome has not been available. Therefore, whole-genome sequencing was performed, and the sequence was compared with that of the related B. subtilis strain NCIB3610.
ANNOUNCEMENT
The Bacillus subtilis subsp. subtilis type strain DSM10 is a generally accessible Bacillus strain from the German Collection of Microorganisms and Cultures GmbH (DSMZ). It has been used as a reference strain for applied biotechnological research (1–4). For instance, the DSM10T strain produces notable amounts of surfactin (2, 5, 6) and secretes proteases (data not shown). Due to its descent from the B. subtilis Marburg strain (7), it is feasible to genetically engineer the DSM10T strain, making it a promising bacterial system for basic research and industrial strain engineering. According to the DSMZ, B. subtilis DSM10T corresponds to B. subtilis strain NCIB3610 (8). Whole-genome sequencing of B. subtilis DSM10T was performed to verify this assumption.
A single colony was inoculated in LB medium and cultivated overnight at 37°C and 120 rpm. Subsequently, chromosomal DNA was extracted with an innuPrep bacterial DNA kit (Analytik Jena, Jena, Germany). Library preparation and whole-genome sequencing were performed by Eurofins Genomics (Ebersberg, Germany). An Illumina HiSeq 4000 system was employed for sequencing, resulting in 2 × 101-bp paired-end read files. The paired-end reads obtained (2 × 35.9 million) were quality analyzed with FastQC v0.11.9 (9). A subset of 2 × 5 million reads, randomly extracted with seqtk v1.3-r106 (10), were used for short-read assembly with SPAdes v3.14.0 (11) with the option “careful.” This resulted in 26 contigs of >0.2 kb with a total size of 4,166,758 bp, an N50 value of 1,014,761 bp, and an N90 value of 240,612 bp. The genomes of the sibling strain B. subtilis NCIB3610 (GenBank accession number CP020102) and its plasmid pBS32 (GenBank accession number CP020103) were used as references to sort and correctly orient the acquired draft genome of B. subtilis DSM10T with the program Mauve v2015-02-13 (http://www.darlinglab.org/mauve). The alignment obtained revealed an almost perfect match of the DSM10T draft genome to the chromosome of NCIB3610. However, contigs resembling pBS32 were not observed, confirming its absence in DSM10T. The DSM10T genome underwent automated gene annotation by the Prokaryotic Genome Annotation Pipeline (PGAP) during uploading to GenBank (12). This process led to the identification of 4,289 genes, of which 4,252 are protein-coding genes and 37 are pseudogenes. Moreover, 42 tRNAs and 5 noncoding RNAs were identified and annotated. The program breseq v0.35.1 (13) was used to identify specific differences by applying DSM10 reads to the NCIB3610 genome. In all, 39 sequence variations could be identified (Table 1). Seventeen single-nucleotide polymorphisms restored a corresponding NCIB3610 pseudogene.
TABLE 1.
Sequence variations between B. subtilis DSM10T and NCIB3610
| Contig accession no. | Nucleotide position | Mutation | Annotationa | Gene | Descriptionb |
|---|---|---|---|---|---|
| JAEPVU010000002.1 | 26036 | C→T | P307L (CCG→CTG) | mfd → | Transcription repair coupling factor |
| JAEPVU010000003.1 | 11953 | −G | Gene-pseudogene | cysE → | Serine O-acetyltransferase |
| JAEPVU010000006.1 | 105978 | (ATGATAGT)1→2 | Intergenic | H9S96_03325 ← / → H9S96_03330 | Catalase/tRNA-Arg |
| JAEPVU010000007.1 | 83281 | (G)7→6 | Gene-pseudogene | yerH → | Hypothetical protein |
| JAEPVU010000007.1 | 85980 | (C)6→5 | Gene-pseudogene | sapB ← | Methyltransferase |
| JAEPVU010000007.1 | 141088 | T→C | V142A (GCG→GTG) | lplB → | Protein lplB |
| JAEPVU010000007.1 | 147278 | (G)6→5 | Intergenic | H9S96_04045 → / → yezD | Sulfate transporter/DUF2292 domain-containing protein |
| JAEPVU010000007.1 | 241945 | (G)6→5 | Gene-pseudogene | acoL → | Dihydrolipoyl dehydrogenase |
| JAEPVU010000009.1 | 94306 | (A)7→6 | Gene-pseudogene | bmrD (yheH) → | Multidrug ABC transporter permease |
| JAEPVU010000009.1 | 234838 | −ATGTAC | Coding | yitS ← | Fatty acid-binding protein DegV |
| JAEPVU010000009.1 | 320518 | (G)6→5 | Gene-pseudogene | manP → | PTS mannose transporter subunit IIABC |
| JAEPVU010000009.1 | 357081 | −G | Gene-pseudogene | uxaB → | Altronate oxidoreductase |
| JAEPVU010000009.1 | 358313 | (G)6→5 | Gene-pseudogene | uxaB → | Altronate oxidoreductase |
| JAEPVU010000009.1 | 375395 | (C)6→5 | Gene-pseudogene | xkdE → | Phage portal protein |
| JAEPVU010000009.1 | 471575 | (TAAT)4→3 | Intergenic | mtnK ← / ← mtnU | S-Methyl-5-thioribose kinase/hydrolase MtnU |
| JAEPVU010000009.1 | 514727 | (T)8→7 | Intergenic | ykwD ← / → pbpH | Hypothetical protein/penicillin-binding protein |
| JAEPVU010000009.1 | 737764 | A→G | I176V (ATC→GTC) | codY → | GTP-sensing pleiotropic transcriptional regulator CodY |
| JAEPVU010000009.1 | 956741 | A→G | S179G (AGT→GGT) | yndE → | Germination protein |
| JAEPVU010000011.1 | 175058 | −C | Coding (131/138 nt) | yotE ← | Hypothetical protein |
| JAEPVU010000011.1 | 222781 | C→A | V91V (GTG→GTT) | yopW ← | Hypothetical protein |
| JAEPVU010000011.1 | 379673 | (C)6→5 | Intergenic | mgsA ← / ← dapB | Methylglyoxal synthase/4-hydroxy-tetrahydrodipicolinate reductase |
| JAEPVU010000011.1 | 396124 | (C)7→6 | Gene-pseudogene | trpD ← | Anthranilate phosphoribosyltransferase |
| JAEPVU010000011.1 | 534030 | (C)7→6 | Gene-pseudogene | mmgA ← | Acetyl-CoA acetyltransferase |
| JAEPVU010000011.1 | 638280 | −C | Gene-pseudogene | yqfA (floA)← | Hypothetical protein |
| JAEPVU010000011.1 | 719953 | (T)8→7 | Gene-pseudogene | yqaD ← | Hypothetical protein |
| JAEPVU010000011.1 | 728398 | (C)8→7 | Intergenic | yrkN → / ← yrkL | N-Acetyltransferase/general stress protein |
| JAEPVU010000011.1 | 749018 | (C)6→5 | Gene-pseudogene | azlD ← | Branched-chain amino acid transporter AzlD |
| JAEPVU010000011.1 | 752943 | (C)6→5 | Intergenic | cypA ← / ← yrdC | Cytochrome P450/cysteine hydrolase |
| JAEPVU010000011.1 | 875960 | A→G | G66G (GGT→GGC) | rplU ← | 50S ribosomal protein L21 |
| JAEPVU010000011.1 | 900227 | (T)7→6 | Coding | engB ← | YihA family ribosome biogenesis GTP-binding protein |
| JAEPVU010000011.1 | 982769 | (C)6→5 | Intergenic | ytxC ← / ← ytxB | Hypothetical protein/TVP38/TMEM64 family membrane protein YtxB |
| JAEPVU010000011.1 | 1079814 | (C)8→7 | Intergenic | ytnP ← / ← trmB | MBL fold metallo-hydrolase/tRNA (guanosine-46-N7)-methyltransferase TrmB |
| JAEPVU010000012.1 | 88 | A→G | Noncoding | H9S96_16680 ← | tRNA-Ala |
| JAEPVU010000013.1 | 61274 | (G)6→5 | Intergenic | malR (yufM) → / → yufN (nupN) | Two-component system response regulator DcuR/BMP family ABC transporter substrate-binding protein |
| JAEPVU010000013.1 | 227225 | (T)6→5 | Gene-pseudogene | yvrE ← | Sugar lactone lactonase YvrE |
| JAEPVU010000013.1 | 317921 | −C | Gene-pseudogene | yvfT ← | Two-component sensor histidine kinase |
| JAEPVU010000013.1 | 401206 | (T)8→7 | Gene-pseudogene | yvzA ← | Hypothetical protein |
| JAEPVU010000013.1 | 414816 | G→A | L256F (CTC→TTC) | lgt ← | Prolipoprotein diacylglyceryl transferase |
| JAEPVU010000014.1 | 21905 | G→T | F118L (TTC→TTA) | rocB ← | Peptidase M20 |
| JAEPVU010000014.1 | 220111 | A→G | L25L (TTG→CTG) | iolG ← | Inositol 2-dehydrogenase |
Variations identified between B. subtilis strain DSM10T and strain NCIB3610. nt, nucleotides.
PTS, phosphotransferase system; CoA, coenzyme A.
In conclusion, B. subtilis DSM10T is genomically very similar to NCIB3610 but is not identical. The absence of pBS32 could explain the ability of DSM10T to develop competence because no plasmid-borne single-pass transmembrane protein ComI is present to downregulate it, as it is for NCIB3610 (14).
Data availability.
The genome sequence of B. subtilis subsp. subtilis strain DSM10T has been deposited in GenBank under the accession number JAEPVU000000000. The raw sequence reads have been submitted to the NCBI Sequence Read Archive (SRA) (15) under the accession number SRR12632401. The BioProject accession number is PRJNA659394, and the BioSample accession number is SAMN15904628.
ACKNOWLEDGMENTS
This project was funded by the German Research Foundation (DFG) (project 398354917). The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.
We thank Eike Grunwaldt and Maliheh Vahidinasab for their technical assistance.
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Associated Data
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Data Availability Statement
The genome sequence of B. subtilis subsp. subtilis strain DSM10T has been deposited in GenBank under the accession number JAEPVU000000000. The raw sequence reads have been submitted to the NCBI Sequence Read Archive (SRA) (15) under the accession number SRR12632401. The BioProject accession number is PRJNA659394, and the BioSample accession number is SAMN15904628.