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Prokaryotes

Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6

Daniel R. Reuß, Andrea Thürmer, Rolf Daniel, Wim J. Quax, Jörg Stülke
Daniel R. Reuß
aDepartment of General Microbiology, Georg-August-University Göttingen, Göttingen, Germany
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Andrea Thürmer
bDepartment of Genomics and Applied Microbiology and Göttingen Genomics Laboratory, Georg-August-University Göttingen, Göttingen, Germany
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Rolf Daniel
bDepartment of Genomics and Applied Microbiology and Göttingen Genomics Laboratory, Georg-August-University Göttingen, Göttingen, Germany
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Wim J. Quax
cGroningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands
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Jörg Stülke
aDepartment of General Microbiology, Georg-August-University Göttingen, Göttingen, Germany
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DOI: 10.1128/genomeA.00759-16
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ABSTRACT

Bacillus subtilis ∆6 is a genome-reduced strain that was cured from six prophages and AT-rich islands. This strain is of great interest for biotechnological applications. Here, we announce the full-genome sequence of this strain. Interestingly, the conjugative element ICEBs1 has most likely undergone self-excision in B. subtilis ∆6.

GENOME ANNOUNCEMENT

Bacillus subtilis ∆6 is a derivative of the laboratory wild-type strain B. subtilis 168, which was cured from six prophages and AT-rich islands. For this purpose, the prophages SPβ and PBSX, the prophage-like elements prophage 1, prophage 3, and skin, as well as the polyketide synthesis operon pks were deleted. Interestingly, this genome reduction by 7.7% did not have a major impact on physiology, metabolic flux patterns, or genetic competence (1).

B. subtilis ∆6 is a promising starting point for further genome reduction. Moreover, it can serve as a chassis strain in the context of biotechnological applications, that is, highly efficient protein secretion and vitamin production (2–4). Indeed, B. subtilis ∆6 has recently been used to obtain a total genome reduction of 13.6% (5). For a better understanding with respect to future projects, we have sequenced the genome of B. subtilis ∆6. The chromosomal DNA was isolated from a stationary phase culture using a commercially available kit (peqGOLD Bacterial DNA Kit, VWR International GmbH). We obtained 6.63 million reads from an Illumina 75-bp single-read run and mapped them to the B. subtilis 168 genome (GenBank accession number NC_000964) (6) using the Geneious Read Mapper (Geneious version 9.0.5 software, Biomatters, Ltd.) (7). The alignment showed a 118-fold average coverage and a 99.5% pairwise identity to the reference genome of B. subtilis 168. The insertion and the correct sequence of the chloramphenicol resistance gene at the pks operon locus were verified by a standard PCR. The final genome sequence of B. subtilis ∆6 has a length of 3,876,919 bp.

We identified 28 variations (single-nucleotide polymorphism, deletion, insertion, and substitution) with a minimal coverage of 25× and a minimum variant frequency of 0.8. Four of these mutations have an effect on the amino acids sequence of the encoded protein (carA, yobM, ywbD, and walH), whereas four mutations are silent (yczC, yjnA, glcF, and amyX). The remaining 20 variants are located in intergenic and RNA-encoding regions. All variations can be requested from the corresponding author. In addition, we could confirm the presence of all six deletions performed by Westers et al. (1). Interestingly, B. subtilis ∆6 contains a seventh large deletion of 20.5 kb (25 genes; genome position: 529,422 to 549,925 bp). This deletion corresponds to the mobile genetic element ICEBs1 (8), which likely has undergone self-excision, as it has been reported for other B. subtilis strains (9). Taken together, B. subtilis ∆6 is lacking 376 genes at seven different locations covering 8.03% of the reference genome of B. subtilis 168. These deletions increased the GC content from 43.5% to 43.9%.

Nucleotide sequence accession number.The genome sequence of B. subtilis subsp. subtilis strain ∆6 is deposited in GenBank under the accession number CP015975.

ACKNOWLEDGMENTS

D.R.R. was supported by the Göttingen Graduate School for Neuroscience and Molecular Biosciences (DFG grant GSC 226/2).

FOOTNOTES

    • Received 6 June 2016.
    • Accepted 10 June 2016.
    • Published 28 July 2016.
  • Copyright © 2016 Reuß et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6
Daniel R. Reuß, Andrea Thürmer, Rolf Daniel, Wim J. Quax, Jörg Stülke
Genome Announcements Jul 2016, 4 (4) e00759-16; DOI: 10.1128/genomeA.00759-16

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Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6
Daniel R. Reuß, Andrea Thürmer, Rolf Daniel, Wim J. Quax, Jörg Stülke
Genome Announcements Jul 2016, 4 (4) e00759-16; DOI: 10.1128/genomeA.00759-16
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