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