Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data

Abstract

BackgroundBacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length.ResultsWe applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1.These are specific for γ-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases.ConclusionsThe determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks. Multiple genome-level comparisons among five closely related Bacillus species were also carried out. The determined genome sequence of B. subtilis natto and gene annotations are available from the Natto genome browser http://natto-genome.org/.

Resolution of a Meningococcal Disease Outbreak from Whole-Genome Sequence Data with Rapid Web-Based Analysis Methods

“…With current technology the whole process can be completed in less than 48 h from the receipt of a clinical specimen, with a large proportion of this time devoted to culturing of the isolate. Illumina sequencing followed by de novo assembly has been shown to be accurate for bacterial genomes (12,19,37,41). The bioinformatics analysis following de novo assembly takes only a few minutes to an hour, depending on the level of comparison required (Fig.…”

Loss of poly-.GAMMA.-glutamic Acid Synthesis of Bacillus subtilis (natto) Due to IS4Bsu1 Translocation to swrA Gene

“…The B. subtilis (natto) genome contains a total of 37 copies of five genes annotated as ISs (Nishito et al, 2010), and at least two of them, IS4Bsu1 and IS256Bsu1, possess an active transposase gene that enables the ISs to translocate in the genome (Kimura and Itoh, 2007;Takahashi et al, 2007). The target sequence duplicated at the newly created IS4Bsu1 insertion site in swrA was 5′-TGAATTAGT-3′, which did not match any of the known IS4Bsu1 target sequences (Kimura and Itoh, 2007;Takahashi et al, 2007).…”

Transposition of insertion sequence IS<i>256Bsu1</i> in <i>Bacillus subtilis</i> 168 is strictly dependent on <i>recA</i>

“…Bacillus subtilis Marburg 168, however, is unique in that it lacks typical ISs (Kunst et al, 1997;Barbe et al, 2009). In contrast, the closely related B. subtilis natto strains, the starter strains for a traditional Japanese fermented soybean food called natto, harbor various IS copies (Nishito et al, 2010;Kamada et al, 2014Kamada et al, , 2015 including IS4Bsu1 and IS256Bsu1 (Kimura and Itoh, 2007). To explore the genetic and evolutionary background of the absence of ISs in B. subtilis 168, we artificially introduced modified IS4Bsu1 into the 168 strain and developed several new assay systems for transposition frequency (Takahashi et al, 2007a(Takahashi et al, , 2007b).…”