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Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

ABSTRACT: Spore-forming Bacillus strains that produce extracellular poly-?-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.

SUBMITTER: Kubo Y

PROVIDER: S-EPMC3187134 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

Kubo Yuji Y Rooney Alejandro P AP Tsukakoshi Yoshiki Y Nakagawa Rikio R Hasegawa Hiromasa H Kimura Keitarou K

Applied and environmental microbiology 20110715 18

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis ...[more]

PMID: 21764950

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Project description: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 gamma-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/.

Project description:Spore-forming probiotic bacteria offer interesting properties as they have an intrinsic high stability, and when consumed, they are able to survive the adverse conditions encountered during the transit thorough the host gastrointestinal (GI) tract. A traditional healthy food, nattō, exists in Japan consisting of soy fermented by the spore-forming bacterium Bacillus subtilis natto. The consumption of nattō is linked to many beneficial health effects, including the prevention of high blood pressure, osteoporosis, and cardiovascular-associated disease. We hypothesize that the bacterium B. subtilis natto plays a key role in the beneficial effects of nattō for humans. Here, we present the isolation of B. subtilis DG101 from nattō and its characterization as a novel spore-forming probiotic strain for human consumption. B. subtilis DG101 was non-hemolytic and showed high tolerance to lysozyme, low pH, bile salts, and a strong adherence ability to extracellular matrix proteins (i.e., fibronectin and collagen), demonstrating its potential application for competitive exclusion of pathogens. B. subtilis DG101 forms robust liquid and solid biofilms and expresses several extracellular enzymes with activity against food diet-associated macromolecules (i.e., proteins, lipids, and polysaccharides) that would be important to improve food diet digestion by the host. B. subtilis DG101 was able to grow in the presence of toxic metals (i.e., chromium, cadmium, and arsenic) and decreased their bioavailability, a feature that points to this probiotic as an interesting agent for bioremediation in cases of food and water poisoning with metals. In addition, B. subtilis DG101 was sensitive to antibiotics commonly used to treat infections in medical settings, and at the same time, it showed a potent antimicrobial effect against pathogenic bacteria and fungi. In mammalians (i.e., rats), B. subtilis DG101 colonized the GI tract, and improved the lipid and protein serum homeostasis of animals fed on the base of a normal- or a deficient-diet regime (dietary restriction). In the animal model for longevity studies, Caenorhabditis elegans, B. subtilis DG101 significantly increased the animal lifespan and prevented its age-related behavioral decay. Overall, these results demonstrate that B. subtilis DG101 is the key component of nattō with interesting probiotic properties to improve and protect human health.

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Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

Publications

Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

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