Probiotic properties of Bacillus subtilis DG101 isolated from the traditional Japanese fermented food natto.

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, natto, exists in Japan consisting of soy fermented by the spore-forming bacterium Bacillus subtilis natto. The consumption of natto 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 natto for humans. Here, we present the isolation of B. subtilis DG101 from natto 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 natto with interesting probiotic properties to improve and protect human health.

Microbial flora, probiotics, Bacillus subtilis and the search for a long and healthy human longevity.

Probiotics are live microorganisms that have beneficial effects on host health, including extended lifespan, when they are administered or present in adequate quantities. However, the mechanisms by which probiotics stimulate host longevity remain unclear and very poorly understood. In a recent study (Nat. Commun. 8, 14332 (2017) doi: 10.1038/ncomms14332), we used the spore-forming probiotic bacterium Bacillus subtilis and the model organism Caenorhabditis elegans to study the mechanism by which a probiotic bacterium affects host longevity. We found that biofilm-proficient B. subtilis colonized the C. elegans gut and extended the worm lifespan significantly longer than did biofilm-deficient isogenic strains. In addition to biofilm proficiency, the quorum-sensing pentapeptide CSF and nitric oxide (NO) represent the entire B. subtilis repertoire responsible for the extended longevity of C. elegans. B. subtilis grown under biofilm-supporting conditions synthesized higher levels of NO and CSF than under planktonic growth conditions, emphasizing the key role of the biofilm in slowing host aging. Significantly, the prolongevity effect of B. subtilis was primarily due to a downregulation of the insulin-like signaling system that precisely is a key partaker in the healthy longevity of human centenarians. These findings open the possibility to test if the regular consumption of B. subtilis incorporated in foods and beverages could significantly extend human life expectancy and contribute to stop the development of age-related diseases.

Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA.

Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway.

Beneficial bacteria have been shown to affect host longevity, but the molecular mechanisms mediating such effects remain largely unclear. Here we show that formation of Bacillus subtilis biofilms increases Caenorhabditis elegans lifespan. Biofilm-proficient B. subtilis colonizes the C. elegans gut and extends worm lifespan more than biofilm-deficient isogenic strains. Two molecules produced by B. subtilis - the quorum-sensing pentapeptide CSF and nitric oxide (NO) - are sufficient to extend C. elegans longevity. When B. subtilis is cultured under biofilm-supporting conditions, the synthesis of NO and CSF is increased in comparison with their production under planktonic growth conditions. We further show that the prolongevity effect of B. subtilis biofilms depends on the DAF-2/DAF-16/HSF-1 signalling axis and the downregulation of the insulin-like signalling (ILS) pathway.

Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency.

Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.

Determination of NO and CSF Levels Produced by Bacillus subtilis.

The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus subtilis cultures have been measured only under planktonic growth conditions. We sought to determine whether NO and/or CSF production is affected in B. subtilis cells that develop as a biofilm. To measure the production levels of the two prolongevity molecules, we grew B. subtilis cells under planktonic and biofilm supporting condition.

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

UNLABELLED: Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. IMPORTANCE: Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective decision to move over or stay attached to a surface controlled? Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question. Remarkably, we discover that sessile biofilm formation and social sliding motility share the same structural components and the Spo0A regulatory network via sensor kinases, KinB and KinC. Potassium, an inhibitor of KinC-dependent biofilm formation, triggers sliding via a potassium-perceiving cytosolic domain of KinB that resembles the selectivity filter of potassium channels. The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness.

Spo0A links de novo fatty acid synthesis to sporulation and biofilm development in Bacillus subtilis.

During sporulation in Bacillus subtilis, the committed-cell undergoes substantial membrane rearrangements to generate two cells of different sizes and fates: the mother cell and the forespore. Here, we demonstrate that the master transcription factor Spo0A reactivates lipid synthesis during development. Maximal Spo0A-dependent lipid synthesis occurs during the key stages of asymmetric division and forespore engulfment. Spo0A reactivates the accDA operon that encodes the carboxylase component of the acetyl-CoA carboxylase enzyme, which catalyses the first and rate-limiting step in de novo lipid biosynthesis, malonyl-CoA formation. The disruption of the Spo0A-binding box in the promoter region of accDA impairs its transcriptional reactivation and blocks lipid synthesis. The Spo0A-insensitive accDA(0A) cells were proficient in planktonic growth but defective in sporulation (σ(E) activation) and biofilm development (cell cluster formation and water repellency). Exogenous fatty acid supplementation to accDA(0A) cells overcomes their inability to synthesize lipids during development and restores sporulation and biofilm proficiencies. The transient exclusion of the lipid synthesis regulon from the forespore and the known compartmentalization of Spo0A and ACP in the mother cell suggest that de novo lipid synthesis is confined to the mother cell. The significance of the Spo0A-controlled de novo lipid synthesis during B. subtilis development is discussed.