Bacillus Subtilis Spores Research Articles

Combined treatment of ε-polylysine and heat damages protective structures and spore inner membranes to inactivate Bacillus subtilis spores

The sterilizing effect of a combination of heat (80, 90, and 100 °C) and ε-polylysine (ε-PL, 0.25 and 1 g/L) treatments on Bacillus subtilis spores was investigated and compared with that of conventional heat sterilization. The inactivation rate of spores and changes in their protective structure were evaluated using different methods and techniques. Changes in cell membrane's fatty acids, cell walls, proteins and nucleic acids were also analyzed. The results showed that the combined heat and ε-PL treatments significantly (p < 0.05) inactivated the Bacillus subtilis spores compared with the single heat treatment. Besides, the inactivation of spores was enhanced as the temperature and ε-PL concentration of combined treatments increased. The inactivation rate was found to be 2.18 log after heating at 90 °C for 60 min combined with the addition of 1 g/L ε-PL. Additionally, the electrical conductivity of spores' suspension and the positive region of flow cytometry significantly (p < 0.05) increased depending on temperature and ε-PL concentration of a combination treatment, indicating significant damage in the cell membranes and increased permeability. Significant changes in the spore morphology were also observed by the microscopy analysis after a combination treatment. Furthermore, the Fourier transform infrared spectra indicated a phase change in the inner membrane and alteration in the structure of peptidoglycan layer, as well as protein and nucleic acids denaturation after combined treatments. Therefore, the combined heat and ε-PL treatments can be suggested as sterilizing alternative to conventional heat sterilization in the food industry.

Copper delivery to an endospore coat protein of Bacillus subtilis.

A family of cytosolic copper (Cu) storage proteins (the Csps) bind large quantities of Cu(I) via their Cys-lined four-helix bundles, and the majority are cytosolic (Csp3s). The presence of Csp3s in many bacteria appears inconsistent with the current dogma that bacteria, unlike eukaryotes, have evolved not to maintain intracellular pools of Cu due to its potential toxicity. Sporulation in Bacillus subtilis has been used to investigate if a Csp3 binds Cu(I) in the cytosol for a target enzyme. The activity of the Cu-requiring endospore multi-Cu oxidase BsCotA (a laccase) increases under Cu-replete conditions in wild type B. subtilis. In the strain lacking BsCsp3 lower BsCotA activity is observed and is unaffected by Cu levels. BsCsp3 loaded with Cu(I) readily activates apo-BsCotA in vitro. Experiments with a high affinity Cu(I) chelator demonstrate that Cu(I) transfer from Cu(I)-BsCsp3 must occur via an associative mechanism. BsCsp3 and BsCotA are both upregulated during late sporulation. We hypothesise that BsCsp3 acquires cuprous ions in the cytosol of B. subtilis for BsCotA.

Effects of pulsed near infrared light (NIR) on Bacillus subtilis spores.

In this study, we develop a characterization of bacterial spore resistance to NIR pulsed light under modalities traditionally used in multiphoton microscopy. Energy dose and laser power are both key parameters in spore and bacterial cell inactivation. Surprisingly, spores and vegetative cells seem to show a similar sensitivity to pulsed NIR, spores being only 2-fold more resistant than their vegetative counterparts. This work enables us to eliminate certain hypotheses concerning the main driver of spore inactivation processes. Our findings suggest that damage leading to inactivation is mainly caused by photochemical reactions characterized by multiple possible pathways, including DNA damage or oxidation processes.

A Systemic Review on Bacillus Subtilis Spore Structure and Its Applications in the Development of Mucosal Vaccines and Adjuvants

Background and Aim Surface display technology enables the binding of proteins and peptides to the surface of living cells, including mammalian cells, yeast, bacteria, and spores. Among the various systems used for surface display, Bacillus subtilis spores have advantages such as resistance to adverse environmental conditions such as heat, radiation and chemicals, safety for humans and no need for the heterologous protein to pass the membranes for binding to spores. Methods &amp; Materials This study is a review that investigates the structure of the Bacillus subtilis spore, spore surface display, and its application in the development of mucosal vaccines and adjuvants. Ethical Considerations All Ethical principles in writing this article have been observed according to the instructions of the National Ethics Committee and the COPE regulations. Results Heterologous proteins can be displayed genetically and non-genetically on the spore surface. The surface display is a promising strategy for the development of whole-cell factories with many industrial and biotechnological applications, leading to significant advances in the production of biocatalysts, the development of live vaccines, bio-adsorbents and sensors, epitope mapping, inhibitor design, and protein/peptide library screening. Conclusion It is hoped that oral vaccines of Bacillus subtilis spores will be of significant help in the prevention and treatment of diseases including COVID-19 in the future.

Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores.

Here, we describe a stress-tolerant, recyclable, and renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on the Bacillus subtilis spore surface. TIED enables high loading density in the range of 106 to 107 recombinant enzymes per spore, robust catalytic activity of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Furthermore, we demonstrate TIED enzymes to be not only recyclable but also fully renewable after the loss of activity through induction of germination and sporulation, enabling perpetual regeneration of these immobilized biocatalysts.

Optimization of Sporulation Conditions for Bacillus subtilis BSNK-5

Bacillus subtilis spores have important biological applications; however, high spore-cell densities and sporulation efficiencies in fermentation is poorly reported. This study systematically analyzed the spore densities and formation efficiency of B. subtilis BSNK-5 in different culture substrates. A response surface regression equation was established based on the results of single factor and Box–Behnken experimental designs. The optimal medium formulation, as predicted from the equation, consisted of soluble starch at 3 g·L−1, soybean flour at 12 g·L−1, and MgSO4 at 5 g·L−1. The spore yield reached 2.43 × 109 CFU·mL−1, and the sporulation rate was 83.3%, which was nearly three times higher than before optimization using an optimized medium at 36 °C and 200 rpm for 60 h.

The sporicidal activity of a disinfectant with peracetic acid against the spores of Bacillus subtilis and Bacillus cereus according to the european standard PN-EN 17126: 2019-01.

The assessment of the sporicidal effectiveness of disinfectants is important from the point of view of the prevention of nosocomial infections and spore contamination of clinical samples, medical equipment and materials used in patient care. The rods of Bacillus spp. cause infections of the digestive system, bloodstream and, less often, respiratory tract. Cases were diagnosed in immunocompromised patients, malignant neoplasms and in neonatal wards. The source of the infection was hospital linen, reusable towels, catheters or milk from the human milk bank. Determination of the minimal sporicidal parameters of a disinfectant containing peracetic acid. The sporicidal activity of a disinfectant containing peracetic acid against Bacillus subtilis and Bacillus cereus spore suspensions was tested in a defined concentration range during a contact time of 15 minutes, in the presence of various interfering substances (clean and dirty conditions) according to the European Standard PN-EN 17126: 2019-01. The disinfecting preparation containing peracetic acid showed sporicidal activity against Bacillus subtilis at a concentration of 1% for 15 minutes under clean and dirty conditions and at concentrations of 0.5%, 1.00% and 1.25% against the Bacillus cereus spores during the same contact time but only under dirty conditions. The preparation showed no sporicidal activity against Bacillus cereus at concentrations of 2%, 3%, 4% and 5% during a contact time of 15 minutes under both dirty and clean conditions. In areas where there is a risk of infecting a patient or contaminating clinical specimens, materials and equipment with spores of Bacillus spp., it is necessary to use disinfectants with sporicidal activity confirmed according to the PN-EN 17126: 2019-01 standard. The sporicidal activity of disinfectants containing peracetic acid may depend on the method of preparing the solutions, their concentration, pH, temperature and the contamination degree of the disinfected surface.

High Pressure Thermal Sterilization and ε-Polylysine Synergistically Inactivate Bacillus subtilis Spores by Damaging the Inner Membrane

This study was conducted to determine the sterilization effect of a combination of high pressure thermal sterilization (HPTS) and ε-polylysine (ε-PL) on Bacillus subtilis spores. The spores were treated with HPTS (550 MPa at 25, 65, and 75°C) and ε-PL at 0.1 and 0.3%. HPTS and ε-PL synergistically decreased the number of surviving spores and increased the release of the intracellular components in the spore suspension, with the maximal effects from treatment with 550 MPa at 75°C plus 0.3% ε-PL. Maximum fluidity and permeability of the cell inner membrane were observed with 550 MPa at 75°C plus 0.3% ε-PL. Changes in membrane lipids were detected from 3,000 to 2,800 cm-1 by Fourier transform infrared spectroscopy. The results provide new insights into the mechanism by which HPTS and ε-PL synergistically sterilize B. subtilis spores.

Investigating the Bioconversion of Tyrosol to Hydroxyltyrosol by Recombinant Bacillus Subtilis Spores and the Anticancer Effects of Hydroxytyrosol on MCF-7

چکیده: مقدمه: تیروزول (2و 4- هیدروکسی فنیل اتانول ) از ترکیبات فنلی طبیعی موجود در زیتون است که اثرات آنتی اکسیدانی و ضد سرطانی آن شناخته شده است. بخش قابل توجهی از این ترکیبات فنلی در فرایند تولید روغن از زیتون خارج شده و وارد آب استفاده شده در فرایند و نهایتا وارد پساب کارخانه می‌شوند. در حالی که می‌توان این ترکیبات را به مواد فراسودمند و مکمل‌های غذایی تبدیل کرد. در این مطالعه تیروزول در اثر تبدیل زیستی با استفاده از آنزیم تیروزیناز تثبیت شده در سطح اسپور به هیدروکسی تیروزول تبدیل و اثر ضد سرطانی ترکیب حاصل بر روی سلول‌های سرطان سینه رده MCF-7 بررسی شد. مواد و روش‌ها: اسپورهای سویه نوترکیب Bacillus subtilis DB104 (pSDJH-cotE-tyr) که در پژوهش قبلی با استفاده از روش‌های مهندسی ‌ژنتیک و تکنیک نمایش پروتئین در سطح ایجاد شده بود، در محیط اسپورزایی تهیه شد. با استفاده از اسپورها واکنش تبدیل زیستی تیروزول به هیدروکسی تیروزول انجام و روند واکنش با استفاده از کروماتوگرافی مایع با فشار بالا مورد ارزیابی قرار گرفت. اثر ضد سرطانی هیدروکسی تیروزول بر روی رده سلولی MCF-7 با استفاده از فلوسیتومتری مطالعه شد. نتایج: نتایج حاصل از کروماتوگرافی مایع با فشار بالا نشان داد که، 1 میلی‌مولار از تیروزول پس از 60 دقیقه توسط تیروزیناز بیان شده در سطح اسپور به 1 میلی مولار هیدروکسی تیروزول تبدیل شد. همچنین اثرات ضدسرطانی هیدروکسی تیروزول حاصل بیش از 80 درصد در سلول‌های سرطانیMCF-7 پس از 48 ساعت مشاهده گردید. بحث و نتیجه گیری: براساس نتایج حاصل، ازتیروزیناز تثبیت شده درسطح اسپور می‌توان به عنوان آنزیمی فعال جهت تولید ترکیبات فرا سودمند با خاصیت ضد سرطانی از مشقات روغن زیتون استفاده کرد. اثرات ضد سرطانی هیدروکسی تیروزول تولید شده در اثر تبدیل زیستی تیرزول بر سلول‌های سرطانی MCF-7 ، بر اساس مطالعات انجام شده بر سایر سلول‌های سرطانی تائید شده است.

Sporicidal mechanism of the combination of ortho-phthalaldehyde and benzyldimethyldodecylammonium chloride as a disinfectant against the Bacillus subtilis spores.

Previous studies have shown that the combination disinfectant, Ortho-phthalaldehyde and benzyldimethyldodecylammonium chloride (ODB), can effectively kill a variety of microorganisms, such as Escherichia coli, Staphylococcus aureus, and Candida albicans. To observe the sporicidal ability and mechanism of ODB for spores, Bacillus subtilis spores were used as the research object in this experiment. TEM images revealed that ODB destroyed the integrity of the coat, cortex, and inner membrane of the spores after 0.5-h treatment, and the nuclear material was also broken and exuded after 4-h treatment. The broken structure led to the release of dipicolinic acid (DPA) in large amount. The results show that B. subtilis spores can be effetely killed by ODB through destroying the structure of the spores.

Evaluating the persistence and stability of a DNA-barcoded microbial system in a mock home environment.

Recent advancements in engineered microbial systems capable of deployment in complex environments have enabled the creation of unique signatures for environmental forensics operations. These microbial systems must be robust, able to thrive in specific environments of interest and contain molecular signatures, enabling the detection of the community across conditions. Furthermore, these systems must balance biocontainment concerns with the stability and persistence required for environmental forensics. Here we evaluate the stability and persistence of a recently described microbial system composed of germination-deficient Bacillus subtilis and Saccharomyces cerevisiae spores containing nonredundant DNA barcodes in a controlled simulated home environment. These spore-based microbial communities were found to be persistent in the simulated environment across 30-day periods and across multiple surface types. To improve the repeatability and reproducibility in detecting the DNA barcodes, we evaluated several spore lysis and sampling processes paired with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR-associated proteins (Cas) detection (Sherlock). Finally, having optimized the detectability of the spores, we demonstrate that we can detect the spores transferring across multiple material types. Together, we further demonstrate the utility of a recently described microbial forensics system and highlight the importance of independent validation and verification of synthetic biology tools and applications. Graphical Abstract.

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission.

Bacterial developmental checkpoint that directly monitors cell surface morphogenesis.

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous "coat" and an inner peptidoglycan "cortex," separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Flower Pollen-Based Photosensitization Process for Enhanced Solar Disinfection of Drinking Water: Reactor Design and Inactivation Mechanisms

Solar disinfection (SODIS) is a chemical-free, low-cost, and user-friendly approach to achieving Sustainable Development Goal No. 6 (SDG 6). Herein, a natural flower pollen-based photosensitizer was fabricated in a facile way and used to enhance the conventional SODIS process. Complete inactivation of five biohazards (Escherichia coli K-12, Spingopyxis sp. BM1-1, Bacillus subtilis spores, Enterococcus faecalis, and tetracycline-resistant E. coli) at their culturable or viable but nonculturable states was achieved by both batch reactors and a flow-through reactor with a standing separation–recycling system. Herein, microorganisms adhered to the photosensitizer’s surface due to electrostatic attraction and topological interactions. The photosensitizer induced the localized heating effect under solar irradiation, and a portion of microorganisms was inactivated by the photothermal effect. Meanwhile, abundant reactive species (•OH and 1O2) were generated and contributed to the photocatalytic inactivation of microorganisms. Synergism between the photothermal and photocatalytic processes enhanced the destruction of cell membranes and the penetration of reactive species. A comprehensive inactivation pathway was proposed and verified. The findings suggested that the natural flower pollen-based photosensitization process is promising to enhance the SODIS for effective disinfection, which assists rural and underdeveloped areas in achieving SDG 6.

Inactivation Mechanism of a Combined Treatment of Ε-Polylysine and Heat Against Bacillus Subtilis Spores

Inactivation Mechanism of a Combined Treatment of Ε-Polylysine and Heat Against Bacillus Subtilis Spores

A microwave-based system combines drying, extraction and sterilization under low-temperature vacuum conditions

This study develops a simultaneous system combining low-temperature drying, extraction, and sterilization under low temperature, by comprising a microwave source and a vacuum chamber. The study commences by investigating the reduction in moisture content under microwave heating in vacuum conditions as a function of the drying time, the material load, and the microwave output power. Subsequently, the study compares the results obtained for the yield and composition of the essential oil extracted from Coleus amboinicus Loureiro leaves using the proposed system with those obtained using a conventional steam distillation technique. Finally, the study investigates the performance of the developed system in inactivating Bacillus subtilis spores using a low-pressure plasma sterilization technique. The experimental results show that the low-temperature vacuum conditions established in the proposed drying system accelerate the rate of moisture removal. Furthermore, the microwaves penetrate the interior of the material, heating the internal moisture directly. This not only reduces the drying time but also preserves the original flavor and appearance of the dried material. The extraction results show no significant difference between the final yield percentages and compositions of the essential oils extracted using the proposed system and the distilled steam technique, respectively. However, the extraction time is reduced from 90 minutes in the distilled steam method to just 30 minutes in the proposed system. Finally, the plasma sterilization results demonstrate that the proposed system reduces the number of colony-forming units of Bacillus subtilis from 170 to 7 within a treatment time of 1 s, and achieves complete sterilization following 180 s.

Paper-based electrochemical peptide sensor for label-free and rapid detection of airborne Bacillus anthracis simulant spores

Bacillus anthracis is a fatal biological warfare agent, with a mortality rate of almost 100% when the bacteria are inhaled. Therefore, developing a biosensor for rapidly detecting B. anthracis spores in the ambient air is critical. Conventional techniques tend to be time-consuming, labor-intensive, and require highly trained personnel, thereby preventing rapid onsite detection. Moreover, low-cost, stable probes are required for continuous monitoring of multiple airborne targets. This study presents a paper-based electrochemical peptide sensor for rapid, label-free, sensitive, and economical quantification of liquid-borne and airborne Bacillus subtilis spores, widely used nonpathogenic B. anthracis simulant spores. These sensors were fabricated using wax printing on Whatman filter paper along with stencil-printed carbon paste electrodes and Ag/AgCl ink, as well as subsequent peptide (NHFLPKV) immobilization. These low-cost quickly fabricated sensors were characterized by differential pulse voltammetry and could detect spore concentrations as low as 6.9 × 102 colony-forming units (CFU) mL-1 in 30 min with high specificity towards the spores and stability. Furthermore, spore concentrations were determined from air samples obtained using the BioSampler, which demonstrated that these measurements agreed with those using the plate-counting assay; therefore, the surface proteins of the spores were intact through sampling with the BioSampler. These paper-based peptide sensors can be used for onsite rapid and continuous airborne spore-monitoring systems.

An Adaptable and Modular Set of Laboratory Exercises Connecting Genotype to Phenotype in Sporulating Bacillus subtilis.

ABSTRACTPractical lab exercises that help students draw connections between genotype and phenotype, and make and test predictions about the identity of mutants, are invaluable in college-level cell biology, genetics, and microbiology courses. While many bacteria are easy to grow and manipulate within the time and resource constraints of a laboratory course, their phenotypes are not always observable or relevant-seeming to college students. Here, we leverage sporulation by the bacterium Bacillus subtilis, a well-characterized and genetically tractable system, to create 5 adaptable lab exercises that can be implemented in different combinations to suit the needs of a variety of courses and instruction modes. Because phenotypic changes during sporulation are striking morphological changes to cells that are easily observable with basic light microscopy, and because spore-forming bacteria related to B. subtilis have clear applications for human and environmental health, these exercises have the potential to engage students’ interest while introducing and reinforcing key concepts in microbiology, cell biology, and genetics.

Enterococcal bacteremia in mice is prevented by oral administration of probiotic Bacillus spores.

Whether and how probiotics promote human health is a controversial issue. Their claimed benefit for counteracting gastrointestinal infection is linked predominantly to reducing pathogen abundance within the intestinal microbiota. Less understood mechanistically is the reported value that probiotics could have in reducing systemic infections. Enterococcus faecalis is an opportunistic pathogen that causes systemic infection after translocation through the intestinal epithelium, particularly in hospitalized and immune-depleted patients receiving antibiotic therapy. In this study, we used an E. faecalis mouse infection model with wild-type and isogenic mutant strains deficient in genes of the E. faecalis Fsr (fecal streptococci regulator) quorum-sensing system. We show that E. faecalis translocation from the mouse gut into the blood is mediated by the Fsr quorum-sensing system through production of the protease GelE, which compromises intestinal epithelium integrity. Furthermore, we demonstrate that orally administered probiotic Bacillus subtilis spores blocked E. faecalis translocation from the gut to the bloodstream and subsequent systemic infection in mice by inhibiting Fsr activity. These findings demonstrate that a key aspect of Enterococcus pathogenesis is controlled by quorum sensing, which can be targeted with probiotic Bacillus spores.

Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling.

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.