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Antibiotic resistance microorganisms (ARMs), particularly gram-negative bacteria, pose a global health threat. The effects of fermentation on phytochemicals are numerous, and exploring this potential is the focus of drug development. The study investigated the role of fermentation in modifying V. amygdalina leaf secondary metabolites as an effective antibiotic against Escherichia. coli, Bacillus subtilis and Salmonella typhi. This work showed that fermentation increased the content of lycopene, flavonoid and carotenoid compounds but decreased chlorophyll, soluble protein and phenol. Pearson's correlation heatmap showed a strong correlation between microbial activities and secondary metabolic changes. The methanolic extract of fermented V. amygdalina leaf pulp (at day 9) showed significant antioxidant and anti-inflammatory activities. The GCMS and FTIR results showed unique compounds and structural modifications at different intervals of the fermentation period. In-vitro and in-silico analyses showed that fermentation did not alter the inhibition rate against B. subtilis; however, E. coli and S. typhi were significantly inhibited by fermented V. amygdalina pulp extracts. In-silico analyses showed that 4,6-Cholestadien-3β-ol– a compound present only on the ninth day of fermentation–was responsible for the inhibition of the gram-negative bacteria via the substitution of multiple non-ionic interactions of some key catalytic site residues with non-ionic types, thereby denying ionisation and salt-bridge properties that porins explore to resist antibiotics; and higher binding affinity to OmpC and OmpF than ampicillin. Therefore, this steroid-derived compound may open a new pipeline for developing ion-independent multi-target antibiotics against broad-spectrum multidrug-resistant gram-positive and gram-negative bacteria in food and pharmaceutical purposes. [ABSTRACT FROM AUTHOR]

The spread of grape leaf diseases has a negative impact on the sustainable development of agriculture. Diseases induced by Uncinula necator significantly affect the quality of grapes. Bacillus biocontrol agents have been proven effective in disease management. However, limited research has been conducted on the impact of biocontrol agents on the assembly and potential functions of plant phyllosphere microbial communities. This study used high-throughput sequencing combined with bioinformatics analysis and culture omics technology for analysis. The results showed that biocontrol bacteria B. subtilis utilized in this study can significantly reduce the disease index of powdery mildew (p<0.05); concurrently, it exhibits a lower disease index compared to traditional fungicides. A comprehensive analysis has revealed that biocontrol bacteria have no significant impact on the diversity of phyllosphere fungi and bacteria, while fungicides can significantly reduce bacterial diversity. Additionally, biocontrol agents can increase the complexity of fungal networks and enhance the degree of modularity and stability of the bacterial network. The results also showed that the biocontrol agents, which contained a high amount of B. subtilis, were able to effectively colonize the grapevine phyllosphere, creating a microenvironment that significantly inhibits pathogenic bacteria on grape leaves while enhancing leaf photosynthetic capacity. In conclusion, biocontrol agents significantly reduce the grape powdery mildew disease index, promote a microenvironment conducive to symbiotic microorganisms and beneficial bacteria, and enhance plant photosynthetic capacity. These findings provide a basis for promoting biocontrol agents and offer valuable insights into sustainable agriculture development. [ABSTRACT FROM AUTHOR]

One of the latest trends in sustainable agriculture is the use of beneficial microorganisms to stimulate plant growth and biologically control phytopathogens. Bacillus subtilis, a Gram-positive soil bacterium, is recognized for its valuable properties in various biotechnological and agricultural applications. This study presents, for the first time, the successful encapsulation of B. subtilis within electrospun poly(3-hydroxybutyrate) (PHB) fibers, which are dip-coated with cellulose derivatives. In that way, the obtained fibrous biohybrid materials actively ensure the viability of the encapsulated biocontrol agent during storage and promote its normal growth when exposed to moisture. Aqueous solutions of the cellulose derivatives—sodium carboxymethyl cellulose and 2-hydroxyethyl cellulose, were used to dip-coat the electrospun PHB fibers. The study examined the effects of the type and molecular weight of these cellulose derivatives on film formation, mechanical properties, bacterial encapsulation, and growth. Scanning electron microscopy (SEM) was utilized to observe the morphology of the biohybrid materials and the encapsulated B. subtilis. Additionally, ATR-FTIR spectroscopy confirmed the surface chemical composition of the biohybrid materials and verified the successful coating of PHB fibers. Mechanical testing revealed that the coating enhanced the mechanical properties of the fibrous materials and depends on the molecular weight of the used cellulose derivatives. Viability tests demonstrated that the encapsulated B. subtilis exhibited normal growth from the prepared materials. These findings suggest that the developed fibrous biohybrid materials hold significant promise as biocontrol formulations for plant protection and growth promotion in sustainable agriculture. [ABSTRACT FROM AUTHOR]

Chitosanase plays a pivotal role in the production of chitooligosaccharide. Nevertheless, there is untapped potential for enhancing both its catalytic efficiency and thermostability, which could significantly bolster its therapeutic and biotechnological applications. In this study, two computer-aided protein design methods, namely Fireprot and PROSS, were utilized to pinpoint 6 single Bacillus subtilis chitosanase BsCsn46A mutants (S126A, D191A, K70A, L159I, K104P, and A129L) as well as 4 multiple mutants (K70A/S126A, K70A/S126A/K104P, K70A/S126A/L159I, and K70A/S126A/K104P/L159). The wild-type (WT) and all 10 BsCsn46A mutants displayed robust adaptability across a broad pH range, exhibiting peak activity within the pH spectrum of 5.5 to 9.5. The results demonstrated that, compared to the WT, 9 out of 10 mutants exhibited significantly heightened chitosanase activity, with the sole exception being the D191A mutant, which displayed activity levels nearly identical to the WT. Notably, the A129L displayed an impressive 20% increase in the enzyme activity at elevated temperatures, specifically in the range of 55–80°C. Assessing protein stability, results indicated that all samples maintained stability when incubated at 30°C for 1 h. However, when subjected to a higher temperature of 40°C for 1 h, only the A129L mutant retained stability, which persisted even after an extended incubation period of 3 h at 40°C. Furthermore, a thermal stability analysis revealed noteworthy differences between the WT and the mutants. The WT chitosanase activity diminished by 50% after brief 30-min incubation at 50°C, whereas the K70A/S126A, K70A/S126A/K104P, and A129L mutants maintained 50% of their activity for approximately 2 h under the same conditions. In summary, the study provides valuable insights into the thermostability and catalytic activity of chitosanase, highlighting promising candidates for industrial chitosanase applications. [ABSTRACT FROM AUTHOR]

The intensified concerns related to agrochemicals' ecological and health risks have encouraged the exploration of microbial agents as eco-friendly alternatives. Some members of Bacillus spp. are potential plant-growth-promoting agents and benefit numerous crop plants globally. This study aimed to explore the beneficial effects of two Bacillus strains (B. subtilis strain IS1 and B. amyloliquificiens strain IS6) capable of alleviating the growth of tomato plants against salinity stress and Fusarium wilt disease. These strains were able to significantly promote the growth of tomato plants and biomass accumulation in pot trials in the absence of any stress. Under salinity stress conditions (150 mM NaCl), B. subtilis strain IS1 demonstrated superior performance and significantly increased shoot length (45.74%), root length (101.39%), fresh biomass (62.17%), and dry biomass (49.69%) contents compared to control plants. Similarly, B. subtilis strain IS1 (63.7%) and B. amyloliquificiens strain IS6 (32.1%) effectively suppressed Fusarium wilt disease and significantly increased plant growth indices compared to the pathogen control. Furthermore, these strains increased the production of chlorophyll, carotenoid, and total phenolic contents. They significantly affected the activities of enzymes involved in antioxidant machinery and the phenylpropanoid pathway. Hence, this study effectively demonstrates that these Bacillus strains can effectively alleviate the growth of tomato plants under multiple stress conditions and can be used to develop bio-based formulations for use in the fields. [ABSTRACT FROM AUTHOR]

BACKGROUND: The demand for animal products is increasing in developing countries due to population growth. However, livestock production contributes significantly to global warming, accounting for 25%. Probiotics can help improve livestock efficiency by enhancing gut microbes and fat metabolism. They can modify rumen populations, enhance fermentation, reduce methane emissions and improve feed digestion. In this study, the goal was to determine the most effective method of reducing methane emissions in the rumen of sheep in vitro by adding different concentrations of Saccharomyces cerevisiae and Bacillus subtilis. RESULTS: Adding 8 × 106 CFU g−1S. cerevisiae during fermentation reduced pH levels after 48 h. This also increased the concentrations of NH3‐N, microbial protein and total gas production. At the same time, it decreased methane emissions. Furthermore, adding 20 × 106 CFU g−1B. subtilis to the mixture increased total gas production (TGP) and methane production, with the highest production observed after 48 h. However, it did not affect pH levels after 48 h. CONCLUSION: It can be concluded that S. cerevisiae had significantly increased microbial protein and NH3‐N concentrations after fermentation without altering pH. Additionally, the addition of S. cerevisiae enhanced TGP and reduced methane emissions. It is worth noting that TGP increased because B. subtilis was added at a concentration of 20 × 106 CFU g−1, with no significant differences between concentrations. Therefore, we recommend adding S. cerevisiae and B. subtilis to the diet at doses of 8 and 20 × 106 CFU g−1, as it resulted in higher TGP and reduced methane emissions. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Milk rice pudding (MRP) is a commercial and popular dairy dessert, but owing to its characteristics and valuable ingredients, it may be contaminated by many pathogenic and spoilage microorganisms. So, this study aimed to improve the quality and safety of MRP by using cinnamon and clove essential oils. Concerning the evaluation of the minimum inhibitory concentration (MIC) for both oils with 0.2, 0.5, and 1% concentrations, B. cereus and B. subtilis were sensitive (+) to cinnamon and clove 0.5%, with inhibition zones of 13.3 and 14 mm for cinnamon and 11.3 and 12 mm for clove EO, respectively. While both bacteria were very sensitive (++) to cinnamon 1% (18.8 and 19.5 mm) and clove 1% (17.3 and 18.7 mm), respectively. Therefore, MRP was prepared by adding cinnamon and clove EOs at 0.6%. Treatments containing EOs showed a significant reduction of tested microorganisms compared to controls. B. cereus wasn’t detected in clove and cinnamon EO treatments at day 21 of the storage period, while B. subtilis vanished on day 14 for the cinnamon treatment and on day 21 for clove MRP. Moreover, the results revealed the enhancement of sensory characteristics of MRP supplemented with EOs without any significant alteration in their pH values. This study recommends the addition of cinnamon and clove EOs (0.6%) to MRP, as it isn’t only an excellent substitution of chemical preservatives with powerful antibacterial efficiency but also improves the overall acceptance of the product.

Biodiversity and the number of bacteria present in the soil are two of the main parameters of soil quality, especially for agricultural purposes. Analysis of the low-fertilized soils suggested that the number and diversity of the bacterial communities in this soil are low. Hence, various methods have been used to stimulate bacterial activity and improve agricultural soil conditions. One of the popular methods is the inoculation of bacteria such as B. subtilis and R. erythropolis. These bacteria are potential species as bio-inoculants in soil management. However, the effectiveness of these bacteria in stimulating the activity of bacterial communities and improving soil properties of the low-fertilized soil is still sparsely explored. Therefore, this study aimed to analyze the impact of the inoculation of B. subtilis and R. erythropolis on the bacterial community structure and soil properties of low-fertilized soil. The soil used is agricultural soil for tobacco farming activities using agrochemicals. Bacterial community structures were analyzed using the environmental DNA (eDNA) method. The soil properties analyzed were total nitrogen, carbon, phosphorous, potassium, and pH. This study suggests that B. subtilis and R. erythropolis may affect the bacterial community structure and increase the number of bacteria to reach the ideal limit for fertile soil. Adding bacterial inoculants could stimulate the growth of bacteria and the nutrient cycle in the soil environment, resulting in improved soil fertility. [ABSTRACT FROM AUTHOR]

Bacillus subtilis was engineered to produce circular subgenomes that are directly transmittable to another B. subtilis. The conjugational plasmid pLS20 integrated into the B. subtilis genome supported not only subgenome replication but also transmission to another B. subtilis species. The subgenome system developed in this study completes a streamlined platform from the synthesis to the transmission of giant DNA by B. subtilis. [ABSTRACT FROM AUTHOR]

Recently, plastic wastes are considered a main environmental problem, and many bacterial isolates were tested to biodegrade them. The low-density polyethylene (LDPE) plastic sheets were tested to be degraded by a marine bacterial consortium. The potent marine plastic degrading isolates were biochemically identified as Bacillus licheniformis, Bacillus subtilis, and Paenibacillus xylanilyticus using the BIOLOG identification system. The identification of the most potent plastic-degrading bacterium was confirmed as Bacillus licheniformis FMMA using the 16S rRNA gene sequence. This bacterial consortium was physiologically adjusted as follows: pH 7, temperature 35°C, inoculum size 4ml/ 100ml (1.0X107CFU/ ml), and an incubation period of 30 days. It led to 34.1% plastic loss of weight. The mechanical properties (maximum force and the elongation% at break) of these treated LDPE plastic sheets showed 7.49N and 112.2%, respectively, compared to that of B. licheniformis FMMA, which showed a 25.5% plastic weight loss, with maximum force and elongation% at a breakpoint of 8.9N and 114.2%, respectively. In addition, the plastic biodegradation was also estimated through a scanning electron microscopy and Fourier transform-infrared (FTIR) spectroscopy, were a great reduction in the intensity of the -CH2 peak appeared at 2900cm-1, and the disappearance of the -OH peak at 3500cm-1 was observed. [ABSTRACT FROM AUTHOR]

Milk rice pudding (MRP) is a commercial and popular dairy dessert, but owing to its characteristics and valuable ingredients, it may be contaminated by many pathogenic and spoilage microorganisms. So, this study aimed to improve the quality and safety of MRP by using cinnamon and clove essential oils. Concerning the evaluation of the minimum inhibitory concentration (MIC) for both oils with 0.2, 0.5, and 1% concentrations, B. cereus and B. subtilis were sensitive (+) to cinnamon and clove 0.5%, with inhibition zones of 13.3 and 14 mm for cinnamon and 11.3 and 12 mm for clove EO, respectively. While both bacteria were very sensitive (++) to cinnamon 1% (18.8 and 19.5 mm) and clove 1% (17.3 and 18.7 mm), respectively. Therefore, MRP was prepared by adding cinnamon and clove EOs at 0.6%. Treatments containing EOs showed a significant reduction of tested microorganisms compared to controls. B. cereus wasn't detected in clove and cinnamon EO treatments at day 21 of the storage period, while B. subtilis vanished on day 14 for the cinnamon treatment and on day 21 for clove MRP. Moreover, the results revealed the enhancement of sensory characteristics of MRP supplemented with EOs without any significant alteration in their pH values. This study recommends the addition of cinnamon and clove EOs (0.6%) to MRP, as it isn't only an excellent substitution of chemical preservatives with powerful antibacterial efficiency but also improves the overall acceptance of the product. [ABSTRACT FROM AUTHOR]

Simple Summary: The experiment was conducted to investigate the effects of Bacillus subtilis on the growth and photosynthetic system of cucumber seedlings under different potassium levels. The pot experiment was conducted with "Xinjin 4" as the test material, and the two-factor experiment was designed. The two factors were different concentrations of potassium ion and Bacillus subtilis treatment. The effects of different treatments on growth, photosynthetic characteristics, root morphology and chlorophyll fluorescence parameters of cucumber seedlings were studied. The results showed that Bacillus subtilis had the most significant effect on cucumber seedling growth and leaf photosynthesis when the concentration of potassium ion was 0.2 g/pot. This study provided a theoretical basis for further utilizing Bacillus subtilis to make microbial fertilizer and improve the nutrient absorption efficiency of cucumber to promote the development of agriculture. Potassium deficiency is one of the important factors restricting cucumber growth and development. This experiment mainly explored the effect of Bacillus subtilis (B. subtilis) on cucumber seedling growth and the photosynthetic system under different potassium levels, and the rhizosphere bacteria (PGPR) that promote plant growth were used to solubilize potassium in soil, providing theoretical support for a further investigation of the effect of biological bacteria fertilizer on cucumber growth and potassium absorption. "Xinjin No. 4" was used as the test material for the pot experiment, and a two-factor experiment was designed. The first factor was potassium application treatment, and the second factor was bacterial application treatment. The effects of different treatments on cucumber seedling growth, photosynthetic characteristics, root morphology, and chlorophyll fluorescence parameters were studied. The results showed that potassium and B. subtilis had obvious promotion effects on the cucumber seedling growth and the photosynthesis of leaves. Compared with the blank control, the B. subtilis treatment had obvious effects on the cucumber seedling height, stem diameter, leaf area, total root length, total root surface area, total root volume, branch number, crossing number, gs, WUE, Ci, and A; the dry weight of the shoot and root increased significantly (p ≤ 0.05). Potassium application could significantly promote cucumber growth, and the effect of B. subtilis and potassium application was greater than that of potassium application alone, and the best effect was when 0.2 g/pot and B. subtilis were applied. In conclusion, potassium combined with B. subtilis could enhance the photosynthesis of cucumber leaves and promote the growth of cucumber. [ABSTRACT FROM AUTHOR]

Chitosan nanoparticles (CSNP) with an average size of 25.67 nm were synthesized via a novel wet chemical route and characterized using scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. Comparative antibacterial assays of Chitosan NP suspensions prepared in water (at neutral pH) and in dilute acetic acid and chitosan gel prepared in dilute acetic acid/hydrochloric acid (all at concentrations up to 1%) were performed against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) bacteria using spread plate method. A parallel viability test was conducted to confirm the presence of surviving cells in the bulk test volume. Minimum bactericidal concentration (MBC) of chitosan gel was 0.5 mg/ml for the bulk chitosan dissolved in 0.05% v/v aq. acetic acid and 0.1 mg/ml for chitosan dissolved in 0.01% v/v aqueous (aq.) hydrochloric acid. In comparison, Chitosan NP were found to be growth promoter at neutral pH and exhibited cell protective efficacy in presence of aq. acetic acid. The biocidal activity of chitosan gel in acidic media was higher when prepared in strong inorganic acid, that is, aq. HCl in comparison with the gel prepared in a relatively weak organic acid that is, aq. CH3COOH at the same concentration. Antibacterial action also showed pH dependence with higher activity at lower pH. However, respective aq. acids also gave comparable bactericidal action; indicating that chitosan may not have any inherent antibacterial property and basically it acts as a growth promoter. [ABSTRACT FROM AUTHOR]

Background: Meju is a base material for making soy sauce, soybean paste, and red chili pepper paste, which are representative ingredients of Korean cuisine. Objectives: This study aimed to isolate a predominant bacterial strain of B. subtilis from meju and to observe its inhibitory effects on an aflatoxigenic mold. Methods: We used yellow soybeans (Glycine max (L.) Merr.) grown in South Korea, and meju was produced according to the recommended methods of the Korea Food Research Institute. The identification of the strain was conducted based on its morphological and biochemical characteristics and 16S rDNA sequence. Evaluation of the bacterial effect against A. parasiticus ATCC 15517 was done in yeast extract–sucrose broth at 28 °C. Its inhibitory effect was evaluated using two approaches: mycelial weight and aflatoxin production. Aflatoxins were determined using high-performance liquid chromatography. Results: In the meju samples fermented for three months, a B. subtilis strain, K-0924 was identified. At the end of the incubation period of A. parasiticus, dry mycelial weight was significantly reduced by more than 80% (p < 0.01) and total aflatoxin production was inhibited by more than 63% (p < 0.05) in the presence of B. subtilis. Conclusions: These results indicate that B. subtilis K-0924 inhibits the growth and aflatoxin production of toxigenic Aspergillus, which can be contaminated with meju. We could expect more inhibition by other bacteria related to fermentation of meju, and further examination is necessary on species other than B. subtilis. [ABSTRACT FROM AUTHOR]

Recovery of rare earth elements (REEs) from wastewater with Bacillus subtilis (B. subtilis) during culture is promising due to its environmental benefits. However, the effects of REEs in the culture media on B. subtilis are poorly understood. This study aims to investigate the effects of the terbium (Tb(III)), a typical rare earth element, on the cell growth, sporulation, and spore properties of B. subtilis. Tb(III) can suppress bacterial growth while enhancing spore tolerance to wet heat. Spore germination and content of dipicolinic acid (DPA) were promoted at low concentrations of Tb(III) while inhibited at a high level, but an inverse effect on initial sporulation appeared. Scanning electron microscope and energy dispersive spectrometer detection indicated that Tb(III) complexed cells or spores and certain media components simultaneously. The germination results of the spores after elution revealed that Tb(III) attached to the spore surface was a key effector of spore germination. In conclusion, Tb(III) directly or indirectly regulated both the nutrient status of the media and certain metabolic events, which in turn affected most of the properties of B. subtilis. Compared to the coat-deficient strain, the wild-type strain grew faster and was more tolerant to Tb(III), DPA, and wet heat, which in turn implied that it was more suitable for the recovery of REEs during cultivation. These findings provide fundamental insights for the recovery of rare earths during the culture process using microorganisms. [ABSTRACT FROM AUTHOR]

Biodegradable gelatin coating embedded with different concentrations (1, 3 and 5%) of silver nanoparticles (AgNPs) designed as an antimicrobial packaging system to improve the quality of toast bread samples and prolong their shelf life when stored at room temperature for a 5-day period. The findings revealed a significant enhancement in the physical (film thickness, solubility, water vapor permeability, oxygen permeability) and mechanical (tensile strength and elongation at break) properties of the gelatin-based films upon the integration of AgNPs up to 3% (P < 0.05). SEM analysis displayed a homogeneous structure in the nanocomposite films when AgNPs were integrated at concentrations ≤ 3%. This was complemented by the confirmation of the interaction between the polymer and nanoparticles, as well as the crystallinity of AgNPs in the film, validated through FT-IR spectroscopy and XRD respectively. The evaluation of physicochemical and microbial characteristics of the coated bread revealed that samples containing 3% of AgNPs effectively preserved moisture content and water activity, minimized bread firmness and staleness, thereby maintaining the lightness index (L*) of the crust/crumb in comparison to the control (gelatin alone). Furthermore, all actively coated treatments, particularly those with AgNPs at concentrations ≥ 3%, demonstrated robust antimicrobial activity by preventing bacterial and mold growth during 5 days. [ABSTRACT FROM AUTHOR]

Peptones are one of the priciest culture components utilized in the cultivation of microorganisms. The objective of this work was to prepare feather hydrolysate peptone from chicken feathers using Bacillus subtilis ES5 (GenBank: OQ034477). The cell growth performances of all investigated microorganisms were shown to exhibit better results with feather hydrolysate peptone (FHP) in comparison to commercial peptone. As per the current findings, microbial-based FHP preparation is significantly less expensive than commercial peptones, and the suggested process provides cost-effective and eco-friendly solutions. Furthermore, the present investigation demonstrated opening the door to the prospect of converting chicken feather waste to peptone.

Background: Meju is a base material for making soy sauce, soybean paste, and red chili pepper paste, which are representative ingredients of Korean cuisine. Objectives: This study aimed to isolate a predominant bacterial strain of B. subtilis from meju and to observe its inhibitory effects on an aflatoxigenic mold. Methods: We used yellow soybeans (Glycine max (L.) Merr.) grown in South Korea, and meju was produced according to the recommended methods of the Korea Food Research Institute. The identification of the strain was conducted based on its morphological and biochemical characteristics and 16S rDNA sequence. Evaluation of the bacterial effect against A. parasiticus ATCC 15517 was done in yeast extract–sucrose broth at 28 °C. Its inhibitory effect was evaluated using two approaches: mycelial weight and aflatoxin production. Aflatoxins were determined using high-performance liquid chromatography. Results: In the meju samples fermented for three months, a B. subtilis strain, K-0924 was identified. At the end of the incubation period of A. parasiticus, dry mycelial weight was significantly reduced by more than 80% (p < 0.01) and total aflatoxin production was inhibited by more than 63% (p < 0.05) in the presence of B. subtilis. Conclusions: These results indicate that B. subtilis K-0924 inhibits the growth and aflatoxin production of toxigenic Aspergillus, which can be contaminated with meju. We could expect more inhibition by other bacteria related to fermentation of meju, and further examination is necessary on species other than B. subtilis.

Combining probiotic bacteria is a promising strategy to increase the effectiveness and avoid side effects of probiotic therapy. Bacteria that find themselves in a common environment are able to both change their properties and show new ones under the influence of each other. The change of colonization and antagonistic properties, which provide bacteria with competitive advantages in the development of new spaces, deserves special attention. The aim of this research was to study the features of the mutual influence of probiotic bacilli: B. clausii, B. coagulans and B. subtilis on growth, swimming and swarming motility when co-cultivated on agar media of different solidity. Materials and methods. The study used commercial strains of bacilli from three probiotic preparations: Enterogermina, Lactovit forte and Subalin. The ability of the studied species of bacilli to influence each other’s growth was investigated by the agar block method (using 1.5 % nutrient agar) and the spot-on-lawn assay (using 0.7 % nutrient agar). The study of the mutual influence on swimming and swarming activity was carried out when bacilli were cultivated on 0.25 % and 0.70 % tryptone agar, respectively. The diameters of swimming halos and migration swarms formed by monoand mixed cultures were measured, and the areas covered by them were compared. The phenotypes of the meeting of swarms formed by cultures spotted on the swarm plates at different locations were also investigated. Results. The studied probiotic species of the bacilli did not show a strong ability to inhibit each other’s growth. The B. clausii culture had no inhibitory effect, and the B. coagulans culture demonstrated a moderate inhibitory influence on the growth of the other two species of bacilli when using both diffusion methods. The B. subtilis culture showed moderate or weak inhibitory activity against the B. clausii culture and weak or no inhibitory activity against the B. coagulans culture using the agar block method or spot-on-lawn assay, respectively. The B. coagulans + B. subtilis, B. clausii + B. coagulans and B. clausii + B. subtilis mixed cultures showed moderate, weak and no inhibitory activity against third cultures, respectively. The studied species of bacilli showed different swimming ability and swarming potential as well as the ability to influence each other’s motility. Swimming halos formed by the B. clausii + B. subtilis and B. clausii + B. сoagulans + B. subtilis mixed cultures covered significantly larger plate areas than the swimming halos formed by each culture separately during the same cultivation time. The highest swarming potential was observed in B. coagulans culture and B. cla usii + B. coagulans + B. subtilis mixed culture. The studied bacilli did not show the ability to merge swarms, but, on the contrary, their swarms at the point of contact formed visible “boundary” or “intermediate” lines, demonstrating the ability to identify nonself. Conclusions. The obtained results indicate the ability of the probiotic species of bacilli: B. clausii, B. coagulans and B. subtilis to mutually influence growth, swimming and swarm motility. The highest indicators of swimming and swarming of the triple mixed culture indicate an increase in the colonization potential of the studied bacilli when they are combined. The hypothesis of mutual induction of inhibitory compounds production by bacilli, which may increase the overall antagonistic potential of the triple mixed culture, is subject to further confirmation.

The intensified concerns related to agrochemicals’ ecological and health risks have encouraged the exploration of microbial agents as eco-friendly alternatives. Some members of Bacillus spp. are potential plant-growth-promoting agents and benefit numerous crop plants globally. This study aimed to explore the beneficial effects of two Bacillus strains (B. subtilis strain IS1 and B. amyloliquificiens strain IS6) capable of alleviating the growth of tomato plants against salinity stress and Fusarium wilt disease. These strains were able to significantly promote the growth of tomato plants and biomass accumulation in pot trials in the absence of any stress. Under salinity stress conditions (150 mM NaCl), B. subtilis strain IS1 demonstrated superior performance and significantly increased shoot length (45.74%), root length (101.39%), fresh biomass (62.17%), and dry biomass (49.69%) contents compared to control plants. Similarly, B. subtilis strain IS1 (63.7%) and B. amyloliquificiens strain IS6 (32.1%) effectively suppressed Fusarium wilt disease and significantly increased plant growth indices compared to the pathogen control. Furthermore, these strains increased the production of chlorophyll, carotenoid, and total phenolic contents. They significantly affected the activities of enzymes involved in antioxidant machinery and the phenylpropanoid pathway. Hence, this study effectively demonstrates that these Bacillus strains can effectively alleviate the growth of tomato plants under multiple stress conditions and can be used to develop bio-based formulations for use in the fields.

One of the latest trends in sustainable agriculture is the use of beneficial microorganisms to stimulate plant growth and biologically control phytopathogens. Bacillus subtilis, a Gram-positive soil bacterium, is recognized for its valuable properties in various biotechnological and agricultural applications. This study presents, for the first time, the successful encapsulation of B. subtilis within electrospun poly(3-hydroxybutyrate) (PHB) fibers, which are dip-coated with cellulose derivatives. In that way, the obtained fibrous biohybrid materials actively ensure the viability of the encapsulated biocontrol agent during storage and promote its normal growth when exposed to moisture. Aqueous solutions of the cellulose derivatives—sodium carboxymethyl cellulose and 2-hydroxyethyl cellulose, were used to dip-coat the electrospun PHB fibers. The study examined the effects of the type and molecular weight of these cellulose derivatives on film formation, mechanical properties, bacterial encapsulation, and growth. Scanning electron microscopy (SEM) was utilized to observe the morphology of the biohybrid materials and the encapsulated B. subtilis. Additionally, ATR-FTIR spectroscopy confirmed the surface chemical composition of the biohybrid materials and verified the successful coating of PHB fibers. Mechanical testing revealed that the coating enhanced the mechanical properties of the fibrous materials and depends on the molecular weight of the used cellulose derivatives. Viability tests demonstrated that the encapsulated B. subtilis exhibited normal growth from the prepared materials. These findings suggest that the developed fibrous biohybrid materials hold significant promise as biocontrol formulations for plant protection and growth promotion in sustainable agriculture.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria. Synopsis: Ribosome stalling in the middle of a translated mRNA leads to collisions and triggers quality control pathways. Using structural and biochemical approaches, this study reveals how MutS2 detects and resolves ribosome collisions in Bacillus subtilis. MutS2 selectively binds collided ribosomes through interactions with its KOW and SMR domains. Unlike its E. coli homolog SmrB, B. subtlis MutS2 does not cleave mRNA on collided ribosomes. MutS2 splits stalled ribosomes into subunits using its ATPase activity. After splitting, the nascent peptide trapped on the 50 S subunit is targeted for degradation by RqcH. Structural and biochemical studies show how the B. subtilis MutS2 protein separates collided ribosomes, revealing striking differences to the actions of its E. coli homolog SmrB. [ABSTRACT FROM AUTHOR]

Antibacterial efficacy of chitosan-capped copper oxide nanoparticles (CuO–CS NPs) was evaluated against the Gram-negative bacteria (E. coli) and Gram-positive bacteria (B. subtilis). Comparative antibacterial activity evaluation was performed for CuO–CS NPs, nano Cu(OH)2, nano silver, CuO, and CuSO4.5H2O salt. Miles and Misra method and the spread plate CFU count method were used to enumerate cell counts for antibacterial activity evaluation. The spread plate colony forming unit (CFU) count method is preferable because of its reliability and accuracy. The minimum bactericidal concentration (MBC) of CuO–CS NPs were determined to be 2 µg/ml for E. coli and 12.5 µg/ml for B. subtilis. The values were comparable to those obtained for AgNPs. Moreover, in kinetic study these NPs showed the ability to decontaminate 2 × 105 CFU/ml of E. coli within 2 h at a concentration of 10 μg/ml and 3 × 105 CFU/ml of B. subtilis within 4 h at a concentration of 20 μg/ml. The potential application of synthesized CuO-CS NPS as an effective antibacterial nanocoating on fabrics was also demonstrated by using both qualitative and quantitative test methods. [ABSTRACT FROM AUTHOR]

Microorganisms are integral components of ecosystems, exerting profound impacts on various facets of human life. The recent United Nations General Assembly (UNGA) Science Summit emphasized the critical importance of comprehending the microbial world to address global challenges, aligning with the United Nations Sustainable Development Goals (SDGs). In agriculture, microbes are pivotal contributors to food production, sustainable energy, and environmental bioremediation. However, decades of agricultural intensification have boosted crop yields at the expense of soil health and microbial diversity, jeopardizing global food security. To address this issue, a study in West Bengal, India, explored the potential of a novel multi-strain consortium of plant growth promoting (PGP) Bacillus spp. for soil bioaugmentation. These strains were sourced from the soil’s native microbial flora, offering a sustainable approach. In this work, a composite inoculum of Bacillus zhangzhouensis MMAM, Bacillus cereus MMAM3), and Bacillus subtilis MMAM2 were introduced into an over-exploited agricultural soil and implications on the improvement of vegetative growth and yield related traits of Gylcine max (L) Meril. plants were evaluated, growing them as model plant, in pot trial condition. The study’s findings demonstrated significant improvements in plant growth and soil microbial diversity when using the bacterial consortium in conjunction with vermicompost. Metagenomic analyses revealed increased abundance of many functional genera and metabolic pathways in consortium-inoculated soil, indicating enhanced soil biological health. This innovative bioaugmentation strategy to upgrade the over-used agricultural soil through introduction of residual PGP bacterial members as consortia, presents a promising path forward for sustainable agriculture. The rejuvenated patches of over-used land can be used by the small and marginal farmers for cultivation of resilient crops like soybean. Recognizing the significance of multi-strain PGP bacterial consortia as potential bioinoculants, such technology can bolster food security, enhance agricultural productivity, and mitigate the adverse effects of past agricultural activities. [ABSTRACT FROM AUTHOR]

A delivery system for vectors for molecular cloning into bacterial cells of the genus Bacillus has been developed. A specific feature of the developed system is the use of the pBS72 plasmid, which provides the conjugative transfer of the conditionally lethal pKS1mob vector obtained into the cells of the studied bacteria. The ability of vector pKS1mob to be replicated in Escherichia coli and B. subtilis cells at a low temperature (30°C) and the presence of two polylinkers around the kanamycin resistance gene makes it possible to clone target gene fragments into its composition using traditional genetic engineering approaches. Inactivation of the rok gene in the pBS72 plasmid made it possible to transform the strain containing it with the constructed vector pKS1mob with high efficiency. Crossing the B. subtilis 168 donor strain, containing the conjugative pBS72 and pKS1mob mobilizable plasmids, with a recipient strain of the genus Bacillus made it possible to introduce the pKS1mob plasmid into it. The possibility of using the created system for inactivation of the codY gene of Bacillus licheniformis was shown. [ABSTRACT FROM AUTHOR]

The demand for soybeans as one of cereal commodities increases every year, however, to meet the production and demand level is still challenging in Indonesia. One of the causes of low soybean production is stress during cultivation, lack of nutrients, or poor quality of soybean seeds. One solution is to increase seed protection by coating the seeds with an environmentally friendly biological seed coating. This study aims to obtain the best biological seed coating for increasing soybean seed viability, vigor, and yield. The research was conducted at the Faculty of Agriculture Experimental Station of Universitas Padjadjaran, Jatinangor from August to November 2021. Two sets of experimental design were employed in this study, a Completely Randomized Design for the viability and vigor parameters in laboratory, and a Randomized Block Design with four replications for evaluation of the yield components in the field. The experiment was carried out with a combination of soybean cultivar and biological seed coating. The cultivars used in this study were Anjasmoro cv. (yellow) and Detam-4 cv. (black) and the biological seed coatings were B. subtilis, Trichoderma sp., and Rhizobium sp. Post-hoc test after F-test used Least Significance Different (LSD) with a significant level of 5%. The results showed that the application of the three biological seed coatings was not significantly affect seed physiological traits. However, the seed coating treatments increased the number of seeds per plant and seed weight per plant in yellow soybeans. Meanwhile, the application of B. subtilis and Rhizobium sp. in black soybeans increased the number of seeds per plant and the weight of seeds per plant. The best biological seed coating in this experiment was B. subtilis. [ABSTRACT FROM AUTHOR]

The present study reports an easy eco-friendly, cost efficient, and rapid method for the synthesis of silver nanoparticles (Ag NPs) using palm sprouts as reducing cum capping agent. Green synthesis of silver nanoparticles was successfully performed using palm sprouts plant extract via a simple and cheaper eco-friendly method. Palm sprouts extract reduces silver nitrate to silver nanoparticles. The resulting materials were analyzed by Fourier-transform infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and X-ray Diffraction (XRD) analysis. FT-IR spectrum confirms the presence of various functional groups in the active biomolecules, it acts as a capping agent for the nanoparticles. The morphology of this sample was analyzed through SEM and the presence of silver was confirmed accordingly. The green synthesized Ag NPs exhibited an excellent antibacterial activity against E. coli and P. aeruginosa and B. subtilis and S. aureus besides imparting efficient antimicrobial activity against pathogenic bacteria as well. [ABSTRACT FROM AUTHOR]

ABSTRACT: In the current study, the proteolytic enzyme (serratiopeptidase) was used to enhance the efficacy of Bacillus subtilis (B. subtilis) probiotic as a growth promotor in broiler chicken. The effects of serratiopeptidase on the efficacy of different levels of B. subtilis as a growth promotor in broiler chicks were evaluated regarding growth performance traits, villus histomorphometric characterization, and intestinal microbiota count. Day-old broiler chicks (n = 120) were allocated into 4 groups having 3 replicates/group. In the control group (C), the basal diet was kept without supplementation. In treatment groups (P100, P150, and P200), the basal diet was supplemented with 100, 150, and 200 mg probiotics, respectively besides 30 mg proteolytic enzyme in the 3 treated groups for 4 wk. The performance parameters were significantly affected by the supplementation of serratiopeptidase to the B. subtilis treatment groups. Feed intake (FI), body weight gain (WG), feed conversion ratio (FCR), and dressing percent were significantly improved in the treatment groups as compared to the control group. Significantly, the lowest feed intake was recorded for the P200 group. The highest body weight gain and dressing percentage were recorded for the P200 group. An improved FCR was recorded in the P200 group (1.7) as compared to the control group. The different levels of B. subtilis supplemented with serratiopeptidase revealed significant improvements (P

Abstract Over 380 host plant species have been known to develop leaf spots as a result of the fungus Alternaria alternata. It is an aspiring pathogen that affects a variety of hosts and causes rots, blights, and leaf spots on different plant sections. In this investigation, the lipopeptides from the B. subtilis strains T3, T4, T5, and T6 were evaluated for their antifungal activities. In the genomic DNA, iturin, surfactin, and fengycin genes were found recovered from B. subtilis bacterium by PCR amplification. From different B. subtilis strains, antifungal Lipopeptides were extracted, identified by HPLC, and quantified with values for T3 (24 g/ml), T4 (32 g/ml), T5 (28 g/ml), and T6 (18 g/ml). To test the antifungal activity, the isolated lipopeptides from the B. subtilis T3, T4, T5, and T6 strains were applied to Alternaria alternata at a concentration of 10 g/ml. Lipopeptides were found to suppress Alternaria alternata at rates of T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). The T6 strain outperformed the other three by having the highest antifungal activity against Alternaria alternata (85.88%).

ABSTRACT The effects of surface mechanical constraints that may promote or prevent bacterial expansion on semi-solid surfaces are largely unknown. In this work, we have manufactured agar surfaces with different viscoelasticity, topography, and roughness. To capture the essential biophysics of the bacterial expansion we have developed a continuum model that faithfully reproduces the main patterns of the short-range and long-range expansion with two critical parameters: local interfacial forces and colony viscosity. Cohesive energy of the bacterial colony that determines the extent of exploration was dependent on agar surface viscoelasticity. On soft surfaces, bacteria produce low viscoelastic colonies that allow guided population of bacteria to traverse distances that are six orders of magnitude larger than the size of the individual bacterium. Bacteria growing on stiff surfaces produce colonies with significantly increased viscoelasticity that prevent bacterial exploration of new territory and allow formation of a very steep cliff at the edge of the colony. Upon flooding of the rough surfaces, we have induced aquaplaning and spreading of bacteria. A layer of water between the bacterium and surface results in a loss of traction allowing bacteria to spread across the otherwise inhibitory rough surface. The results shed new light on the bacterial ability to rapidly colonize new territories. IMPORTANCE How bacterial cells colonize new territory is a problem of fundamental microbiological and biophysical interest and is key to the emergence of several phenomena of biological, ecological, and medical relevance. Here, we demonstrate how bacteria stuck in a colony of finite size can resume exploration of new territory by aquaplaning and how they fine tune biofilm viscoelasticity to surface material properties that allows them differential mobility. We show how changing local interfacial forces and colony viscosity results in a plethora of bacterial morphologies on surfaces with different physical and mechanical properties.

Microorganisms are integral components of ecosystems, exerting profound impacts on various facets of human life. The recent United Nations General Assembly (UNGA) Science Summit emphasized the critical importance of comprehending the microbial world to address global challenges, aligning with the United Nations Sustainable Development Goals (SDGs). In agriculture, microbes are pivotal contributors to food production, sustainable energy, and environmental bioremediation. However, decades of agricultural intensification have boosted crop yields at the expense of soil health and microbial diversity, jeopardizing global food security. To address this issue, a study in West Bengal, India, explored the potential of a novel multi-strain consortium of plant growth promoting (PGP) Bacillus spp. for soil bioaugmentation. These strains were sourced from the soil’s native microbial flora, offering a sustainable approach. In this work, a composite inoculum of Bacillus zhangzhouensis MMAM, Bacillus cereus MMAM3), and Bacillus subtilis MMAM2 were introduced into an over-exploited agricultural soil and implications on the improvement of vegetative growth and yield related traits of Gylcine max (L) Meril. plants were evaluated, growing them as model plant, in pot trial condition. The study’s findings demonstrated significant improvements in plant growth and soil microbial diversity when using the bacterial consortium in conjunction with vermicompost. Metagenomic analyses revealed increased abundance of many functional genera and metabolic pathways in consortium-inoculated soil, indicating enhanced soil biological health. This innovative bioaugmentation strategy to upgrade the over-used agricultural soil through introduction of residual PGP bacterial members as consortia, presents a promising path forward for sustainable agriculture. The rejuvenated patches of over-used land can be used by the small and marginal farmers for cultivation of resilient crops like soybean. Recognizing the significance of multi-strain PGP bacterial consortia as potential bioinoculants, such technology can bolster food security, enhance agricultural productivity, and mitigate the adverse effects of past agricultural activities.

The uncertainty associated with the impact of a bioinoculant on soil microbial community and, as a consequence, on soil quality, as well as the need to define its persistence, has prompted the demand for an accurate detection and tracking of the presence and the quantification of a target microbial inoculant in soil. Although DNA or RNA-based molecular detection are well established and commonly applied in this regard, alternative ligands such as DNA-aptamers have several advantages over them, such as low cost, ease of modification, ease of immobilisation on lab-on-chip or nanosensors, high stability and not thermolability. In this study, we used a toggle-cell SELEX method to isolate, select and characterise ssDNA (single-strand DNA) aptamers to detect a Bacillus subtilis strain which is being tested as a plant growth promoting rhizobacteria (PGPR) formulation. Two ssDNA aptamers (patenting application n.102022000022590) showed strong affinity and specificity for B. subtilis strains, with values of the kinetic parameters Kd (dissociation constant) in the nanomolar range and Bmax (maximum intensity of binding) around 1. Validation of the suitability of the aptamers was validated on three inoculated soils characterised by different chemical-physical features and in soil from a field trial with the formulated B. subtilis PCM/B 00105 strain. These are considered significant features to monitor B. subtilis strains in soil, practical to optimise bioinoculant application methods, support regulatory processes and foster the shift of agricultural production toward more sustainable cropping systems. Key points: • First DNA aptamers binding a B. subtilis strain included in a bioinoculum formulation. • First DNA aptamer binding B. subtilis in soil. • Aptamer may be a method for microbial inoculant detection in soil. [ABSTRACT FROM AUTHOR]

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications. [ABSTRACT FROM AUTHOR]

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill‐defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch‐to‐batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96‐well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL‐based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL‐glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated. [ABSTRACT FROM AUTHOR]

The functional characteristics of the Rap‒Phr quorum sensing signal system of the plasmid pBS72 were investigated. Phylogenetic relationship was revealed between Rap phosphatase coded by the plasmid pBS72 and the homologous polypeptides RapP and RapI determined by the plasmid pBS32 (68.4% identity) and the ICEBs1 conjugative transposon (36.9% identity). Similar to the phylogenetically related phosphatases, the studied Rap protein had a negative effect on sporulation. Unlike the known signal systems, Rap‒Phr proteins were found to affect the viability of plasmid-bearing donor bacteria in the course of conjugative transfer of the plasmid pBS72 in the isogenic system. Impaired rap-phr genes resulted in a 10-fold decrease in the number of viable donor cells with the mutant plasmid after 3 and 4 h, and in a 100-fold decrease after 24 h. The number of formed transconjugants remained almost the same. Our results provide the basis for investigation of the mechanisms responsible for the effect of extrachromosomal genetic elements on the donor bacteria, providing for the propagation of the pBS72-like plasmids in natural environments. [ABSTRACT FROM AUTHOR]

The structural features of bacterial cellulose (BC) make it very popular to use it for creating compos- ite materials. Immobilization of bacteria of the Bacillus genus in BC polymer gives the carrier matrix new functional properties that help restore and maintain a favorable balance of microflora in the gastrointesti- nal tract. The addition of sodium alginate had a positive effect on the synthesis of BC by K. xylinus C-3, providing a polymer yield of 12.11 g/l. The "adsorption-incubation" method was used for surface and spatial immobilization of Bacillus subtilis, which provides a high concentration of cells -- 108 CFU per 1 g of carrier. The BC/Bacillus biocomposite inhibited the growth of E. coli, S. typhi, S. typhimurium, Citr. aerogenes, and P. vulgaris up to 85.4% after 24 hours of contact, and after 72 hours completely inhibited the growth and viability of test organisms in vitro. Immobilized probiotics significantly exceeded the suspension of free microbial ells in terms of resistance to gastric juice. The activity of probiotics in vivo determined on models of experimental dysbiosis. Probiotic microgranules of BC/B.subtilis can be used to correct the microbiocenosis of the large intestine. [ABSTRACT FROM AUTHOR]

Simple Summary: The objective of this study was to test the hypothesis that B. subtilis and B. licheniformis supplementation in a negative control diet (in comparison to a standard control diet) has the potential to improve performance and nutrient digestibility of growing–finishing pigs. Three fattening pig groups were used: standard diet, negative control diet (5% soybean meal replaced by 5% rapeseed meal), or the negative control diet supplemented with a probiotic. The use of a probiotic preparation containing specific Bacillus strains as a feed additive for growing–finishing pigs resulted in improved growth performance and faecal digestibility, and was able to counteract the lower nutritional level of a negative control diet. The objective of the present study was to test the hypothesis of B. subtilis and B. licheniformis supplementation to a negative control diet in comparison to a standard control diet, had the potential to improve the performance and nutrient digestibility of growing–finishing pigs. For this purpose, 384 fattening pigs of 85 d of age were allotted to three treatments: a standard diet, a negative control (NC) diet (5% soybean meal replaced by 5% rapeseed meal), or a NC diet + probiotic. After reaching a body weight of approximately 110 kg, all animals going to the slaughterhouse (87% of total pigs) were selected to measure carcass quality. Moreover, the apparent total tract digestibility of protein was evaluated at the end of the grower period. The results of this study indicate that supplementation of the tested Bacillus-based probiotic significantly improved average daily gain (ADG, +14.6%) and Feed:gain ratio (F:G, −9.9%) during the grower phase compared to the NC diet. The improvement observed during the grower phase was maintained for the whole fattening period (ADG, +3.9%). Probiotic supplementation significantly improved the total apparent faecal digestibility of dry matter and crude protein in pigs at the end of the grower period. The improvements observed with the additive tested could indicate that supplementation of the Bacillus-based probiotic was able to counteract the lower level of crude protein and standardised ileal digestible amino acids in the NC diet by means of improved protein digestibility. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV)-excited visible fluorescence is an attractive option for low-cost, low-complexity, rapid imaging of bacterial and fungal samples for imaging diagnostics in the biomedical community. While several studies have shown there is potential for identification of microbial samples, very little quantitative information is available in the literature for the purposes of diagnostic design. In this work, two non-pathogenic bacteria samples (E. coli pYAC4, and B. subtilis PY79) and a wild-cultivated green bread mold fungus sample are characterized spectroscopically for the purpose of diagnostic design. For each sample, fluorescence spectra excited with low-power near-UV continuous wave (CW) sources, and extinction and elastic scattering spectra are captured and compared. Absolute fluorescence intensity per cell excited at 340 nm is estimated from imaging measurements of aqueous samples. The results are used to estimate detection limits for a prototypical imaging experiment. It was found that fluorescence imaging is feasible for as few as 35 bacteria cells (or ∼ 30 µm3 of bacteria) per pixel, and that the fluorescence intensity per unit volume is similar for the three samples tested here. A discussion and model of the mechanism of bacterial fluorescence in E. coli is provided. [ABSTRACT FROM AUTHOR]

Abstract Commercial carotenoid production is dominated by chemical synthesis and plant extraction, both of which are unsustainable and can be detrimental to the environment. A promising alternative for the mass production of carotenoids from both an ecological and commercial perspective is microbial synthesis. To date, C30 carotenoid production in Bacillus subtilis has been achieved using plasmid systems for the overexpression of biosynthetic enzymes. In the present study, we employed a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system to develop an efficient, safe, and stable C30 carotenoid-producing B. subtilis strain, devoid of plasmids and antibiotic selection markers. To this end, the expression levels of crtM (dehydrosqualene synthase) and crtN (dehydrosqualene desaturase) genes from Staphylococcus aureus were upregulated by the insertion of three gene copies into the chromosome of B. subtilis. Subsequently, the supply of the C30 carotenoid precursor farnesyl diphosphate (FPP), which is the substrate for CrtMN enzymes, was enhanced by expressing chromosomally integrated Bacillus megaterium-derived farnesyl diphosphate synthase (FPPS), a key enzyme in the FPP pathway, and abolishing the expression of farnesyl diphosphate phosphatase (YisP), an enzyme responsible for the undesired conversion of FPP to farnesol. The consecutive combination of these features resulted in a stepwise increased production of C30 carotenoids. For the first time, a B. subtilis strain that can endogenously produce C30 carotenoids has been constructed, which we anticipate will serve as a chassis for further metabolic engineering and fermentation optimization aimed at developing a commercial scale bioproduction process.

Treatment of wastewater has been a hotspot of research since ages. Emerging technologies and methodologies have been postulated to resolve the issue worldwide. Almost 97.2% of our earth is covered with water bodies, out of which 12,500 water bodies are situated in India. As per the statistics, nearly 70% water bodies are polluted in Southern Asia. Presence of emerging pollutants exacerbate the quality of flowing water. Amongst all possible ways, microbial bioremediation has been considered one of the most thriving methods to treat wastewater. This research will manifest about the isolation of B. subtilis from soil followed by its characterisation and action in treatment of wastewater which was collected from industry. From the study, it was concluded that B. subtilis holds the potential of degradation. Significant decrease in values of BOD and COD were achieved.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMβ) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that S. felis antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.

The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.

Potassium deficiency is one of the important factors restricting cucumber growth and development. This experiment mainly explored the effect of Bacillus subtilis (B. subtilis) on cucumber seedling growth and the photosynthetic system under different potassium levels, and the rhizosphere bacteria (PGPR) that promote plant growth were used to solubilize potassium in soil, providing theoretical support for a further investigation of the effect of biological bacteria fertilizer on cucumber growth and potassium absorption. “Xinjin No. 4” was used as the test material for the pot experiment, and a two-factor experiment was designed. The first factor was potassium application treatment, and the second factor was bacterial application treatment. The effects of different treatments on cucumber seedling growth, photosynthetic characteristics, root morphology, and chlorophyll fluorescence parameters were studied. The results showed that potassium and B. subtilis had obvious promotion effects on the cucumber seedling growth and the photosynthesis of leaves. Compared with the blank control, the B. subtilis treatment had obvious effects on the cucumber seedling height, stem diameter, leaf area, total root length, total root surface area, total root volume, branch number, crossing number, gs, WUE, Ci, and A; the dry weight of the shoot and root increased significantly (p ≤ 0.05). Potassium application could significantly promote cucumber growth, and the effect of B. subtilis and potassium application was greater than that of potassium application alone, and the best effect was when 0.2 g/pot and B. subtilis were applied. In conclusion, potassium combined with B. subtilis could enhance the photosynthesis of cucumber leaves and promote the growth of cucumber.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease that causes significant yield losses in wheat production worldwide, including in Egypt. The use of biocontrol agents is among the best eco-friendly management strategies to control this disease, as they are more sustainable and environmentally friendly than traditional chemical control methods. In a comparative analysis, antioxidant enzyme activity and various management approaches were compared with two bacterial biocontrol agents, Bacillus subtilis and Pseudomonas putida. This study showed the remarkable efficacy of endophytic bacteria, B. subtilis and P. putida, in mitigating wheat stripe rust infection across three wheat varieties, namely Misr1, Gimmeiza11, and Sids12. B. subtilis exhibited superior performance compared to P. putida, resulting in infection types of 1 and 2.66, respectively, following inoculation. The highest reduction rate was observed with Tilit fungicide (500 ppm), followed by B. subtilis and Salicylic acid (1000 ppm), respectively. Variations in wheat varieties' response to Pst infection were observed, with Misr1 exhibiting the lowest infection and Sids12 showing high susceptibility. Among the tested inducers, Salicylic acid demonstrated the greatest reduction in disease infection, followed by Indole acetic acid, while Oxalic acid exhibited the lowest decrease. Additionally, the study evaluated the activities of five antioxidant enzymes, including Catalase, Ascorbate peroxidase (APX), glutathione reductase (GR), Superoxide dismutase (SOD), and peroxidase (POX), in the wheat-stripe rust interaction under different integrated management approaches. The wheat variety Misr1 treated with Tilit (500 ppm), B. subtilis, Salicylic acid, Montoro (500 ppm), and P. putida exhibited the highest increase in all enzymatic activities. These findings provide valuable insights into the effectiveness of B. subtilis and P. putida as biocontrol agents for wheat stripe rust control in Egypt, emphasizing their potential role in sustainable, integrated, and environmentally friendly management practices. [ABSTRACT FROM AUTHOR]

Over 380 host plant species have been known to develop leaf spots as a result of the fungus Alternaria alternata. It is an aspiring pathogen that affects a variety of hosts and causes rots, blights, and leaf spots on different plant sections. In this investigation, the lipopeptides from the B. subtilis strains T3, T4, T5, and T6 were evaluated for their antifungal activities. In the genomic DNA, iturin, surfactin, and fengycin genes were found recovered from B. subtilis bacterium by PCR amplification. From different B. subtilis strains, antifungal Lipopeptides were extracted, identified by HPLC, and quantified with values for T3 (24 g/ml), T4 (32 g/ml), T5 (28 g/ml), and T6 (18 g/ml). To test the antifungal activity, the isolated lipopeptides from the B. subtilis T3, T4, T5, and T6 strains were applied to Alternaria alternata at a concentration of 10 g/ml. Lipopeptides were found to suppress Alternaria alternata at rates of T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). The T6 strain outperformed the other three by having the highest antifungal activity against Alternaria alternata (85.88%). [ABSTRACT FROM AUTHOR]

Bacillus subtilis is traditionally classified as a PGPR that colonizes plant roots through biofilm formation. The current study focused on investigating the influence of various factors on bacilli biofilm formation. In the course of the study, the levels of biofilm formation by the model strain B. subtilis WT 168 and on its basis created regulatory mutants, as well as strains of bacilli with deleted extracellular proteases under conditions of changes in temperature, pH, salt and oxidative stress and presence of divalent metals ions. B. subtilis 168 forms halotolerant and oxidative stress-resistant biofilms at a temperature range of 22 °C–45 °C and a pH range of 6–8.5. The presence of Ca2+, Mn2+ and Mg2+ upsurges the biofilm development while an inhibition with Zn2+. Biofilm formation level was higher in protease-deficient strains. Relative to the wild-type strain, degU mutants showed a decrease in biofilm formation, abrB mutants formed biofilms more efficiently. spo0A mutants showed a plummeted film formation for the first 36 h, followed by a surge after. The effect of metal ions and NaCl on the mutant biofilms formation is described. Confocal microscopy indicated that B. subtilis mutants and protease-deficient strains differ in matrix structure. The highest content of amyloid-like proteins in mutant biofilms was registered for degU-mutants and protease-deficient strains. [ABSTRACT FROM AUTHOR]

Introduction: The number of published biofilm studies and novel ways for studying them has risen dramatically in recent years, ow-ing to the broad application of biofilms in medicine. Some bacteria develop biofilms that are highly resistant to antimicrobial agents, resulting in persistent infections. This necessitates the development of alternative methods for combating biofilms. In this regard, the application of cationic polymers is a good candidate for realization of this strategy. Aim: The aim of our study was to investigate the potential of a newly synthesized covalently attached star copolymer of N,N’-dimeth-ylaminoethyl methacrylate and hydroxyl-bearing oligo(ethylene glycol) methacrylate [P(DMAEMA-co-HOEGMA)] to silica surfaces and its quaternized version [P(QDMAEMA-co-HOEGMA)] for destruction of biofilms formed by Bacillus subtilis or Pseudomonas aeruginosa. Materials and methods: Model strains representing different genera and taxonomic groups were selected for the study. The anti-biofilm activities of two different newly synthesized cationic polymers were investigated by observation (live/dead staining) of the viability of bacterial cells within the biofilm.Results: The results obtained by the live/dead labeling of bacterial biofilms show a substantial decrease in the viability of population in the presence of cationic polymers, better expressed at B. subtilis.Conclusions: The studied two immobilized on silica wafers newly synthesized star copolymers exhibited potential for anti-biofilm effects. The results demonstrated combined potential for reducing the viability of bacterial cells within the biofilms and probably for loosening the biofilm matrix. The effect was better expressed in B. subtilis.

Abstract Background Despite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming. Results In this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes. Conclusions The novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.