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Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq's utility is demonstrated, with future applications in ecology, health, and engineering.

UNLABELLED: Bacillus subtilis is an important industrial and environmental microorganism known to occupy many niches and produce many compounds of interest. Although it is one of the best-studied organisms, much of this focus including the reconstruction of genome-scale metabolic models has been placed on a few key laboratory strains. Here, we substantially expand these prior models to pan-genome-scale, representing 481 genomes of B. subtilis with 2,315 orthologous gene clusters, 1,874 metabolites, and 2,239 reactions. Furthermore, we incorporate data from carbon utilization experiments for eight strains to refine and validate its metabolic predictions. This comprehensive pan-genome model enables the assessment of strain-to-strain differences related to nutrient utilization, fermentation outputs, robustness, and other metabolic aspects. Using the model and phenotypic predictions, we divide B. subtilis strains into five groups with distinct patterns of behavior that correlate across these features. The pan-genome model offers deep insights into B. subtilis metabolism as it varies across environments and provides an understanding as to how different strains have adapted to dynamic habitats. IMPORTANCE: As the volume of genomic data and computational power have increased, so has the number of genome-scale metabolic models. These models encapsulate the totality of metabolic functions for a given organism. Bacillus subtilis strain 168 is one of the first bacteria for which a metabolic network was reconstructed. Since then, several updated reconstructions have been generated for this model microorganism. Here, we expand the metabolic model for a single strain into a pan-genome-scale model, which consists of individual models for 481 B. subtilis strains. By evaluating differences between these strains, we identified five distinct groups of strains, allowing for the rapid classification of any particular strain. Furthermore, this classification into five groups aids the rapid identification of suitable strains for any application.

Traditionally, diseases have been managed with synthetic chemicals, but concerns over their environmental and health impacts have led to increased interest in biological control methods as sustainable alternatives. This study explores the potential of the seed-borne endophytes for ecofriendly biological control agent against brown spot Bipolaris oryzae (Breda de Haan) and sheath blight disease (Rhizoctonia solani Kuhn) in rice. The findings revealed that B. subtilis RE-1 effectively reduced the mycelial growth of brown spot and sheath blight pathogen up to 46.27% and 40.37%, respectively under in vitro conditions. Endophytes produced extracellular hydrolytic enzymes viz., amylase, protease, cellulase and lipase that degrade fungal mycelia. Additionally, B. subtilis RE-1 synthesized antimicrobial peptides (AMPs) including Iturin (C, D), Bacilysin, Fengycin D, Mycosubtilin, Bacillomycin (A, D) and hydrolytic enzymes viz., b-glucanase, which collectively demonstrated potent antimicrobial and antifungal activities. The identification of six antimicrobial lipopeptides genes and one hydrolytic enzyme gene in seed-borne endophytes is a novel finding of this study, highlighting its comprehensive disease-fighting potential. Furthermore, B. subtilis enhances seed germination rates to 96% and increases the vigor index to 1574.784, compared to the control seeds with 92% germination and a vigor index of 1393.984. The augmentation of seedling growth can be attributed to the release of IAA, siderophore and ammonia utilization. These findings underscore B. subtilis as a promising endophyte that could enhance rice production sustainably. [ABSTRACT FROM AUTHOR]

This study investigates the effect of dietary Bacillus subtilis and Lactobacillus on the growth performance, serum biochemistry, nutrient apparent digestibility, and cecum flora of broilers under heat stress (HS) and provides a theoretical basis for the application of probiotic additives to alleviate the stress of poultry under HS. A total of 200 Cobb broilers were randomly assigned to four replicates of 10 broilers in each of the five groups. The growth performance, serum biochemistry, nutrient apparent digestibility, and cecum flora of broilers were detected on the 28th, 35th, and 42nd days, respectively. Results revealed that HS can affect the growth performance and serum biochemical indexes of broilers, lowered the number of intestinal bifidobacteria and Lactobacillus, and increase the number of Escherichia coli in comparsion to the CON group. Compared with the HS group, the ADFI of HS broilers in the BS group and the combined group significantly increased (P < 0.05) at 22–28 days of age, and the serum calcium and phosphorus increased (P < 0.05) significantly at 42 days of age. Meanwhile, the number of Lactobacillus in the BS group and LAB group increased significantly at 42 days of age (P < 0.05). The number of Escherichia coli in the LAB group and combination group decreased significantly at 35 days of age (P < 0.01). The present study revealed that the addition of Bacillus subtilis or Lactobacillus to diets increased ADFI, increased probiotic counts, and lowered Escherichia coli counts in HS broilers, while probiotics alone work well. [ABSTRACT FROM AUTHOR]

Marine bacteria release exopolymeric substances (EPS) molecules released in response to the harsh environmental conditions of the environment as a survival strategy. Nitrogen or carbon sources affects the metabolic synthesis of EPS in microorganisms. In this study, 2 different nutritional sources were used to extract EPS from a marine bacterium associated with the sponge Hyrtios erectus collected from the Red Sea. The antibacterial activity and chemical composition of the EPS from Bacillus subtilis M5 grown in Zobell marine broth (ZBE) and nutrient broth (NBE) was studied. The organism demonstrated potential activity against tested pathogenic bacteria. We observed differences in the antibacterial activity and the chemical composition of EPS polymers isolated from the organism. Fourier transform infrared (FTIR) spectrum revealed the peaks for carbonyl, alkyl and hydroxyl functional groups confirming the presence of functional groups characteristic of polysaccharides. Minor differences was observed in the relative intensities and slight peak shifts between the two spectra may be attributed to variations in the specific EPS compositions or extraction conditions. Scanning electron microscopic images revealed crystalline and amorphous characteristics of EPS for ZBE and NBE respectively. Only little differences were observed between the two EPS biopolymers both in antibacterial activity and chemical composition. [ABSTRACT FROM AUTHOR]

Camellia oleifera is an important edible oil woody plant in China. Anthracnose is a serious disease of C. oleifera, causing severe economic losses and posing a huge threat to the C. oleifera industry. The fungi Colletotrichum siamense is one of the main pathogens causing anthracnose of C. oleifera. In this study, antifungal activity of microorganisms isolated from garbage enzyme against C. siamense were investigated for the first time. 8 strains were isolated and purified from garbage enzyme. By morphological observation, gene sequence analysis, among the strains, 4 isolates were identified as Trichoderma harzianum and 4 isolates were identified as Bacillus subtilis. The result showed that 8 isolates displayed a relatively good potential on inhibiting the mycelial growth in C. siamense, but T. harzianum was more effective than B. subtilis as a biocontrol agent, with the best efficiency of 76.5%. In the vitro treatments, the combination of T. harzianum and B. subtilis were the most effective treatments in reducing the incidence and severity of anthracnose in C.oleifera, with the highest control efficiency of 77.56%. It can be concluded that T. harzianum and B. subtilis are promising biocontrol agents to manage anthracnose. These promising results provided valuable information on using garbage enzyme of wastes as a new source of antagonists to control fungi disease. [ABSTRACT FROM AUTHOR]

In the present work, new heterobimetallic complexes with the general formula [Zn(C16H32N8)M2(R)4Cl2], where M = Sn (IV) and Si (IV) and R = ‐CH3 and ‐C6H5, were synthesized by reacting the methanolic solution of the monometallic complex [Zn(C16H36N8)Cl2] with group 14 organometallic dichloride [(CH3)2SnCl2/(C6H5)2SnCl2/(CH3)2SiCl2/(C6H5)2SiCl2]. The structures of the synthesized complexes were determined using several spectrometric techniques, including FT‐IR, 1H NMR, 13C NMR, 119Sn NMR, 29Si NMR, ESI‐MS, PXRD, and molar conductivity measurements. Density functional theory (DFT) calculations were carried out to investigate the quantum chemical characteristics of the newly synthesized Zn (II) complexes. The antimicrobial potential of the monometallic and heterobimetallic complexes was evaluated against two Gram‐positive bacteria (Bacillus subtilis and Bacillus cereus), two Gram‐negative bacteria (Xanthomonas campestris and Pseudomonas aeruginosa), and two fungal strains (Aspergillus flavus and Fusarium oxysporum) by the agar well diffusion method, with heterobimetallic complex [Zn(C16H32N8)Sn2(C6H5)4Cl2] as the most potent complex against microbes. The complex [Zn(C16H32N8)Sn2(C6H5)4Cl2] exhibited significant potential in inhibiting inflammation, demonstrating an IC50 value of 9.75 μg/mL, which closely rivals that of the standard drug sodium diclofenac (IC50 = 4.59 μg/mL). Furthermore, all the synthesized compounds were assessed for their anticancer potential against three cancer cell lines (HCT‐116, MCF‐7, and HeLa) by the MTT assay. The results indicated that the complex [Zn(C16H32N8)Sn2(C6H5)4Cl2] exhibited the most pronounced cytotoxic effects, with values of 4.25 ± 0.15 μM, 4.90 ± 0.18 μM, and 5.20 ± 0.24 μM observed for HCT‐116, MCF‐7, and HeLa cancer cell lines, respectively. [ABSTRACT FROM AUTHOR]

In our work, we have successfully synthesized twelve diorganotin(IV) complexes (4–15) from different hydrazone ligands and diorganotin(IV) dichlorides. The complexes have a general formula [R2SnL1–3], where R = Et, Me, Bu, and Ph groups. The hydrazone ligands (1–3) were synthesized through a condensation reaction involving 3‐ethoxysalicylaldehyde with different benzhydrazide derivatives. All the synthesized hydrazone ligands and their complexes underwent screening by employing numerous spectroscopic and physicochemical techniques, such as molar conductance measurements, mass spectrometry, (1H, 119Sn, and 13C) NMR, SEM‐EDAX, and FT‐IR. Spectroscopic analysis revealed that the hydrazone ligands were attached to tin atoms in a tridentate fashion via ONO donor atoms, suggesting a pentacoordinated geometry for the complexes. Furthermore, the X‐ray crystallography analysis for Complex 6 revealed the distorted square pyramidal geometry around the tin atom. Moreover, a DFT study was also carried out for Ligand 3 and its complexes (12–15) by employing B3LYP/LanL2DZ theory level (Gaussian 9 software package) to obtain the optimized geometry and global reactivity descriptors, structural behavior, and their efficacy against different microbes. To assess the biological efficacy of synthesized compounds, an in vitro antimicrobial assay was conducted against different fungal (Aspergillus niger and Candida albicans) and bacterial (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa) strains. The results of the antimicrobial assay demonstrated that Complexes 7, 11, and 15 depicted better results against E. coli strain and C. albicans strain, implying that the higher lipophilic character of these complexes facilitates their easy passage through the cell membrane of microbes. Furthermore, an ADMET study was carried out to evaluate the toxicity score of synthesized compounds. [ABSTRACT FROM AUTHOR]

Pistacia vera L. hull, a the major byproduct of pistachio processing, is a source of functional compounds with antioxidant and antimicrobial activities. The extraction of these natural compounds from pistachio hulls and their use instead of synthetic chemicals has gained great attention. In this work, the phytochemical contents and antioxidant and antimicrobial activities of pistachio hull ethanolic (PVE) and aqueous (PVD) extracts obtained by microwave-assisted extraction (MAE) were investigated. Gallic acid (1.9 and 1.5 mg/g dw), quercetin (0.025 and 0.009 mg/g dw), total phenolic (23.3 and 14.7 mg GAE/g dw) and flavonoid (5.0 and 2.9 mg QE/g dw) contents and antioxidant activities (SC50 0.63 and 0.56 mg/mL) of PVE and PVD extracts were determined, respectively. The extracts exhibited antimicrobial effects against Enterococcus faecalis, Staphylococcus aureus, Streptococcus uberis, Bacillus cereus, and Bacillus subtilis. Minimal inhibitory concentrations (MICs, 0.8–49.0 and 9.6–82.5 mg/mL) and the minimal bactericidal concentrations (MBCs, 1.3–99.1 and 15.5–150.0 mg/mL) of PVE and PVD extracts were determined, respectively. Kill curves revealed that PVE and PVD extracts could inhibit the growth of bacteria. It was shown that PVE and PVD extracts could represent a good economical source of functional and bioactive compounds. [ABSTRACT FROM AUTHOR]

Probiotics can enhance broiler chicken health by improving intestinal microbiota, potentially replacing antibiotics. They protect against bacterial diseases like necrotic enteritis (NE) in poultry. Understanding their role is crucial for managing bacterial diseases, including NE. This study conducted a meta-analysis to assess the effects of Bacillus subtilis probiotic supplementation on feed conversion ratio (FCR), NE lesion score, and mortality. Additionally, a systematic review analysed gut microbiota changes in broilers challenged with Clostridium perfringens with or without the probiotic supplementation. Effect sizes from the studies were estimated in terms of standardized mean difference (SMD). Random effect models were fitted to estimate the pooled effect size and 95% confidence interval (CI) of the pooled effect size between the control [probiotic-free + C. perfringens] and the treatment [Bacillus subtilis supplemented + C. perfringens] groups. Overall variance was computed by heterogeneity (Q). The meta-analysis showed that Bacillus subtilis probiotic supplementation significantly improved FCR and reduced NE lesion score but had no effect on mortality rates. The estimated overall effects of probiotic supplementation on FCR, NE lesion score and mortality percentage in terms of SMD were −0.91 (CI = −1.34, −0.49; P < 0.001*); −0.67 (CI = −1.11, −0.22; P = 0.006*), and −0.32 (CI = −0.70, 0.06; P = 0.08), respectively. Heterogeneity analysis indicated significant variations across studies for FCR (Q = 69.66; P < 0.001*) and NE lesion score (Q = 42.35; P < 0.001*) while heterogeneity was not significant for mortality (Q = 2.72; P = 0.74). Bacillus subtilis probiotic supplementation enriched specific gut microbiota including Streptococcus, Butyricicoccus, Faecalibacterium, and Ruminococcus. These microbiotas were found to upregulate expression of various genes such as TJ proteins occluding, ZO-1, junctional adhesion 2 (JAM2), interferon gamma, IL12-β and transforming growth factor-β4. Moreover, downregulated mucin-2 expression was involved in restoring the intestinal physical barrier, reducing intestinal inflammation, and recovering the physiological functions of damaged intestines. These findings highlight the potential benefits of probiotic supplementation in poultry management, particularly in combating bacterial diseases and promoting intestinal health. [ABSTRACT FROM AUTHOR]

Amylases are highly needful product in food, beverage, and detergent industry. With the objective of isolating α-amylases producing strains, samples were collected from local bakery chimneys and kitchens, Chennai, Tamil Nadu, India. The potential α-amylase producers were isolated and screened by Starch-Iodine plate assay method. The optimized parameters such as 1.5% of starch concentration in the media with 7.2-pH at 35 °C for 36 h enhanced the production of α-amylase. The strain showing high potential, identified by 16s rRNA sequencing was found to be Bacillus subtilis. The Bacillus subtilis strain produced extracellular α-amylase with activity of 1330.14 U/mL. Further partial purification by ammonium sulphate precipitation followed by dialysis and column chromatography enhanced the specific activity to 1695.306 U/mg. The molecular weight of enzymes was found to be around 36 kDa. The enzyme stability and activity characterization revealed that the enzyme was thermotolerant which was able to retain more than 70% activity at 70 °C, 80 °C and 90 °C. The purified enzyme was also found to be Ca2+ independent. The hydrolysis of raw starch from rice, wheat and corn flour using the purified α-amylase from Bacillus subtilis showed significant activity of 72.8%, 76.1% and 65.2% with 4 h incubation respectively. The potentiality of the enzyme as an additive with commercial detergent was also determined to be effective. This study favourably attracts the attention towards industrial applications. [ABSTRACT FROM AUTHOR]

The rational use of chemical fungicides in chilli pepper (Capsicum annuum L.) for controlling Phytophthora capsici Leo. should include molecules that promote induced systemic resistance in the plant. The study objective was to assess how the application of fungicides, bio-controllers and resistance inducers affects bioactive compounds production and oxidative metabolism in pepper plants when inoculated with P. capsici zoospores. Statistical analyses show significant differences in total flavonoids, total phenols and salicylic acid concentrations in plants treated with Trichoderma harzianum, Acibenzolar-S-Methyl (ASM) and phosphonic acid. In addition, the application of ASM and Bacillus subtilis showed the highest values of antioxidant capacity. On the other hand, ASM application favoured increases in superoxide dismutase and phenyl ammonium lyase activity with values of 4.42 ± 0.016 (units · min−1 · g−1) and 3.90 ± 0.22 U · g−1, respectively. However, hydrogen peroxide production was similar to that of phosphonic acid treatments. Similarly, plants treated with B. subtilis and Metalaxyl-M showed increases in peroxidase enzyme activity with values ranging from 2.86 ± 0.05 to 4.52 ± 0.02 (μmol · [mg protein]−1 · min−1). Likewise, phosphonic acid-treated plants exhibited high catalase enzyme activity with values ranging from 2.38 ± 0.032 μmol H2O2 · min−1 · g−1 to 4.30 ± 0.024 μmol H2O2 · min−1 · g−1. The complementary use of products with biological and resistance-inducing functions could help to mitigate the environmental impacts that occur due to the use of chemical fungicides for controlling P. capsici on chilli pepper. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of different concentrations of fructooligosaccharide (FOS) instead of glucose (GLU) as the carbon source in the biofloc system. GLU was used as the carbon source in the control group, and 2.5%, 5.0%, 7.5%, and 10.0% FOS were used instead of GLU as the carbon source in experimental groups. Ammonium chloride was used the nitrogen source with a C/N ratio of 15. Bacillus subtilis was supplemented to promote biofloc formation. The results showed that the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total nitrogen were reduced effectively in all groups. High-throughput 16S rRNA sequencing revealed that the abundance of Flavobacterium was significantly increased in the 2.5% FOS group (P < 0.05), while Acinetobacter was significantly increased in the 10.0% FOS group on the 14th day (P < 0.05), the abundance of Aeromonadaceae was significantly reduced in all experimental groups (P < 0.05). Compared with the control group, the abundance of Aeromonadaceae was significantly decreased in 2.5% FOS on the 28th day (P < 0.05). Correlation analysis between microbial community composition and environmental factors indicated Flavobacterium and norank_f__norank_o__Saccharimonadales were negatively associated with three forms of nitrogen. In summary, the substitution of GLU with 2.5% FOS as the carbon source in the biofloc system significantly increased the abundance of core bacteria (Flavobacterium and Acinetobacter) and significantly reduced harmful bacteria (Aeromonadaceae), thereby stabilizing bacterial communities within the aquatic environment. The positive effects exhibited by 10.0% FOS were similar to 2.5% FOS. Based on experimental results and cost, the recommended proportion of FOS as a substitute for GLU is 2.5%. This research provides a foundation for the application of FOS as a carbon source in biofloc system and serves as a reference for future studies in this field. [ABSTRACT FROM AUTHOR]

Farmers usually prefer to sell Chinese mitten crabs (Eriocheir sinensis) until around the Spring Festival for greater economic benefits. However, the hoarding stages in winter and cold chain logistics would reduce the vitality of crabs, which would affect the economic benefits. Therefore, this experiment investigated the effects of adding Bacillus subtilis B10 on their health and vitality status after transportation, as well as the immunity and lipid metabolism in cold. The results showed that adding 1 × 109 CFU/kg B. subtilis B10 in feed could improve the vitality of crabs and reduce feet breakage caused by transportation. In male crabs, it could promote growth, and improve antioxidant capacity by increasing the expression of antioxidant gene including SOD and PRX1, increasing the activity of CuZn-SOD and T-SOD, and regulate fat metabolism by decreasing the relative expression of FAD6 and FAD9 thus adapting to environmental changes. In female crabs, it could not promote growth, but it could improve antioxidant capacity by increasing the activity of T-AOC, CuZn-SOD, and T-SOD, and regulate the expression of immune-related and apoptosis-related genes including CASP1, AKT, and AQP and regulate fat metabolism by decreasing the relative expression of FAD6, FAD9, and ELOVL6, thus adapting to environmental changes. This experiment provided a reference basis for reducing economic losses in the hoarding stage and transportation, promoting breeding methods and nutritional requirements of single-sex farming models, and application of probiotics in the field of crab farming. [ABSTRACT FROM AUTHOR]

Considering the growing environmental concerns, bioutilization of keratin waste with keratinase has emerged as a potential solution to alleviate the significant burden of poultry processing waste. The main objectives of the study were to isolate and screen keratinase-producing bacteria and to optimize the production conditions of the enzyme using a statistical approach. A promising bacterium, Bacillus subtilis GH2, was isolated from a traditional leather tanning pond. This bacterium was identified using 16 S rDNA gene sequencing. By optimizing the medium components and culture conditions, a significant increase in keratinase activity of 421.65 U was obtained at a medium pH of 7.5 and temperature of 38 °C. This corresponds to a 1.96-fold increase compared to the first screening stage. The optimal activity of the enzyme was obtained at a pH of 7 and a temperature of 45 °C. Ca+2 showed the highest improved keratinase activity. Furthermore, the bacterium showed promising potential for feather biodegradation. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of dietary Bacillus subtilis supplementation on gut microbiota diversity, digestive enzyme activity, and Toll-like receptor (TLR) expression in broiler chickens. A total of 240 "817" crossbred broiler chickens were randomly assigned to four groups: control (basal diet, BD), group I (BD + 300 g/d B. subtilis at 1.08 × 107 CFU/kg), group II (BD + 600 g/d B. subtilis at 2.16 × 107 CFU/kg), and group III (BD + 900 g/d B. subtilis at 3.24 × 107 CFU/kg). Gut microbiota analysis revealed significant improvements in the abundance of specific microorganisms in the treatment groups, with distinct variations in the core microorganisms between the groups. Notably, protease activity in the ileum was significantly increased in groups II (22.59%; p < 0.01) and III (14.49%; p < 0.05) compared to that in the control group. Moreover, significant up-regulation of TLR1A and TLR7 expression was observed in jejunum and cecum of the treated groups. Additionally, the TLR1B expression in the ileum was significantly increased. Furthermore, TLR2A and MyD88 transcription levels were significantly elevated in the jejunum, liver, spleen, and kidneys of experimental groups. Modulations in the expression of various TLR's (TLR2B, TLR3, TLR4, TLR15, and TLR21) were also observed in different organs. The spleen and kidney of B. subtilis-supplemented chickens exhibited upregulated expression of the proinflammatory cytokine IL-1β. Dietary supplementation with B. subtilis in broiler chickens improved the gut microbiota diversity and significantly upregulated TLR's expression in various organs. B. subtilis could be a valuable feed additive, contributing to improved disease management and overall health in broiler chickens. [ABSTRACT FROM AUTHOR]

BACKGROUND: In terms of crystalline nanoporous materials, metal–organic frameworks (MOFs) are relatively new. They are self‐assembling structures made of organic ligands that serve as linkers between metal centers and metal ions that function as coordination centers. Due to MOFs' high porosity, absence of nonaccessible bulk volume, vast surface areas and variety of pore sizes and topologies, drug delivery via them is becoming more and more common. RESULTS: Zn‐MOF and Zn‐MOF@drug were produced using a solvothermal approach in this study and characterized using a variety of methods, including Fourier transform infrared spectroscopy, powder X‐ray diffraction and scanning electron microscopy. Utilizing the zone of inhibition and minimum inhibitory concentration approaches, Zn‐MOF and Zn‐MOF@drug were evaluated for their antibacterial capability against Escherichia coli and Bacillus subtilis, two types of bacteria. CONCLUSION: The antibacterial potential of Zn‐MOF@drug was greater than that of the metal salt, commercially available ZnO, Zn‐MOF and ligand alone. The mechanism of antibacterial activity of Zn‐MOF@drug was also discussed. © 2024 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600—1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921. [ABSTRACT FROM AUTHOR]

Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance. [ABSTRACT FROM AUTHOR]

Legume seeds account for one-fifth of the plant proteins consumed worldwide, and are divided into 3 groups: pulses, oilseeds and pulses with pods. Traditional fermented products based on legume seeds are an integral part of the diet of many populations, and are generally obtained from oilseeds, such as Glycine max. Fermented seeds are known to have better digestibility, lower amounts of anti-nutritional factors and isoflavones, courtesy of the numerous steps involved in their traditional processing. The present review was therefore undertaken to provide a comprehensive assessment of the contribution of each of the main processing steps, i.e. cooking and fermentation, on the degradation of compounds with anti-nutritional activities. The data presented in this review show that the cooking operation degrades almost all thermolabile anti-nutritional factors (ANFs). Some thermostable ANFs are also eliminated by hydrolysis and diffusion into the cooking water or in the soaking water (in case soaking is performed), especially when the seed/water ratio is sufficient for the cooking or soaking water to be subsequently discarded. Fermentation dominated by Bacillus subtilis or Rhizopus oligosporus is an interesting option when the aim is to further reduce thermostable antinutritional factors that were only partially reduced during cooking. [ABSTRACT FROM AUTHOR]

Due to population growth and climate changes, there is a rising need for alternative food and protein sources to reduce protein scarcity and the environmental impact of food industries. Single‐cell proteins (SCPs) have the potential to partially or fully substitute plant‐ and animal‐derived dietary proteins. Bacillus subtilis is an appealing bacterium for SCP production because of its fast growth and ability to obtain high protein and essential amino acid (AA) content in its biomass. It is also capable of utilizing a wide range of substrates. B. subtilis attractiveness and efficiency can be further enhanced using mutagenesis. In this study, a novel approach to creating mutant strains with enhanced protein and AA content was experimentally validated. The method is based on the application of AA inhibitors for selective pressure to ensure the growth of mutants with enhanced protein and/or AA synthesis capacity. For AA inhibitors, three herbicides were used: glufosinate–ammonium (GA), L‐methionine sulfoximine (MSO), and S‐(2‐aminoethyl)‐L‐cysteine (AEC). Initially, AA inhibitor doses for the complete inhibition of wild‐type (WT) B. subtilis strain were determined. Then, B. subtilis was treated with EMS chemical mutagen and created mutants were cultivated on a medium containing inhibitory dose of AA inhibitors. Growing samples were selected, analyzed, and compared. The optimal inhibitory concentrations of herbicides for mutant selection were 0.05–0.4 M for GA, 0.01–0.05 M for MSO, and 0.2 M for AEC. The best‐performing mutants were selected when using GA—improvement of 7.1 times higher biomass content, 1.5 times higher protein concentration, 1.2 times higher AA content, and 1.2 times higher essential AA index was achieved in comparison with WT B. subtilis. Enhanced mutants were also successfully selected when using MSO and AEC. This study demonstrates the potential of using AA inhibitors for the selection of mutants with improved protein and AA profiles. [ABSTRACT FROM AUTHOR]

Cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerium (FOC), is a prevalent soil-borne disease. In this study, Bacillus subtilis JNF2, isolated from the high incidence area of cucumber Fusarium wilt in Luoyang, demonstrated significant inhibitory effects on FOC and promoted cucumber seedling growth. The biocontrol mechanism of strain JNF2 were elucidated through morphological observation, physiological and biochemical experiments, and whole genome sequence analysis. Pot experiments revealed an 81.33 ± 0.21% control efficacy against Fusarium wilt, surpassing the 64.10 ± 0.06% efficacy of hymexazol. Seedlings inoculated with JNF2 exhibited enhanced stem thickness and leaf area compared to control and hymexazol-treated plants. Physiological tests confirmed JNF2's production of indole-3-acetic acid (IAA), siderophores, and hydrolytic enzymes, such as β-1,3-glucanase, amylase, and protease, which inhibited FOC growth and promoted plant development. Genome analysis identified genes encoding antimicrobial peptides and hydrolases, as well as a novel glycocin synthetic gene cluster. These findings underscore B. subtilis JNF2's potential as a biocontrol agent for sustainable cucumber cultivation. [ABSTRACT FROM AUTHOR]

ABSTRACT The present research was aimed to isolate potential probiotic organisms from dairy products locally made in and around the Saurashtra region of Gujarat. A total of 224 colonies were screened for primary attributes. Based on the results, 70 isolates were carried further for secondary screening. Out of these, only 23 isolates were further tested for antioxidant activities. Only 6 potential probiotic strains were found to have all the probiotic attributes. These isolates demonstrated survivability up to 4 h at pH ≤ 3, bile concentration ≥ 1.5%, autoaggregation ability ≥ 81.08%, and cell surface hydrophobicity more than 70% while using toluene as the test hydrocarbon. The promising six isolates were subjected to 16S rRNA sequencing for species‐level identification and found to be belonging to the genus Bacillus, Enterococcus, and Lactobacillus. The isolates demonstrated higher antioxidant potential as determined by ABTS, DPPH, and FRAP methods. For all three methods, L. rhamnosus was taken as a positive control that showed 85.61%, 39.56%, and 78.18% reduction of free radicals as determined by the ABTS, DPPH, and FRAP methods, respectively. Compared to this, Limosilactobacillus fermentum BAB 7912 demonstrated the highest reduction of ABTS radicals (83.45%), while Bacillus subtilis BAB 7918 reduced 29.95% DPPH free radicals and Bacillus spizizenii BAB 7915 reduced 80.93% ferric ions as determined by the FRAP method. Isolates were subjected to 16S rRNA sequencing for species‐level identification and found to be belonging to genus Bacillus, Enterococcus, and Lactobacillus. [ABSTRACT FROM AUTHOR]

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD 600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis. [Display omitted] • Utilizing Bacillus subtilis as a cell factory for the production of taxadiene. • Additional expression of DXS from MEP pathway can boost taxadiene production. • Application of fused prenyltransferases can enhance the yield of taxadiene. • The highest taxadiene production so far in B. subtilis has been achieved. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the impact of yeast cell wall prebiotics and multispecies probiotics on the gut microbiota, immune response, and growth performance of weaned piglets, as alternatives to antibiotics as growth promoters (AGPs). A randomized complete block design was employed, involving 160 piglets divided into four treatment groups during the nursery phase. The treatments applied throughout the experimental period were as follows: CONT+ = basal diet with halquinol (AGP); YCW = basal diet with yeast cell wall (cell wall of Saccharomyces cerevisiae yeast); SIM+ = basal diet with yeast cell wall + multispecies probiotic (Bacillus subtilis (2.0 x 109 CFU/g), Bacillus coagulans (5.0 x 108 CFU/g), Clostridium butyricum (5.0 x 107 CFU/g), and Bacillus licheniformis (2.0 x 109 CFU/g)); SIM- = basal diet with yeast cell wall + multispecies probiotic (half dose). The parameters assessed included daily feed intake, weight gain, feed conversion ratio (FCR), diarrhea score, serum cytokine levels, and chemokine concentrations, as well as microbiota analysis. During the 21 to 63-day study period, only FCR differed significantly (p = 0.0076). CONT+ and PREB had superior FCRs of 1.543 and 1.585, while SIM- had the least favorable FCR at 1.654. At 35 days, IL-10 levels were greater in the SIM- group, showing a 271.25% increase over those in the other groups. By 49 days, the IL-8 concentration was lower in the PREB group than in the CONT+ group, with a reduction of 247%, while the IL-8 concentrations in the SIM+ and SIM- groups were not significantly different from those in the other groups. The Firmicutes/Bacteroidetes (F/B) ratio in the CONT+ group was lower than that in the PREB, SIM+, and SIM- treatment groups. The Lactobacillaceae family was more abundant in the SIM+ treatment, followed by the SIM- and PREB treatments. The CONT+ treatment had the lowest abundance. The abundance of the genus Lactobacillus differed between the CONT+ group and the PREB, SIM+, and SIM- treatment groups. Prebiotics, used either alone or combined with probiotics, serve as effective substitutes for AGPs, boosting piglets' health and performance throughout the nursery phase. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION There are few studies on the effects of electrically stimulated bacteria on anti‐nutritional factors and microbial communities in mulberry leaf silage. This study aimed to examine the impact of the combined use of electrically stimulated Bacillus subtilis (EB) and Lactobacillus casei (LC) on the quality and degradation of anti‐nutritional factors in mulberry leaf silage.The results revealed that the synergistic effect of EB and LC significantly enhanced the nutritional value of mulberry leaves, as evidenced by the promotion of lactic acid synthesis, the reduction of anti‐nutritional factors, and the augmentation of lactic acid bacteria following a 60‐day silage period. Moreover, the EB + LC co‐inoculation resulted in the highest quality of mulberry leaf silage, with the degradation rates of tannin and phytic acid at 38.8% and 47.1%, respectively. The combination of EB + LC also enhanced lactic acid content, along with significant reductions in ammonia nitrogen (NH3‐N), soluble protein, and non‐protein nitrogen (NPN) (P < 0.05). Spearman correlation analysis showed that Lactobacillus in the silage was significantly positively correlated with crude protein (CP) and lactic acid, while negatively correlated with water‐soluble carbohydrates, pH and NPN (P < 0.05). In contrast, Weissella was significantly negatively correlated with CP and lactic acid.This study represents a pioneering application of electro‐stimulation in the field of feed silage, offering a scientifically substantiated approach to degrading anti‐nutritional factors in mulberry leaves for livestock feeding. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Milk allergy commonly occurs in children, mainly caused by bovine-derived casein (CAS) protein. Neutrophil-activating protein (NAP) of Helicobacter pylori plays an immunomodulatory role with potential to suppress Th2-type immune responses. Bacillus subtilis (B. subtilis) spores are commonly used as oral vectors for drug delivery. Objective: To investigate whether recombinantly expressed NAP on B. subtilis spores could be an effective treatment for CAS allergy in mouse. Methods: After CAS sensitization, mice were orally administered B. subtilis spores expressing recombinant NAP for 6 weeks. Allergic symptoms and parameters were evaluated after CAS challenge oral gavage, including allergic inflammation, splenic cytokines, and serum-specific antibodies. Protein levels of Toll-like receptor 2 (TLR2) and c-JUN in the jejunum tissue were measured by western blot. Bone marrow-derived macrophages (BMDMs) were stimulated with inactivated NAP spores to measure the influence on cytokine profiles in vitro. Results: NAP recombinant spore treatment significantly reduced allergic symptoms and intestinal inflammation. Interleukin-12 and interferon-gamma levels increased, whereas serum CAS-specific IgG1 and IgE levels decreased. TLR2 and c-JUN expression levels were elevated in the jejunal tissue. Inactivated NAP spores polarized BMDMs to the M1 phenotype and enhanced cytokine expression, which were inhibited by a TLR2 neutralizing antibody. Conclusion: NAP offers a new strategy in the treatment of CAS allergy by inhibiting the Th2 response, while eliciting macrophages to promote Th1 immune responses. Oral treatment of recombinant Bacillus subtilis spore was found effective against casein allergy, which delivered neutrophil-activating protein (NAP) of Helicobacter pylori. We explored that recombinant CotC-NAP spores could elicit macrophages M1 polarization through TLR2-dependent signaling pathway, which activate phosphorylation of NF-κB and c-JUN, further release cytokines like interleukin-12 and interferon-gamma to rectify the balance of Th1/Th2 and regulate the level of Tregs. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant ribosome variants arise spontaneously in bacterial populations; however, their impact on the overall bacterial physiology remains unclear. We studied the naturally arising antibiotic-resistant L22* ribosome variant of Bacillus subtilis and identified a Mg2+-dependent physiological cost. Coculture competition experiments show that Mg2+ limitation hinders the growth of the L22* variant more than the wild type (WT), even under antibiotic pressure. This growth disadvantage of L22* cells is not due to lower ribosome abundance but rather due to reduced intracellular Mg2+ levels. Coarse-grained elastic-network modeling of ribosome conformational dynamics suggests that L22* ribosomes associate more tightly with Mg2+ when compared to WT. We combined the structural modeling and experimental measurements in a steady-state model to predict cellular adenosine 5'-triphosphate (ATP) levels, which also depend on Mg2+. Experiments confirmed a predicted ATP drop in L22* cells under Mg2+ limitation, while WT cells were less affected. Intracellular competition for a finite Mg2+ pool can thus suppress the establishment of an antibiotic-resistant ribosome variant. [ABSTRACT FROM AUTHOR]

This study compares chemical and green synthesized zinc sulfide nanoparticles for multiple biological properties. A chemical synthesis approach is utilized to synthesize PEGylated ZnS‐NPs while the bulbs of Fritillariae cirrhosae bulbus are utilized for green synthesis. After extensive characterization, the NPs are examined for multifunctional biological properties such as antimicrobial, antioxidant, in vitro anticancer, and blood compatibility. Our study found that chemical synthesis results in higher yield and better dispersion behavior. However, the green ZnS‐NPs possessed increased antibacterial and antiparasitic potential against Leishmania tropica as the NPs resulted in significant inhibition against Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) with minimum inhibitory concentration (MIC) of 0.156 and 0.312 mg/mL, respectively while IC50 of 115.21 and 128.5 µg/mL were observed for the promastigote and amastigote forms of parasite. Furthermore, Fritillaria cirrhosa (F. cirrhosa) synthesized NPs possessed slightly improved antioxidant properties and in vitro anticancer potential against MCF‐7 breast cancer cells. Despite differences in the biological properties, the NPs did not result in hemolytic behavior thus demonstrating the compatible nature against blood cells. It was thus evident from the present study that the chemical and green synthesis techniques not only result in NPs with different physicochemical characteristics but also significantly influence the bio‐interaction of the obtained ZnS‐NPs. [ABSTRACT FROM AUTHOR]

Background: Hypoxanthine, prevalent in animals and plants, is used in the production of food additives, nucleoside antiviral drugs, and disease diagnosis. Current biological fermentation methods synthesize quantities insufficient to meet industrial demands. Therefore, this study aimed to develop a strain capable of industrial-scale production of hypoxanthine. Results: De novo synthesis of hypoxanthine was achieved by blocking the hypoxanthine decomposition pathway, thus alleviating transcriptional repression and multiple feedback inhibition, and introducing a purine operon from Bacillus subtilis to construct a chassis strain. The effects of knocking out the IMP(Inosine 5'-monophosphate) branch on the growth status and titer of the strain were then investigated, and the effectiveness of adenosine deaminase and adenine deaminase was verified. Overexpressing these enzymes created a dual pathway for hypoxanthine synthesis, enhancing the metabolic flow of hypoxanthine synthesis and preventing auxotrophic strain formation. Introducing IMP-specific 5' -nucleotidase addressed the issue of adenylate accumulation. In addition, the metabolic flow of the guanine branch was dynamically regulated by the guaB gene. The supply of glutamine and aspartic acid precursors was enhanced by introducing an exogenous glnA mutant gene, overexpressing aspC, and replacing the weaker promoter to regulate the aspartic acid branching pathway. Ultimately, fermentation in a 5 L bioreactor for 48 h produced 30.6 g/L hypoxanthine, with a maximum real-time productivity of 1.4 g/L/h, the highest value of hypoxanthine production by microbial fermentation reported so far. Conclusions: The intracellular purine biosynthesis pathway is extensive and regulated at multiple levels in cells. The IMP branch in the hypoxanthine synthesis pathway has a higher metabolic flux. The current challenge lies in systematically allocating the metabolic flux within the branch pathway to achieve substantial product accumulation. In this study, E. coli was used as the chassis strain to construct a dual pathway for IMP and AMP(Adenosine 5'-monophosphate) synergistic hypoxanthine synthesis and dynamically regulate the guanine branch pathway. Overall, our experimental efforts culminated in a high-yield, plasmid- and defect-free engineered hypoxanthine strain. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION Ginkgo biloba seeds (GBS) are rich in flavonoids, proteins and reducing sugar, and have been consumed as food and medicinal nuts for thousands of years. However, the presence of ginkgotoxins and their poor palatability limit people's consumption of them.This study used solid‐state fermentation with Bacillus subtilis natto and Lactobacillus plantarum to enhance the safety and benefits of GBS. Optimized fermentation conditions increased the content of beneficial components like total flavonoids, soluble protein and reducing sugar while eliminating unpleasant odors (isoamyl aldehyde and hexanal) and reducing the toxin 4′‐O‐methylpyridoxine by 91.17%. Fermentation of GBS powder can significantly enhance its anti‐inflammatory and antioxidant activities in vitro (P < 0.001). Furthermore, it exhibits a dose‐dependent effect within a certain concentration range. Mixed fermentation (FBnLp) was evaluated for its effects on obesity and metabolic syndrome in mice fed a high‐fat diet. FBnLp significantly reduced body and liver weight gain, prevented dyslipidemia and decreased inflammatory and oxidative stress compared to unfermented GBS. Histological analysis showed that FBnLp improved liver health by reducing fat accumulation and preventing non‐alcoholic fatty liver disease. Meanwhile, it was found that feeding FBnLp increased the expression of CPT‐1α, which regulates energy expenditure and fat breakdown, and downregulated the expression of SREBP‐1c, FAS and ACC, which regulate fat synthesis.This research provides new insights and technological support for the application and development of FBnLp as a functional product, addressing key issues in its use and industry growth. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) poses a significant challenge in the global swine industry, leading to substantial economic losses and reproductive and respiratory complications. The causative agent, PRRS virus (PRRSV), with its high mutation rate, complicates the development of universally effective vaccines. Furthermore, current PRRS vaccines are limited by high costs and complex administration methods. Therefore, in this study, we aimed to develop an innovative Bacillus subtilis spore-based oral vaccine targeting PRRS. Their oral administration was evaluated in mice and pigs, and blood, saliva, feces, and bronchoalveolar lavage fluid samples were collected for further analyses. Our vaccine induced IgG and IgA immune responses in both models, with swine demonstrating considerable increase in specific antibody and cytokine levels. These results indicate a high potential for more effective and economically viable control of PRRS in commercial pig farming. The ease of administration and cost-effectiveness of the vaccine also offer a feasible option for widespread application. Our results suggest a new direction in veterinary vaccine development, underscoring the potential of B. subtilis spores in creating effective vaccines for large-scale, real-world applications in animal health management. [ABSTRACT FROM AUTHOR]

Given the extensive heterogeneity and variability, understanding cellular functions and regulatory mechanisms through the analysis of multi‐omics datasets becomes extremely challenging. Here, a comprehensive modeling framework of multi‐omics machine learning and metabolic network models are proposed that covers various cellular biological processes across multiple scales. This model on an extensive normalized compendium of Bacillus subtilis is validated, which encompasses gene expression data from environmental perturbations, transcriptional regulation, signal transduction, protein translation, and growth measurements. Comparison with high‐throughput experimental data shows that EM_iBsu1209‐ME, constructed on this basis, can accurately predict the expression of 605 genes and the synthesis of 23 metabolites under different conditions. This study paves the way for the construction of comprehensive biological databases and high‐performance multi‐omics metabolic models to achieve accurate predictive analysis in exploring complex mechanisms of cell genotypes and phenotypes. [ABSTRACT FROM AUTHOR]

Background: Vitamin B12 is a crucial B-group vitamin, first isolated from the liver due to its role in combating pernicious anemia. It is distinguished by its unique and complex structure, which makes its chemical synthesis challenging and expensive. Consequently, vitamin B12 is alternatively obtained through microbial fermentations. Molasses, an affordable and safe agro-industrial waste, can be used as a carbon source for vitamin B12 production, offering a cost-effective alternative to expensive sugars in the production medium. Results: A total of 87 yeast, actinomycete, and bacterial isolates were screened for vitamin B12 production, with 15 isolates showing high productivity. Bacillus isolates were selected for further analysis using MALDI-TOF and molecular identification. These isolates were identified as four strains of Bacillus subtilis (MZ08, JT10, BY11, and JT17), one strains of Bacillus sp. (CB09), and one strain of Peribacillus acanthi (MZ01). Genetic circuits associated with vitamin B12 production were demonstrated in a closely related strain of Peribacillus acanthi MZ01 strain. Three strains (MZ01, MZ08, and JT17) were selected for further evaluation of vitamin B12 productivity under different sugar types (glucose, sucrose, fructose, lactose, and galactose) and varying inoculum sizes. The inoculum size significantly impacted vitamin B12 production, with an increase from 5 to 10% enhancing yields. The ability of the strains to produce vitamin B12 varied depending on the type of sugar used. Peribacillus acanthi MZ01 strain showed the highest productivity and subsequently, selected for optimizing vitamin B12 production conditions using response surface methodology. Furthermore, the optimized conditions were then applied to molasses-based medium to achieve high vitamin B12 yields by MZ01 strain. Conclusion: In this study, Peribacillus acanthi was characterized for the first time as a vitamin B12 producer, demonstrating high productivity among various tested strains. The optimization of production conditions using response surface methodology, further enhanced vitamin B12 yields, showcasing the strain's efficiency in microbial fermentations. This research also highlights the potential of using molasses as a cost-effective alternative carbon source, significantly reducing production costs. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In recent years, the use of fish-derived probiotics in aquaculture has become more widespread. However, research on Anguilla japonica -derived probiotics is still limited. To evaluate the potential of probiotics for disease control in eel aquaculture, isolates were obtained from the intestinal tract of healthy Anguilla japonica. These isolates were assessed for their adhesion properties, inhibition of pathogen adhesion, and hydrolytic enzyme production. Morphological characteristics and 16S rRNA sequence analysis were used for identification. Results showed that the AJQ03 strain adhered to the intestinal mucus and inhibited common pathogenic bacteria through adhesion inhibition, and further produced amylase, lipase, protease, and cellulase. Based on morphological characteristics and 16S rRNA sequencing, AJQ03 was identified as Bacillus subtilis. The strain demonstrated tolerance to various extreme conditions, as well as survival in simulated gastrointestinal fluids and superior growth in intestinal fluid compared to Luria-Bertani (LB) broth. In vitro safety tests showed that AJQ03 was not resistant to 32 antibiotics and exhibited γ hemolysis on blood plate. In vivo safety tests demonstrated a 100% survival rate for the fish, with stable organ indices, reduced bacterial loads in the liver and spleen, and complete bacterial clearance by day 7 without residue. Intestinal bacterial load results confirmed effective colonization by strain AJQ03. Analysis of the impact of AJQ03 on the gut microbiota of A. japonica revealed a significant increase in the relative abundance of Bacillus at the genus level, corroborating the colonization efficiency of AJQ03. Additionally, the relative abundances of Klebsiella , Pseudomonas , and Aeromonas were significantly lower compared to the controls, indicating that strain AJQ03 effectively reduced harmful bacteria and improved gut microbiota composition. This study confirms that B. subtilis AJQ03, isolated from the intestine of A. japonica , can serve as a probiotic candidate in A. japonica aquaculture. [ABSTRACT FROM AUTHOR]

We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: A hydrolytic enzyme called protease from microbial sources has expansive applications in leather as well as tannery industries and thus leaves no choice but to discover a potential candidate for efficient yield of protease with eccentric characteristics. Methods and results: In the current study, using skim milk agar medium, three bacterial strains (Pro SS14, Pro NTL1, Pro SM) were identified for protease production out of thirty tannery waste samples. The bacterial isolates were identified through morphological, biochemical and molecular basis and the highest protease producer (134 U/mL) having the paramount protein content (8.75%) with the greatest biomass (absorbance 1.7 at 600 nm) was further bioinformatically identified as Bacillus subtilis Pro NTL1. Moreover, the cultural conditions—pH, temperature, incubation period and salinity were tested to get the optimized condition for the three isolates where Bacillus subtilis Pro NTL1 gave best growth at pH 9 demonstrating alkaline protease producing capability, at 45 °C for 72 h proving its thermotolerance characteristics and at 3% NaCl concentration declaring its halotolerance property. Furthermore, following Kirby-Bauer agar disc diffusion technique, the antimicrobial susceptibility pattern was investigated showing Bacillus subtilis Pro NTL1 was resistant to ampicillin, penicillin, sulfafurazole and sensitive to amoxicillin, gentamycin, cefotaxime, kanamycin, imipenem, tetracycline. Additionally, Bacillus subtilis Pro NTL1 showed antagonistic activity against three pathogens named Bacillus cereus, Bacillus megaterium and Escherichia coli. Conclusions: Thus, these findings suggest that Bacillus subtilis Pro NTL1 is a newly isolated strain with unique features which might help to contribute in several biotechnological purposes with significant industrial benefits. [ABSTRACT FROM AUTHOR]

Phytoremediation is a strategy for the amelioration of soil heavy metal contamination that aligns with ecological sustainability principles. Among the spectrum of phytoremediation candidates, woody plants are considered particularly adept for their substantial biomass, profound root systems, and non-participation in the food chain. This study used Eucalyptus camaldulensis —a tree species characterized for its high biomass and rapid growth rate—to assess its growth and metal uptake in mining tailings. The results were as follows: exposure to heavy metals reduced the E. camaldulensis uptake of potassium (K), phosphorus (P), and calcium (Ca). Heavy metal stress negatively affected the biomass of E. camaldulensis. Lead (Pb) primarily accumulated in the roots, while cadmium (Cd) predominantly accumulated in the stems. The application of organic fertilizers bolstered the stress tolerance of E. camaldulensis , mitigating the adverse impacts of heavy metal stress. A synergistic effect occurred when organic fertilizers were combined with bacterial fertilizers. The plant's enrichment capacity for Cd and its tolerance to Pb was augmented through the concurrent application of bacterial and organic fertilizers. Collectively, the application of organic fertilizers improved the heavy metal tolerance of E. camaldulensis by enhancing the uptake of K, P, and Ca and elevating the content of glutathione peroxidase (GPX) and gibberellin acid (GA) in roots. These findings provided nascent groundwork for breeding E. camaldulensis with enhanced heavy metal tolerance. Moreover, this proved the potentiality of E. camaldulensis for the management of heavy metal-contaminated tailings and offers a promising avenue for future environmental restoration. [ABSTRACT FROM AUTHOR]

The aim of this study was to explore whether and how Bacillus subtilis KC1 can enhance the growth performance of Mycoplasma gallisepticum (MG)-infected broilers. Broilers were randomly divided into 4 groups: the control group (basal diet), the MG group (basal diet + MG challenge), the KC group (basal diet + B. subtilis KC1 supplementation), the KC + MG group (basal diet + B. subtilis KC1 supplementation + MG challenge). The results showed that, compared to the control group, MG group exhibited significantly reduced body weight and average daily gain, and increased feed conversion ratio of broilers. However, compared to the MG group, the B. subtilis KC1 + MG group exhibited significantly improved above indicators of growth performance. In addition, compared to the MG group, B. subtilis KC1 + MG group exhibited increased superoxide dismutase levels and reduced levels of malondialdehyde, interleukin-1β, and tumor necrosis factor- α of broilers. Furthermore, metabolomics and transcriptomics analyses indicated that MG infection disrupted amino acid metabolism in broilers, whereas B. subtilis KC1 supplementation alleviated the abnormal amino acid metabolism caused by MG. These results suggested that B. subtilis KC1 may alleviate the poor growth performance caused by MG infection in broilers by improving amino acid metabolism. [ABSTRACT FROM AUTHOR]

The identification of perilous metal ions and the treatment of diseases induced by microorganisms are critical areas requiring attention. We report herein the design and development of an acetylene‐functionalized Schiff base compound 4, using a low‐cost and straightforward approach. This compound 4 exhibits dual functions in the detection of harmful metal ions, particularly Al(III), as well as in the prevention of disorders caused by bacteria, specifically Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis). It was well characterized through the utilization of diverse spectroscopic techniques, such as NMR (1H and 13C), FT‐IR, and mass spectrometry. Furthermore, the complete structure clarification of the Schiff base compound 4 was revealed through the utilization of X‐ray crystallography. The limit of detection (LOD) for Al(III) is 35.5 × 10−8 M, surpassing the WHO drinking water standard of 26.7 µM, underscoring the high sensitivity of compound 4. The antibacterial activity of compound 4 has also been effectively evaluated on Gram‐positive (S. aureus and B. subtilis) bacteria. Furthermore, compound 4 was docked to S. aureus and B. subtilis proteins, which represents outstanding outcomes with binding energies of −7.31 kcal/mol and −7.87 kcal/mol, respectively. The antibacterial activity of the compound 4 in both in vitro and docking studies suggests that it possesses the potential to serve as an antibacterial agent in the future. [ABSTRACT FROM AUTHOR]

Background RESULTS Conclusion Lignin peroxidase is closely related to agriculture and food as it improves the quality of feedstuffs, facilitates the degradation of lignin in agricultural wastes, and degrades azo dyes that have similar complex structures to lignin. However, the current status of homologous or heterologous expression of lignin peroxidase is unsatisfactory and needs to be modified with the help of immobilization and directed evolution to maximize its potential. Directed evolution technology is an effective strategy for designing and improving enzyme characteristics, and Bacillus subtilis spore surface display technology is an efficient method for preparing immobilized enzymes.A colorimetric dye decolorization assay using Congo red as a substrate was developed and optimized for high‐throughput screening of spore surface display in a 96‐well plate. After two rounds of screening, a superior mutant strain was selected from 2700 mutants. Its highest catalytic activity was 196.36%. The amino acid substitution sites were identified as N120D and I242T.The mechanism for the enhanced catalytic activity was explained using protein modeling and functional analysis software. This study provides insights into the rational design of lignin peroxidase and its application in food and agriculture. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

[Objectives] The study aimed to investigate the effects of Bacillus subtilis fermentation broth components on the growth of tomato seedlings, optimize fermentation conditions for effective metabolites, and provide technical support for the preparation of efficient microbial fertilizers. [Methods] Bacillus subtilis subsp. subtilis NRCB002 was used as the test strain. A pot experiment was conducted to evaluate the impact of NRCB002 and its fermentation broth components on tomato seedlings. Biomass of NRCB002 and the concentration of acetoin were used as indicators, and single-factor experiments were conducted to optimize type and concentration of carbon and nitrogen sources, the composition of inorganic salts, as well as fermentation temperature and time. The composition of inorganic salt was further optimized by orthogonal experimental design and range analysis. [Results] Compared with control using deionized water and fermentation medium, the NRCB002 fermentation broth supernatant, NRCB002 fermentation broth, resuspended NRCB002 bacteria, and resuspended NRCB002 bacteria with acetoin significantly increased tomato growth parameters, including plant height, stem diameter, leaf area, root-shoot ratio, total root length, total root surface area, total root volume, number of lateral roots, dry weights of tomato shoot and root. This suggested that NRCB002 and its fermentation broth had favorable promoting effects on tomato growth. Furthermore, the tomato shoot dry weights of fermentation broth supernatant, fermentation broth, and resuspended NRCB002 bacteria with acetoin were significantly higher than those of NRCB002 bacteria alone. The biomass and acetoin concentration of NRCB002 strain were higher when using 80 g·L-1 sucrose as carbon source and 25 g·L-1 yeast extract as nitrogen source. The optimal combination of inorganic salt was 1.5 g·L-1 KH2PO4, 1.0 g·L-1 MgSO4·7H2O, and 0.2 g·L-1 MnSO4·H2O. The optimal fermentation temperature was 37 ℃, and the fermentation time was 96 h. Using the optimized fermentation conditions and 1 L shake flasks for fermentation, the NRCB002 biomass(D600)was 15.70, and acetoin concentration was 29.65 g·L-1. These values were 3.4 times and 11.8 times higher than those of the initial fermentation medium, respectively. [Conclusions] The NRCB002 fermentation broth promoted tomato growth, and acetoin was a key promoting substance in the broth. [ABSTRACT FROM AUTHOR]

Formaldehyde is a known toxic compound, and functional formaldehyde detoxification is crucial for the survival of all living cells. Such detoxification systems are of particular importance for methylotrophic microorganisms that rely on formaldehyde as a central metabolite in their one-carbon metabolism. Understanding formaldehyde dissimilation pathways in non-methylotrophic industrial microorganisms is necessary for ongoing research aiming at engineering methylotrophy into their metabolism (synthetic methylotrophy). There is a variety of formaldehyde dissimilation pathways across microorganisms, often based on the activity of formaldehyde dehydrogenases. In this study, we investigated the role of the yycR gene of Bacillus subtilis putatively encoding a novel, uncharacterized zinc-type alcohol dehydrogenase-like protein. We showed that the B. subtilis ΔyycR mutant displayed a reduced formaldehyde tolerance level and confirmed the enzymatic activity of recombinantly produced and purified YycR as formaldehyde dehydrogenase in vitro. Biochemical analyses demonstrated that YycR activity is optimal at 40°C, with the highest measured activity at pH 9.5, formaldehyde is the preferred substrate, and the kinetic constants are Km of 0.19 ± 0.05 mM and Vmax of 2.24 ± 0.05 nmol min−1. Altogether, we showed that YycR is a novel formaldehyde dehydrogenase with a role in formaldehyde detoxification in B. subtilis, providing valuable insights for future research on synthetic methylotrophy in this organism. IMPORTANCE Formaldehyde is a key metabolite in methanol assimilation for many methylotrophic microorganisms, and at the same time, it is toxic to all living cells, which means its intracellular concentrations must be tightly controlled. An in-depth understanding of methanol detoxification systems in industrially relevant microorganisms is a prerequisite for the introduction of methanol utilization pathways into their metabolism (synthetic methylotrophy). Bacillus subtilis, an industrial workhorse conventionally used for the production of enzymes, is known to possess two formaldehyde detoxification pathways. Here, we identify a novel formaldehyde dehydrogenase in this bacterium as a path towards creating innovative prospect strategies for strain engineering towards synthetic methylotrophy [ABSTRACT FROM AUTHOR]

Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in Bacillus subtilis are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates. Which cells express and deploy the various T7SS substrates is still unknown. To identify which cells express at least one of the T7SS substrates, we investigated the yfj operon. The yfjABCDEF operon encodes at least one predicted T7SS substrate. Starting with an in silico analysis of the yfj operon promoter region, we identified potential regulatory sequences. Using a yfj promoter-reporter fusion, we then identified several regulators that impact expression of the operon, including a regulator of biofilm formation, DegU. In a degU deletion mutant, yfj expression is completely abolished. Mutation of predicted DegU binding sites also results in a significant reduction in yfj reporter levels. Further analysis of yfj regulation reveals that deletion of spo0A has the opposite effect of the degU deletion. Following the yfj reporter by microscopy of cells harvested from biofilms, we find that the yfj operon is expressed specifically in the subset of cells undergoing sporulation. Together, our results define cells entering sporulation as the subpopulation most likely to express products of the yfj operon in B. subtilis. IMPORTANCE Differential expression of genes in a bacterial community allows for the division of labor among cells in the community. The toxin substrates of the type VII secretions system (T7SS) are known to be active in Bacillus subtilis biofilm communities. This work describes the expression of one of the T7SS-associated operons, the yfj operon, which encodes the YFJ toxin, in the sporulating subpopulation within a biofilm. The evidence that the YFJ toxin may be deployed specifically in cells at the early stages of sporulation provides a potential role for deployment of T7SS in community-associated activities, such as cannibalism. [ABSTRACT FROM AUTHOR]

Two‐component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In Bacillus subtilis, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the des gene, encoding the sole desaturase in these bacteria, Δ5‐Des. Additionally, B. subtilis possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work, we show that YvfT/YvfU controls the expression of the yvfRS operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to des, yvfRS transcription is induced at 37°C and not at 25°C. Our in vivo and in vitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over yvfRS expression in its unphosphorylated state. Our study uncovers an intriguing case of cross‐regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability. [ABSTRACT FROM AUTHOR]

Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72℃, and γ-PGA yield of 36.31 g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38 h at 60℃, the yield of γ-PGA increased to 48.88 g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08 g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries. [ABSTRACT FROM AUTHOR]

A major unknown in the field of planetary protection is the degree to which natural atmospheric processes remove terrestrial microorganisms from robotic and crewed spacecraft that could potentially contaminate Mars (i.e., forward contamination). We present experiments in which we measured the removal rate of Bacillus subtilis HA101 spores from aluminum surfaces under the bombardment of naturally rounded sand grains. To simulate grain impacts, we constructed a pneumatic sand-feed system and gun to accelerate grains to a desired speed, with independent control of impacting grain mass, flux, and angle. Spore counts of the resulting bombarded surfaces when using scanning electron microscopy indicate that although spores directly impacted by sand grains would likely be killed, those immediately adjacent to grain impacts might be released into the environment intact. The experiments demonstrate a linear relationship between the fractional dislodgement rate of spores and grain impact speed, which can be used to estimate input to microbial transport models (e.g., using numerical models of saltation). Even the slowest grain impacts (∼2.7 m/s) dislodged spores. Such slow events may be common and widespread on Mars, which suggests that microbial dislodgement by slow saltation near the surface is largely unavoidable. [ABSTRACT FROM AUTHOR]

Here we present a "breathing" vessel consisting of expanded polytetrafluoroethylene, which allows gas exchange but no liquid permeation. The bacterial culture inside needs only agitation to promote air supply. Using this setup, a Bacillus subtilis cell factory for scyllo -inositol production grew to produce scyllo -inositol efficiently. The results indicate that our approach represents a sustainable "greener" approach for the cell factory. [ABSTRACT FROM AUTHOR]