Lactobacillus acidophilus and Bacillus subtilis significantly change the growth performance, serum immunity and cecal microbiota of Cherry Valley ducks during the fattening period.

This study explored the effects of a Bacillus subtilis and Lactobacillus acidophilus mixture containing the co-fermented products of the two probiotics on growth performance, serum immunity and cecal microbiota of Cherry Valley ducks. This study included 480 one-day-old Cherry Valley ducks divided into four feeding groups: basal diet (control group) and basal diet supplemented with 300, 500, or 700 mg/kg of the probiotic powder; the ducks were raised for 42 days. Compared with the control group, body weight on day 42 and the average daily gain on days 15-42 significantly increased (p < 0.05), and the feed conversion rate significantly decreased (p < 0.05) in the experimental groups. Furthermore, the serum immunoglobulin (Ig) A, IgG, IgM, and interleukin (IL)-4 levels increased significantly (p < 0.05), and IL-1ß, IL-2, and tumor necrosis factor-α decreased significantly (p < 0.05) in the experimental groups. Finally, Sellimonas, Prevotellaceae NK3B31 group, Lachnospiraceae NK4A136 group and Butyricoccus played an important role in the cecal microbiota of the experimental group. Thus, the probiotic powder has impacts on the growth performance, serum immunity and cecal microbiota of Cherry Valley Ducks.

Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani.

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Enhancing effect of oregano essential oil and Bacillus subtilis on broiler immune function, intestinal morphology and growth performance.

The present study evaluated the effect of two categories of feed additives on chicken performance through immunological and intestinal histo-morphometric measurements. A total of 150 one-day-old male broiler chicks (Cobb) were randomly assigned to three groups. Group I received a non-supplemented basal diet. While groups II and III were treated with a basal diet supplemented with oregano essential oil (OEO) and Bacillus subtilis, respectively, in water for 28 days. Blood samples were taken at 6, 18 and 28 days for hematological analysis, phagocytosis, lymphocyte proliferation and measuring antibody responses. Additionally, growth performance indices were recorded weekly. The results showed that groups supplemented with OEO and B. subtilis improved growth performance expressed by a significant increase in weight gain (P < 0.05), with a significant reduction (P < 0.05) in feed conversion ratio (FCR). Hematological findings indicated a significant increase in blood parameters as well as a significant increase in phagocytic % & phagocytic index at all time points with a greater probiotic effect. On the other hand, OEO produced a significant increase in lymphocyte proliferation at 18 & 28 days. Humoral immunity revealed a significant increase in serum antibody titer phytobiotic & probiotic-fed groups at time points of 18 & 28 days with a superior phytobiotic effect. The histological examination showed a significant increase in villi length, villi width, crypt depth & V/C ratio. In conclusion, these results indicated positive effects of B. subtilis & OEO on both growth and immunity and could be considered effective alternatives to the antibiotic.

Progress in Identification of UDP-Glycosyltransferases for Ginsenoside Biosynthesis.

Ginsenosides, the primary pharmacologically active constituents of the Panax genus, have demonstrated a variety of medicinal properties, including anticardiovascular disease, cytotoxic, antiaging, and antidiabetes effects. However, the low concentration of ginsenosides in plants and the challenges associated with their extraction impede the advancement and application of ginsenosides. Heterologous biosynthesis represents a promising strategy for the targeted production of these natural active compounds. As representative triterpenoids, the biosynthetic pathway of the aglycone skeletons of ginsenosides has been successfully decoded. While the sugar moiety is vital for the structural diversity and pharmacological activity of ginsenosides, the mining of uridine diphosphate-dependent glycosyltransferases (UGTs) involved in ginsenoside biosynthesis has attracted a lot of attention and made great progress in recent years. In this paper, we summarize the identification and functional study of UGTs responsible for ginsenoside synthesis in both plants, such as Panax ginseng and Gynostemma pentaphyllum, and microorganisms including Bacillus subtilis and Saccharomyces cerevisiae. The UGT-related microbial cell factories for large-scale ginsenoside production are also mentioned. Additionally, we delve into strategies for UGT mining, particularly potential rapid screening or identification methods, providing insights and prospects. This review provides insights into the study of other unknown glycosyltransferases as candidate genetic elements for the heterologous biosynthesis of rare ginsenosides.

Biosynthesis and assessment of antibacterial and antioxidant activities of silver nanoparticles utilizing Cassia occidentalis L. seed.

This research explores the eco-friendly synthesis of silver nanoparticles (AgNPs) using Cassia occidentalis L. seed extract. Various analytical techniques, including UV-visible spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX), were employed for comprehensive characterization. The UV-visible spectra revealed a distinct peak at 425 nm, while the seed extract exhibited peaks at 220 and 248 nm, indicating the presence of polyphenols and phytochemicals. High-resolution TEM unveiled spherical and oval-shaped AgNPs with diameters ranging from 6.44 to 28.50 nm. The SEM exhibiting a spherical shape and a polydisperse nature, thus providing insights into the morphology of the AgNPs. EDX analysis confirmed the presence of silver atoms at 10.01% in the sample. XRD results unequivocally confirm the crystalline nature of the AgNPs suspension, thereby providing valuable insights into their structural characteristics and purity. The antioxidant properties of AgNPs, C. occidentalis seed extract, and butylated hydroxytoluene (BHT) were assessed, revealing IC50 values of 345, 500, and 434 µg/mL, respectively. Antibacterial evaluation against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli demonstrated heightened sensitivity of bacteria to AgNPs compared to AgNO3. Standard antibiotics, tetracycline, and ciprofloxacin, acting as positive controls, exhibited substantial antibacterial efficacy. The green-synthesized AgNPs displayed potent antibacterial activity, suggesting their potential as a viable alternative to conventional antibiotics for combating pathogenic bacterial infections. Furthermore, potential biomedical applications of AgNPs were thoroughly discussed.

Bacillus subtilis SOM8 isolated from sesame oil meal for potential probiotic application in inhibiting human enteropathogens.

BACKGROUND: While particular strains within the Bacillus species, such as Bacillus subtilis, have been commercially utilised as probiotics, it is critical to implement screening assays and evaluate the safety to identify potential Bacillus probiotic strains before clinical trials. This is because some Bacillus species, including B. cereus and B. anthracis, can produce toxins that are harmful to humans. RESULTS: In this study, we implemented a funnel-shaped approach to isolate and evaluate prospective probiotics from homogenised food waste - sesame oil meal (SOM). Of nine isolated strains with antipathogenic properties, B. subtilis SOM8 displayed the most promising activities against five listed human enteropathogens and was selected for further comprehensive assessment. B. subtilis SOM8 exhibited good tolerance when exposed to adverse stressors including acidity, bile salts, simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and heat treatment. Additionally, B. subtilis SOM8 possesses host-associated benefits such as antioxidant and bile salt hydrolase (BSH) activity. Furthermore, B. subtilis SOM8 contains only haemolysin toxin genes but has been proved to display partial haemolysis in the test and low cytotoxicity in Caco-2 cell models for in vitro evaluation. Moreover, B. subtilis SOM8 intrinsically resists only streptomycin and lacks plasmids or other mobile genetic elements. Bioinformatic analyses also predicted B. subtilis SOM8 encodes various bioactives compound like fengycin and lichendicin that could enable further biomedical applications. CONCLUSIONS: Our comprehensive evaluation revealed the substantial potential of B. subtilis SOM8 as a probiotic for targeting human enteropathogens, attributable to its exceptional performance across selection assays. Furthermore, our safety assessment, encompassing both phenotypic and genotypic analyses, showed B. subtilis SOM8 has a favourable preclinical safety profile, without significant threats to human health. Collectively, these findings highlight the promising prospects of B. subtilis SOM8 as a potent probiotic candidate for additional clinical development.

Responses of growth performance, immunity, disease resistance of shrimp and microbiota in Penaeus vannamei culture system to Bacillus subtilis BSXE-1601 administration: Dietary supplementation versus water addition.

This study evaluated the effects of Bacillus subtilis BSXE-1601, applied either as dietary supplementation or water addition, on growth performance, immune responses, disease resistance of Penaeus vannamei, and microbiota in shrimp gut and rearing water. During the 42-day feeding experiment, shrimp were fed with basal diet (CO and BW group), basal diet supplemented with live strain BSXE-1601 at the dose of 1 × 109 CFU kg-1 feed (BD group) and 15 mg kg-1 florfenicol (FL group), and basal diet with strain BSXE-1601 added to water at the concentration of 1 × 107 CFU L-1 every five days (BW group). Results showed that dietary supplementation of strain BSXE-1601 significantly promoted growth performance of shrimp, both in the diet and water, enhanced disease resistance against Vibrio parahaemolyticus (P < 0.05). The BD and BW groups exhibited significant increases in acid phosphatase, alkaline phosphatase, lysozyme, peroxidase, superoxide dismutase activities, phenonoloxidase content in the serum of shrimp compared to the control (P < 0.05). Meanwhile, the expression of immune-related genes proPO, LZM, SOD, LGBP, HSP70, Imd, Toll, Relish, TOR, 4E-BP, eIF4E1α, eIF4E2 were significantly up-regulated compared to the control (P < 0.05). When added in rearing water, strain BSXE-1601 induced greater immune responses in shrimp than the dietary supplement (P < 0.05). Chao1 and Shannon indices of microbiota in rearing water were significantly lower in BD group than in the control. The microbiota in rearing water were significantly altered in BD, BW and FL groups compared to the control, while no significant impacts were observed on the microbiota of shrimp gut. When supplemented into the feed, strain BSXE-1601 obviously reduced the number of nodes, edges, modules in the ecological network of rearing water. The results suggested that dietary supplementation of BSXE-1601 could be more suitable than water addition in the practice of shrimp rearing when growth performance, non-specific immunity, disease resistance against V. parahaemolyticus in shrimp were collectively considered.

Expression of Multicopy Tandem Recombinant Ginseng Hexapeptide in Bacillus subtilis and the Evaluation of Antiaging Activity.

Ginseng oligopeptides are naturally occurring small-molecule peptides extracted from ginseng that exhibit positive effects on health and longevity. However, the current industrial production of ginseng oligopeptides primarily relies on plant extraction and chemical synthesis. In this study, we proposed a novel genetic engineering approach to produce active ginseng peptides through multicopy tandem insertion (5 and 15 times). The recombinant ginseng peptides were successfully produced from engineered Bacillus subtilis with an increasing yield from 356.55 to 2900 mg/L as the repeats multiple. Additionally, an oxidative stress-induced aging model caused by H2O2 was established to evaluate whether the recombinant ginseng peptides, without enzymatic hydrolysis into individual peptides, also have positive effects on antiaging. The results demonstrated that all two kinds of recombinant ginseng peptides could also delay cellular aging through various mechanisms, such as inhibiting cell cycle arrest, suppressing the expression of pro-inflammatory factors, and enhancing cellular antioxidant capacity.

Regulation mechanism and bioactivity characteristic of surfactin homologues with C14 and C15 fatty acid chains.

BACKGROUND: Surfactin, a green lipopeptide bio-surfactant, exhibits excellent surface, hemolytic, antibacterial, and emulsifying activities. However, a lack of clear understanding of the synthesis regulation mechanism of surfactin homologue components has hindered the customized production of surfactin products with different biological activities. RESULTS: In this study, exogenous valine and 2-methylbutyric acid supplementation significantly facilitated the production of C14-C15 surfactin proportions (up to 75% or more), with a positive correlation between the homologue proportion and fortified concentration. Subsequently, the branched-chain amino acid degradation pathway and the glutamate synthesis pathway are identified as critical pathways in regulating C14-C15 surfactin synthesis by transcriptome analysis. Overexpression of genes bkdAB and glnA resulted in a 1.4-fold and 1.3-fold increase in C14 surfactin, respectively. Finally, the C14-rich surfactin was observed to significantly enhance emulsification activity, achieving an EI24 exceeding 60% against hexadecane, while simultaneously reducing hemolytic activity. Conversely, the C15-rich surfactin demonstrated an increase in both hemolytic and antibacterial activities. CONCLUSION: This study presents the first evidence of a potential connection between surfactin homologue synthesis and the conversion of glutamate and glutamine, providing a theoretical basis for targeting the synthesis regulation and structure-activity relationships of surfactin and other lipopeptide compounds.

Bacterial crude oil and polyaromatic hydrocarbon degraders from Kazakh oil fields as barley growth support.

Bacterial strains of the genera Arthrobacter, Bacillus, Dietzia, Kocuria, and Micrococcus were isolated from oil-contaminated soils of the Balgimbaev, Dossor, and Zaburunye oil fields in Kazakhstan. They were selected from 1376 isolated strains based on their unique ability to use crude oil and polyaromatic hydrocarbons (PAHs) as sole source of carbon and energy in growth experiments. The isolated strains degraded a wide range of aliphatic and aromatic components from crude oil to generate a total of 170 acid metabolites. Eight metabolites were detected during the degradation of anthracene and of phenanthrene, two of which led to the description of a new degradation pathway. The selected bacterial strains Arthrobacter bussei/agilis SBUG 2290, Bacillus atrophaeus SBUG 2291, Bacillus subtilis SBUG 2285, Dietzia kunjamensis SBUG 2289, Kocuria rosea SBUG 2287, Kocuria polaris SBUG 2288, and Micrococcus luteus SBUG 2286 promoted the growth of barley shoots and roots in oil-contaminated soil, demonstrating the enormous potential of isolatable and cultivable soil bacteria in soil remediation. KEY POINTS: • Special powerful bacterial strains as potential crude oil and PAH degraders. • Growth on crude oil or PAHs as sole source of carbon and energy. • Bacterial support of barley growth as resource for soil remediation.

Growth performance, organs weight, intestinal histomorphology, and oocyst shedding in broiler chickens offered novel single strain Bacillus subtilis isolated from camel dung and challenged with Eimeria.

We evaluated a single strain Bacillus subtilis BS-9 direct-fed microbial (BSDFM) isolated from camel dung in Eimeria challenged broiler chickens. Seven-hundred d-old Ross 708 male chicks were placed in pens (25 birds/pen) and allocated to 2 treatments (n = 14). From d 0 to 13, control pens received untreated water (-BSDFM), and 2 treated pens received water and 2 mL x 108 colony forming unit/bird/d (+BSDFM); daily water intake (WI) was recorded. On d 9, birds in half (+Eimeria) of pens per treatment received of 1 mL of Eimeria maxima and Eimeria acervulina oocysts orally, and the other half (-Eimeria) sterile saline solution. Birds had ad libitum access to feed and a water line from d 14. Feed intake (FI), body weight (BW) and mortality were recorded for calculating BW gain (BWG) and feed conversion ratio (FCR). On d 14 and 35, samples of birds were necropsied for organ weight and intestinal measurements. Excreta samples were collected from d 14 to 19 for oocyst count. There was no treatment effect (P > 0.05) on growth performance or WI on d 0 to 9. There were interactions between BSDFM and Eimeria on d 19 (P = 0.014) and 29 (P = 0.036) BW with unchallenged +BSDFM birds being heavier than birds in the other treatments. The main effects (P < 0.05) on d 10 to 35 FI, BW, and BWG were such that +BSDFM increased and Eimeria decreased (P < 0.01) these parameters. There was interaction (P = 0.022) between BSDFM and Eimeria on d 10 to 35 FCR such that the FCR of challenged -BSDFM birds was poor than that of unchallenged counterparts, but none differed with +BSDFM birds. There was an interaction (P = 0.039) between BSDFM and Eimeria on d 14 bursa weight with challenged birds exhibiting heavier bursa than unchallenged +BSDFM birds. Eimeria reduced (P = 0.01) and BSDFM (P = 0.002) increased the villi height to crypt depth ratio. Results showed that BSDFM supplementation via water can support the growth performance of broiler chickens challenged with Eimeria and may be a strategy to reduce adverse effects of coccidiosis.

Effects of crude protease produced by Bacillus subtilis (MAFIC Y7) on growth performance, immune indices, and anti-inflammatory responses of broilers fed soybean meal- or cottonseed meal-based diets.

A strain of Bacillus subtilis (MAFIC Y7) was isolated from the intestine of Tibetan pigs and was able to express high protease activity. The aim of this study was to characterize the proteases produced by MAFIC Y7, and to investigate the effects of protease addition on growth performance, ileal amino acid digestibility, and serum immunoglobulin and immune factors of broilers fed SBM-based diets, or on growth performance, carcass characteristics, and intestinal morphology of broilers fed CSM-based diets. B. subtilis (MAFIC Y7) expressed protease showed its optimal enzyme activity at 50 °C and pH 7.0. The coated crude enzyme (CCE) showed greater stability at pH 3.0 than its uncoated counterpart. Experiment 1 was conducted with six diets based on three levels of crude protein (CP)-CPlow, CPmedium, and CPhigh-with or without CCE. In CPlow, CCE increased gain:feed (G:F) (days 1 to 21, days 1 to 42) by 8%, 3%, respectively, and enhanced apparent ileal digestibility (AID) of crude protein and lysine (on day 42) by 8.8%, 4.6%, respectively, compared with diets containing no CCE (P < 0.05). CCE increased G:F from days 1 to 21 from 0.63 to 0.68, improved G:F and average daily gain (ADG) during days 1 to 42, and enhanced AID of crude protein, lysine, cysteine, and isoleucine on day 42 compared with the unsupplemented treatments (in CPmedium, P < 0.05). CCE increased serum IgA (on day 21), serum IgA and IgG and increased serum IL-10 (on day 42), but decreased serum tumor necrosis factor-α (TNF-α; on day 21), and serum IL-8 and TNF-α (on day 42) compared with unsupplemented treatments. At CPhigh, CCE decreased serum levels of IL-6 and TNF-α (on day 21), and IL-8 and TNF-α (on day 42) compared with unsupplemented treatments (in CPhigh, P < 0.05). In experiment 2, CSM-based diets with two lysine-to-protein ratios (5.2% or 5.5%) with or without CCE. In the high Lys diet (5.5% Lys:protein), CCE increased ADG and G:F, increased carcass, but decreased abdominal fat compared with the unsupplemented treatment (P < 0.05). In the 5.2% Lys:protein dietary treatment, CCE improved duodenal villus height compared with the unsupplemented treatment (P < 0.05). Supplementation of protease produced by MAFIC Y7 was associated with lower inflammatory responses in SBM diets (CPmedium or CPhigh) and improved ADG in broilers fed CPmedium or CPhigh. The proteases improved ADG and the efficiency of CSM use when the ratio of Lys to protein was 5.5%.

The aim of this study was to investigate the effects of Bacillus subtilis (MAFIC Y7)-expressed protease on reducing inflammatory responses of soybean meal (SBM) diets and improving the efficiency of cottonseed meal (CSM) in broilers. Experiment 1 was conducted with six dietary treatments based on three levels of crude protein (CP)­CPlow, CPmedium, and CPhigh­without or with proteases (0 or 4,000 U/kg). Supplementation of proteases significantly improved growth performance, gain:feed (G:F), and apparent ileal digestibility of crude protein and amino acids (cysteine, isoleucine, and histidine) in broilers fed CPmedium treatment (P < 0.05). Proteases inhibited inflammatory responses in SBM-based diets by decreasing serum tumor necrosis factor-α (TNF-α) (in CPmedium and CPhigh), and interleukin (IL)-6 (in CPhigh); and IL-8 and TNF-α (in CPmedium and CPhigh) on day 21. In experiment 2, broilers were fed with CSM-based diets with two ratios of lysine-to-protein (5.2% or 5.5%) with or without proteases. Proteases in the diet of 5.5% Lys to protein ratio increased growth performance and G:F compared to diets without proteases (P < 0.05). Proteases produced by MAFIC Y7 improved growth performance and G:F in CPmedium. Supplementation of protease was associated with lower inflammatory responses of broilers fed SBM-based diets (CPmedium or CPhigh) and improved the efficiency of CSM use when the ratio of lysine-to-protein was 5.5%.

Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Antimicrobial and antibiofilm properties of selenium-chitosan-loaded salicylic acid nanoparticles for the removal of emerging contaminants from bacterial pathogens.

In this study salicylic acid loaded containing selenium nanoparticles was synthesized and called SA@CS-Se NPs. the chitosan was used as a natural stabilizer during the synthesis process. Fourier transforms infrared spectroscopy (FTIR), Powder X-ray diffraction (XRD), field emission electron microscopy (FESEM), and transmission electron microscopy (TEM) were used to describe the physicochemical characteristics of the SA@CS-Se NPs. The PXRD examination revealed that the grain size was around 31.9 nm. TEM and FESEM techniques showed the spherical shape of SA@CS-Se NPs. Additionally, the analysis of experiments showed that SA@CS-Se NPs have antibacterial properties against 4 ATCC bacteria; So that with concentrations of 75, 125, 150, and 100 µg/ml, it inhibited the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus respectively. Also, at the concentration of 300 µg/ml, it removed 22.76, 23.2, 10.62, and 18.08% biofilm caused by E. coli, P. aeruginosa, B. subtilis, and S. aureus respectively. The synthesized SA@CS-Se NPs may find an application to reduce the unsafe influence of pathogenic microbes and, hence, eliminate microbial contamination.

Disparate Effects of Two Clerodane Diterpenes of Giant Goldenrod (Solidago gigantea Ait.) on Bacillus spizizenii.

New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring minimal bactericidal concentration (MBC), minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50). Two of them, Sg3a (a dialdehyde) and Sg6 (solidagoic acid B), were proved to be the most effective and were selected for further study. Bacillus spizizenii was incubated with the two diterpenes for shorter (1 h) or longer (5 h) periods and then subjected to genome-wide transcriptional analyses. Only a limited number of common genes (28 genes) were differentially regulated after each treatment, and these were mainly related to the restoration of cell membrane integrity and to membrane-related transports. Changes in gene activity indicated that, among other things, K+ and Na+ homeostasis, pH and membrane electron transport processes may have been affected. Activated export systems can be involved in the removal of harmful molecules from the bacterial cells. Inhibition of bacterial chemotaxis and flagellar assembly, as well as activation of genes for the biosynthesis of secondary metabolites, were observed as a general response. Depending on the diterpenes and the duration of the treatments, down-regulation of the protein synthesis-related, oxidative phosphorylation, signal transduction and transcription factor genes was found. In other cases, up-regulation of the genes of oxidation-reduction processes, sporulation and cell wall modification could be detected. Comparison of the effect of diterpenes with the changes induced by different environmental and nutritional conditions revealed several overlapping processes with stress responses. For example, the Sg6 treatment seems to have caused a starvation-like condition. In summary, there were both common and diterpene-specific changes in the transcriptome, and these changes were also dependent on the length of treatments. The results also indicated that Sg6 exerted its effect more slowly than Sg3a, but ultimately its effect was greater.

In Silico Safety Assessment of Bacillus Isolated from Polish Bee Pollen and Bee Bread as Novel Probiotic Candidates.

Bacillus species isolated from Polish bee pollen (BP) and bee bread (BB) were characterized for in silico probiotic and safety attributes. A probiogenomics approach was used, and in-depth genomic analysis was performed using a wide array of bioinformatics tools to investigate the presence of virulence and antibiotic resistance properties, mobile genetic elements, and secondary metabolites. Functional annotation and Carbohydrate-Active enZYmes (CAZYme) profiling revealed the presence of genes and a repertoire of probiotics properties promoting enzymes. The isolates BB10.1, BP20.15 (isolated from bee bread), and PY2.3 (isolated from bee pollen) genome mining revealed the presence of several genes encoding acid, heat, cold, and other stress tolerance mechanisms, adhesion proteins required to survive and colonize harsh gastrointestinal environments, enzymes involved in the metabolism of dietary molecules, antioxidant activity, and genes associated with the synthesis of vitamins. In addition, genes responsible for the production of biogenic amines (BAs) and D-/L-lactate, hemolytic activity, and other toxic compounds were also analyzed. Pan-genome analyses were performed with 180 Bacillus subtilis and 204 Bacillus velezensis genomes to mine for any novel genes present in the genomes of our isolates. Moreover, all three isolates also consisted of gene clusters encoding secondary metabolites.

Phosphate-Solubilizing Bacillus sp. Modulate Soil Exoenzyme Activities and Improve Wheat Growth.

Phosphorus (P) is a vital mineral nutrient in agriculture and its deficiency results in reduced growth, yield, and grain quality in cereals. Much of the applied P in agriculture becomes fixed in soils, limiting its accessibility to plants. Thus, investigating sustainable strategies to release fixed P resources and enhance plant uptake is crucial. This study explored how plant-associated bacteria employ phosphate solubilizing mechanisms to improve P availability. The growth patterns of four bacterial strains, namely Bacillus subtilis ZE15 and ZR3, along with Bacillus megaterium ZE32 and ZR19, were examined in Pikovskaya's broth culture with and without the addition of insoluble phosphorus (P). In the absence of P amendment, most strains reached a stationary growth phase by the fourth day. However, their responses diverged when exposed to P-amended media. Particularly, ZE15 demonstrated the highest P solubilization capability, achieving up to 130 µg mL-1 solubilization in vitro. All strains produced organic acids in Pikovskaya's broth culture. A comparison of the influence of Ca3(PO4)2 revealed significantly greater organic acid quantities in the presence of insoluble P. Notably, strain ZE15 exhibited the highest phosphate esterase activity (3.65 nmol g-1 dry matter), while strain ZE32 showed the highest ß-D glucosidase activity (2.81 nmol g-1 dry matter) in the presence of insoluble P. The ability of Bacillus species to solubilize P in combination with increased exoenzyme activity in the rhizosphere could be used in future studies to support P uptake through enhanced solubilization and mineralization.

Effects of Bacillus subtilis as a single strain probiotic on growth, disease resistance and immune response of striped catfish (Pangasius hypophthalmus).

The present study investigated the potential role of Bacillus subtilis as probiotic in striped catfish (Pangasius hypophthalmus). Fish (initial weight = 150.00±2.63g n = 180) were stocked in circular tanks. Four isonitrogenous (30%) and isolipidic (3.29%) diets were formulated having supplementation of B. subtilis at four different levels (P0; 0, P1: 1×106, P2: 1×108 and P3: 1×1010 CFU/g). Each treatment had three replicates, while each replicate had fifteen fish. The trial started on second week of July and continued for eight weeks. Growth, feed conversion ratio, crude protein content, the concentration of amylase and protease, the profile of both dispensable and non-dispensable amino acids in all four dietary groups increased with a gradual increase of B. subtilis in the diet. At the end of growth experiment, fish in all four groups were exposed to Staphylococcus aureus (5×105 CFU/ml). After S. aureus challenge, fish fed with B. subtilis responded better to damage caused by reactive oxygen species and lipid peroxidation and better survival rate. The catalase and superoxide dismutase level also increased in response to bacterial challenge in B. subtilis fed groups. On the other hand, the concentration of malondialdehyde gradually decreased in these groups (+ve P0 >P1>P2>P3). It is concluded that supplementation of B. subtilis as a probiotic improved the growth, protein content, antioxidant response and immunocompetency against S. aureus in striped catfish. The optimum dosage of B. subtilis, at a concentration of 1×1010 CFU/g, resulted in the most favorable outcomes in striped catfish. This single bacterial strain can be used as an effective probiotic in large scale production of aquafeed for striped catfish. Future studies can investigate this probiotic's impact in the intensive culture of the same species.

Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design.

Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts are still unsatisfactory and far from being ready for industrial application. Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric excess (ee) value of the product generated by pnbA was only approximately 80%, with a high conversion rate (47.8%) of d,l-menthyl acetate with the help of a cosolvent, indicating high catalytic activity but low enantioselectivity (E = 19.95). To enhance the enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed mutagenesis was performed based on the results of molecular docking. The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11% ee and 30.58% conversion of d,l-menthyl acetate. To further improve the properties of pnbA, additional mutants were constructed based on the structure-guided triple-code saturation mutagenesis strategy. Finally, four mutants were screened for the best enantioselectivity (ee > 99%, E > 300) and catalytic efficiency at a high substrate concentration (200 g/L) without a cosolvent. This work provides several generally applicable biocatalysts for the industrial production of l-menthol.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.