Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Multiple Perspectives of Study on the Potential of Bacillus amyloliquefaciens JB20221020 for Alleviating Nutrient Stress in Lettuce.

Soil nutrient deficiency has become a key factor limiting crop growth. Plant growth-promoting rhizobacteria (PGPR) are vital in resisting abiotic stress. In this study, we investigated the effects of inoculation with Bacillus amyloliquefaciens JB20221020 on the physiology, biochemistry, rhizosphere microorganisms, and metabolism of lettuce under nutrient stress. Pot experiments showed that inoculation with B. amyloliquefaciens JB20221020 significantly promoted lettuce growth under nutrient deficiency. At the same time, the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase and the content of proline increased, and the content of Malondialdehyde decreased in the lettuce inoculated with B. amyloliquefaciens JB20221020. Inoculation with B. amyloliquefaciens JB20221020 altered the microbial community of the rhizosphere and increased the relative abundances of Myxococcales, Deltaproteobacteria, Proteobacteria, Devosia, and Verrucomicrobia. Inoculation also altered the rhizosphere metabolism under nutrient deficiency. The folate metabolism pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes enrichment analysis. This study explored the interaction between plants and microorganisms under nutrient deficiency, further explained the critical role of rhizosphere microorganisms in the process of plant nutrient stress, and provided a theoretical basis for the use of microorganisms to improve plant resistance.

Hypocholesterolemic potential of Bacillus amyloliquefaciens KAVK1 modulates lipid accumulation on 3T3-L1 adipose cells and high fat diet-induced obese rat model.

The significance of microorganisms occurring in foods is predominantly targeted due to their application for identifying a novel range of the bacterial spectrum. Diverse microbial species are capable of exhibiting potential pharmacological activities like antimicrobial and anticancer. Microbial strains capable of reducing obesity-related syndromes have also been reported. In the present study, the hypocholesterolemic efficacy of Bacillus amyloliquefaciens isolated from dairy products was scrutinised by in vitro (3T3-L1 adipose cells) and in vivo (high-fat diet-induced obese Wistar albino rats) methods. Potential cholesterol-lowering isolates were screened using a plate assay method and optimised by physical parameters. Molecular identification of the topmost five cholesterol-lowering isolates was acquired by amplification of the 16 S rRNA gene region. Bacillus amyloliquefaciens strain KAVK1, followed by strains KAVK2, KAVK3, KAVK4, and KAVK5 were molecularly determined. Further, cholesterol-lowering strains degraded the spectral patterns determined by the side chain of a cholesterol molecule. The anti-lipase activity was demonstrated using the porcine pancreatic lipase inhibitory method and compared with the reference compound Atorvastatin. Lyophilised strain KAVK1 revealed maximum pancreatic lipase inhibition. Strain KAVK1 attenuated lipid accumulation in 3T3-L1 adipose cell line predicted by Oil Red O staining method. Significant reduction of body weight and change in lipid profile was recognised after the supplement of KAVK1 to obese rats. Histopathological changes in organs were predominantly marked. The result of this study implies that the cholesterol-lowering B. amyloliquefaciens KAVK1 strain was used to treat hypercholesterolemia.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Osmolyte-producing microbial biostimulants regulate the growth of Arachis hypogaea L. under drought stress.

Globally, drought stress poses a significant threat to crop productivity. Improving the drought tolerance of crops with microbial biostimulants is a sustainable strategy to meet a growing population's demands. This research aimed to elucidate microbial biostimulants' (Plant Growth Promoting Rhizobacteria) role in alleviating drought stress in oil-seed crops. In total, 15 bacterial isolates were selected for drought tolerance and screened for plant growth-promoting (PGP) attributes like phosphate solubilization and production of indole-3-acetic acid, siderophore, hydrogen cyanide, ammonia, and exopolysaccharide. This research describes two PGPR strains: Acinetobacter calcoaceticus AC06 and Bacillus amyloliquefaciens BA01. The present study demonstrated that these strains (AC06 and BA01) produced abundant osmolytes under osmotic stress, including proline (2.21 and 1.75 µg ml- 1), salicylic acid (18.59 and 14.21 µg ml- 1), trehalose (28.35 and 22.74 µg mg- 1 FW) and glycine betaine (11.35 and 7.74 mg g- 1) respectively. AC06 and BA01 strains were further evaluated for their multifunctional performance by inoculating in Arachis hypogaea L. (Groundnut) under mild and severe drought regimes (60 and 40% Field Capacity). Inoculation with microbial biostimulants displayed distinct osmotic-adjustment abilities of the groundnut, such as growth parameters, plant biomass, photosynthetic pigments, relative water content, proline, and soluble sugar in respective to control during drought. On the other hand, plant sensitivity indexes such as electrolyte leakage and malondialdehyde (MDA) contents were decreased as well as cooperatively conferred plant drought tolerance by induced alterations in stress indicators such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). Thus, Acinetobacter sp. AC06 and Bacillus sp. BA01 can be considered as osmolyte producing microbial biostimulants to simultaneously induce osmotic tolerance and metabolic changes in groundnuts under drought stress.

Investigation into the potential mechanism of Bacillus amyloliquefaciens in the fermentation of broad bean paste by metabolomics and transcriptomics.

Pixian broad bean paste is a renowned fermented seasoning. The fermentation of broad bean is the most important process of Pixian broad bean paste. To enhance the flavor of tank-fermented broad bean paste, salt-tolerant Bacillus amyloliquefaciens strain was inoculated, resulting in an increase in total amount of volatile compounds, potentially leading to different flavor characteristics. To investigate the fermentation mechanism, monoculture simulated fermentation systems were designed. Metabolomics and transcriptomics were used to explore Bacillus amyloliquefaciens' transcriptional response to salt stress and potential aroma production mechanisms. The results highlighted different metabolite profiles under salt stress, and the crucial roles of energy metabolism, amino acid metabolism, reaction system, transportation system in Bacillus amyloliquefaciens' hypersaline stress response. This study provides a scientific basis for the industrial application of Bacillus amyloliquefaciens and new insights into addressing the challenges of poor flavor quality in tank fermentation products.

Screening signal peptidase based on split-GFP assembly technology to promote the secretion of alkaline protease AprE in Bacillus amyloliquefaciens.

Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.

A Novel Bacillus amyloliquefaciens Specifically Improving the Solubility and Antioxidant Activities of Edible Bird's Nest.

Edible bird's nest (EBN), a most highly priced and valuable foodstuff, contains high percentage of proteins and carbohydrates. However, proteins adhering to these carbohydrates make the EBN hard and tough, which need to be boiled as the bird's nest soup to make the Chinese cuisine. To overcome the hard and tough texture of EBN and improve the digestion degrees, the present study screened and identified a probiotic strain Bacillus amyloliquefaciens YZW02 from 5-year stored EBN sample completely solubilizing EBN for the first time. The 24-h B. amyloliquefaciens fermented EBN contained 20.30-21.48 mg/mL of the soluble protein contents with a recovery rate of 98-100%, DPPH radical scavenging rate of 84.76% and ABTS radical scavenging capacity of 41.05%. The mixed fermentation of B. amyloliquefaciens YZW02 and Bacillus natto BN1 were further applied to improve the low-MW peptide percentages and antioxidant activities. The mixed-fermentation of B. natto BN1 with 4-h cultured B. amyloliquefaciens YZW02 had the lowest percentage (82.23%) of >12-kDa proteins/peptides and highest percentages of 3-12 kDa, 1-3 kDa and 0.1-1 kDa peptides of 8.6% ± 0.08, 7.57% ± 0.09, 1.77% ± 0.05 and 0.73% ± 0.05, with the highest DPPH, ABTS and •OH scavenging capacity of 90.23%, 46.45% and 49.12%, respectively. These findings would provide an efficient strategy for improving the solubility and antioxidant activities of EBNs.

Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

Improving salt-tolerant artificial consortium of Bacillus amyloliquefaciens for bioconverting food waste to lipopeptides.

High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.

Improvement of branched-chain amino acid production by isolated high-producing protease from Bacillus amyloliquefaciens NY130 on isolated soy/whey proteins and their muscle cell protection.

Branched-chain amino acids (BCAAs) are vital components of human and animal nutrition that contribute to the building blocks of proteins. In this study, 170 protease-producing strains were isolated and screened from soy-fermented foods. Bacillus amyloliquefaciens NY130 was obtained from Cheonggukjang with high production of BCAAs. Optimal production of protease from B. amyloliquefaciens NY130 (protease NY130) was achieved at 42 °C and pH 6.0 for 21 h. It was purified and determined as 27- and 40 kDa. Protease NY130 showed maximum activity at pH 9.0 and 45 °C with Km value of 10.95 mg for ISP and 1.69 mg for WPI. Protease-treated ISP and WPI showed increased sweetness and saltiness via electronic tongue analysis and enhanced the protective effect against oxidative stress in C2C12 myocytes by increasing p-mTOR/mTOR protein expression to 160%. This work possesses potential in producing BCAAs by using protease for utilization in food.

Metagenomic analysis of the intestinal microbiome reveals the potential mechanism involved in Bacillus amyloliquefaciens in treating schistosomiasis japonica in mice.

Schistosomiasis japonica is one of the neglected tropical diseases characterized by chronic hepatic, intestinal granulomatous inflammation and fibrosis, as well as dysbiosis of intestinal microbiome. Previously, the probiotic Bacillus amyloliquefaciens has been shown to alleviate the pathological injuries in mice infected with Schistosoma japonicum by improving the disturbance of the intestinal microbiota. However, the underlying mechanisms involved in this process remain unclear. In this study, metagenomics sequencing and functional analysis were employed to investigate the differential changes in taxonomic composition and functional genes of the intestinal microbiome in S. japonicum-infected mice treated with B. amyloliquefaciens. The results revealed that intervention with B. amyloliquefaciens altered the taxonomic composition of the intestinal microbiota at the species level in infected mice and significantly increased the abundance of beneficial bacteria. Moreover, the abundance of predicted genes in the intestinal microbiome was also significantly changed, and the abundance of xfp/xpk and genes translated to urease was significantly restored. Further analysis showed that Limosilactobacillus reuteri was positively correlated with several KEGG Orthology (KO) genes and metabolic reactions, which might play important roles in alleviating the pathological symptoms caused by S. japonicum infection, indicating that it has the potential to function as another effective therapeutic agent for schistosomiasis. These data suggested that treatment of murine schistosomiasis japonica by B. amyloliquefaciens might be induced by alterations in the taxonomic composition and functional gene of the intestinal microbiome in mice. We hope this study will provide adjuvant strategies and methods for the early prevention and treatment of schistosomiasis japonica. IMPORTANCE: Targeted interventions of probiotics on gut microbiome were used to explore the mechanism of alleviating schistosomiasis japonica. Through metagenomic analysis, there were significant changes in the composition of gut microbiota in mice infected with Schistosoma japonicum and significant increase in the abundance of beneficial bacteria after the intervention of Bacillus amyloliquefaciens. At the same time, the abundance of functional genes was found to change significantly. The abundance of genes related to urease metabolism and xfp/xpk related to D-erythrose 4-phosphate production was significantly restored, highlighting the importance of Limosilactobacillus reuteri in the recovery and abundance of predicted genes of the gut microbiome. These results indicated potential regulatory mechanism between the gene function of gut microbiome and host immune response. Our research lays the foundation for elucidating the regulatory mechanism of probiotic intervention in alleviating schistosomiasis japonica, and provides potential adjuvant treatment strategies for early prevention and treatment of schistosomiasis japonica.

Bacillus amyloliquefaciens SC06 Relieving Intestinal Inflammation by Modulating Intestinal Stem Cells Proliferation and Differentiation via AhR/STAT3 Pathway in LPS-Challenged Piglets.

Bacillus amyloliquefaciens is a well-accepted probiotic, with many benefits for both humans and animals. The ability of intestinal stem cells (ISCs) to develop into several intestinal epithelial cell types helps accelerate intestinal epithelial regeneration. Limited knowledge exists on how bacteria regulated ISCs proliferation and regeneration. Our study investigated the effects of Bacillus amyloliquefaciens supplementation on ISC proliferation and regeneration and intestinal mucosal barrier functions in piglets exposed to lipopolysaccharide (LPS). Eighteen piglets (male, 21 days old) were randomly split into 3 clusters: CON cluster, LPS cluster, and SC06+LPS cluster. On day 21, 100 µg/kg body weight of LPS was intraperitoneally administered to the SC06+LPS and LPS groups. We found SC06 supplementation maintained the intestinal barrier integrity, enhanced intestinal antioxidant capacity, reduced generation of inflammatory response, and suppressed enterocyte apoptosis against the deleterious effects triggered by LPS. In addition, our research indicated that the SC06 supplementation not only improved the ISC regeneration, but also resulted in upregulation of aryl hydrocarbon receptor (AhR) in LPS-challenge piglets. Further studies showed that SC06 also induced ISC differentiation toward goblet cells and inhibited their differentiation to intestinal absorptive cells and enterocytes. The coculture system of SC06 and ileum organoids revealed that SC06 increased the growth of ISCs and repaired LPS-induced organoid damage through activating the AhR/STAT3 signaling pathway. These findings showed that SC06, possibly through the AhR/STAT3 pathway, accelerated ISC proliferation and promoted epithelial barrier healing, providing a potential clinical treatment for IBD. Our research demonstrated that SC06 is effective in preventing intestinal epithelial damage after pathological injury, restoring intestinal homeostasis, and maintaining intestinal epithelial regeneration.

Crude Lipopeptides Produced by Bacillus amyloliquefaciens Could Control the Growth of Alternaria alternata and Production of Alternaria Toxins in Processing Tomato.

Alternaria spp. and its toxins are the main contaminants in processing tomato. Based on our earlier research, the current study looked into the anti-fungal capacity of crude lipopeptides from B. amyloliquefaciens XJ-BV2007 against A. alternata. We found that the crude lipopeptides significantly inhibited A. alternata growth and reduced tomato black spot disease incidence. SEM analysis found that the crude lipopeptides could change the morphology of mycelium and spores of A. alternata. Four main Alternaria toxins were detected using UPLC-MS/MS, and the findings demonstrated that the crude lipopeptides could lessen the accumulation of Alternaria toxins in vivo and in vitro. Meanwhile, under the stress of crude lipopeptides, the expression of critical biosynthetic genes responsible for TeA, AOH, and AME was substantially down-regulated. The inhibitory mechanism of the crude lipopeptides was demonstrated to be the disruption of the mycelial structure of A. alternata, as well as the integrity and permeability of the membrane of A. alternata sporocytes. Taken together, crude lipopeptides extracted from B. amyloliquefaciens XJ-BV2007 are an effective biological agent for controlling tomato black spot disease and Alternaria toxins contamination.

Inclusion of a Bacillus-based probiotic in non-starch polysaccharides-rich broiler diets.

This study examined the effects of a 3-strain Bacillus-based probiotic (BP; Bacillus amyloliquefaciens and two Bacillus subtilis) in broiler diets with different rye levels on performance, mucus, viscosity, and nutrient digestibility. We distributed 720 one-d-old female broilers into 72 pens and designed nine diets using a 3 × 3 factorial approach, varying BP levels (0, 1.2 × 106, and 1.2 × 107 CFU/g) and rye concentrations (0, 200, 400 g/kg). On d 35, diets with 200 or 400 g/kg rye reduced broiler weight gain (BWG). Diets with 400 g/kg rye had the highest FCR, while rye-free diets had the lowest (p ≤0.05). Adding BP increased feed intake and BWG in weeks two and three (p ≤0.05). It should be noted that the overall performance fell below the goals of the breed. Including rye in diets reduced the coefficient of apparent ileal digestibility (CAID) for protein, ether extract (EE), calcium, phosphorus, and all amino acids (p ≤0.05). Rye-free diets exhibited the highest CAID for all nutrients, except for methionine, EE, and calcium, while diets with 400 g/kg of rye demonstrated the lowest CAID (p ≤0.05). BP in diets decreased phosphorus CAID (p ≤0.05). Diets containing 1.2 × 107 CFU/g (10X) of BP exhibited higher CAID of methionine than the other two diets (p ≤0.05). Diets containing 10X of BP showed higher CAID of cysteine than diets with no BP (p ≤0.05). Ileal viscosity increased as the inclusion level of rye in the diets increased (p ≤0.05). The ileal concentration of glucosamine in chickens fed diets with 400 g/kg of rye was higher than in those fed diets with no rye (p ≤0.05). Furthermore, ileal galactosamine concentrations were elevated in diets with 200 and 400 g/kg of rye when compared to rye-free diets (p ≤0.05). However, BP in diets had no impact on ileal viscosity, galactosamine, or glucosamine (p > 0.05). In conclusion, the applied Bacillus strains appeared to have a limited capacity to produce arabinoxylan-degrading enzymes and were only partially effective in mitigating the negative impacts of rye arabinoxylans on broilers.

Bacillus amyloliquefaciens SC06 alleviated intestinal damage induced by inflammatory via modulating intestinal microbiota and intestinal stem cell proliferation and differentiation.

The aim of our research was to investigate the effects of Bacillus amyloliquefaciens SC06 on growth performance, immune status, intestinal stem cells (ISC) proliferation and differentiation, and gut microbiota in weaned piglets. Twelve piglets (male, 21 days old, 6.11 ± 0.12 kg) were randomly allocated to CON and SC06 (1 × 108 cfu/kg to diet) groups. This experiment lasted three weeks. Our results showed that SC06 increased (P < 0.05) growth performance and reduced the diarrhea rate in weaned piglets. In addition, SC06 increased intestinal morphology and interleukin (IL)-10 levels, and decreased (P < 0.01) necrosis factor (TNF-α) levels in jejunum and serum. Moreover, weaning piglets fed SC06 had a better balance of colonic microbiota, with an increase in the abundance of Lactobacillus. Furthermore, SC06 enhanced ISCs proliferation and induced its differentiation to goblet cells via activating wnt/ß-catenin pathway in weaned piglets and intestinal organoid. Taken together, SC06 supplementation improved the growth performance and decreased inflammatory response of piglets by modulating intestinal microbiota, thereby accelerating ISC proliferation and differentiation and promoting epithelial barrier healing.

Bioactivity and genome analysis of Bacillus amyloliquefaciens GL18 isolated from the rhizosphere of Kobresia myosuroides in an alpine meadow.

The unique eco-environment of the Qinghai-Tibet Plateau breeds abundant microbial resources. In this research, Bacillus amyloliquefaciens GL18, isolated from the rhizosphere of Kobresia myosuroides from an alpine meadow, and the antagonistic activity, bacteriostatic hydrolase activity, and low temperature, salt, and drought resistance of it were determined and analysed. The seedlings of Avena sativa were root-irrigated using bacteria suspensions (cell concentration 1 × 107 cfu/mL) of GL18, and the growth-promoting effect of GL18 on it was determined under cold, salt and drought stress, respectively. The whole genome of GL18 was sequenced, and its functional genes were analysed. GL18 presented significant antagonistic activity to Fusarium graminearum, Fusarium acuminatum, Fusarium oxysporum and Aspergillus niger (inhibition zone diameter > 17 mm). Transparent zones formed on four hydrolase detection media, indicating that GL18 secreted cellulase, protease, pectinase and ß-1,3-glucanase. GL18 tolerated conditions of 10 °C, 11% NaCl and 15% PEG-6000, presenting cold, salt and drought resistance. GL18 improved the cold, salt and drought tolerance of A. sativa and it showed significant growth effects under different stress. The total length of the GL18 genome was 3,915,550 bp, and the number of coding DNA sequence was 3726. Compared with the clusters of orthologous groups of proteins, gene ontology and kyoto encyclopedia of genes and genomes databases, 3088, 2869 and 2357 functional genes were annotated, respectively. GL18 contained gene clusters related to antibacterial substances, functional genes related to the synthesis of plant growth-promoting substances, and encoding genes related to stress resistance. This study identified an excellent Bacillus strain and provided a theoretical basis for improving stress resistance and promoting the growth of herbages under abiotic stress.

An oxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupin gene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Potentiation effect of the AI-2 signaling molecule on postharvest disease control of pear and loquat by Bacillus amyloliquefaciens and its mechanism.

An autoinducer-2 (AI-2) signaling molecule from Bacillus was synthesized, and its mechanism on the biofilm formation and biocontrol ability of B. amyloliquefaciens was verified in vitro and in vivo. The 16S/ITS amplicon sequencing was used to analyze the effect of B. amyloliquefaciens B4 with or without AI-2 on the microflora of pears during storage. The results showed that B. amyloliquefaciens B4 secreted AI-2, which promoted biofilm formation. Additionally, AI-2 at a concentration of 40 µmol/L enhanced the biocontrol ability of B. amyloliquefaciens B4 on postharvest pear and loquat fruits. Finally, amplicon sequencing demonstrated that the addition of AI-2 increased the abundance of B. amyloliquefaciens B4 in fruit by stimulating the growth and biofilm formation of this bacterium.