• Microbiology and Biotechnology Letters
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• v.51 no.3
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• pp.289-292
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• 2023

Thermotolerant Bacillus subtilis NIB353 was isolated from Nuruk, a traditional Korean fermentation starter. The complete B. subtilis NIB353 genome sequence was obtained using MinION and Illumina (MiSeq) platforms. The B. subtilis NIB353 genome sequence was 4,247,447 bp with a GC content of 43%. The B. subtilis NIB353 strain exhibited orthologous average nucleotide identity values of 98.39% and 98.38% with B. subtilis 168 and B. subtilis ATCC6051a, respectively. The genome has been deposited in GenBank under the accession number NZ_CP089148.1.

• Journal of Animal Science and Technology
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• v.64 no.2
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• pp.291-301
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• 2022

The objective of this study was to evaluate the effects of different mixing ratios of Bacillus licheniformis and Bacillus subtilis in diets on nutrient digestibility, fecal microflora, and odor gas emissions of growing pigs. A total of four crossbred ([Landrace × Yorkshire] × Duroc) barrows with average body weight (BW) of 41.2 ± 0.7 kg were randomly allotted four diets over four periods in a 4 × 4 Latin square design. Treatments were as follows: Control (CON, basal diet), CON + 0.2% probiotic complex (L4S6, B. licheniformis and B. subtilis at a 4:6 ratio), CON + 0.2% probiotic complex (L5S5, B. licheniformis and B. subtilis at a 5:5 ratio), CON + 0.2% probiotic complex (L6S4, B. licheniformis and B. subtilis at a 6:4 ratio). Dietary probiotic supplementation showed higher crude protein (CP) digestibility values and lower Escherichia coli counts in fecal samples than the CON group (p < 0.05). There was no significant difference in NH₃ or H₂S emission until day 3. The positive effect of H₂S and NH₃ emissions was detected earlier with the L4S6 and L5S5 compared to the L6S4, which had a lower ratio of B. subtilis. Both the L4S6 and L5S5 probiotic complexes significantly decreased the fecal H₂S and NH₃ emission in days 4 and 6 (p < 0.05). On day 7, all probiotic complexes decreased (p < 0.05) H₂S and NH₃ emissions than the CON group. Our results agreed that the dietary supplementation of Bacillus licheniformis and Bacillus subtilis complexes in growing pigs can significantly improve CP digestibility and reduce fecal E. coli counts, NH₃ and H₂S emissions. Notably, the higher mixing ratio of Bacillus subtilis in probiotic supplementation is more effective in reducing the odor of manure.

• The Korean Journal of Mycology
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• v.49 no.4
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• pp.441-454
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• 2021

Fungal diseases including anthracnose, stem rot, blight, wilting, and root rot of crops are caused by phytopathogens such as Colletotrichum species, Sclerotinia sclerotiorum, Phytophthora species, and Fusarium oxysporum and F. solani which threaten the production of chili pepper. In this study, to identify biological control agents (BCAs) of phytopathogenic fungi, potentially useful Bacillus species were isolated from the field soils. We screened out five Bacillus strains with antagonistic capacity that are efficiently inhibiting the growth of phytopathogenic fungi. Bacillus species were characterized by the production of extracellular enzymes, siderophores, and indole-3-acetic acid (IAA). Furthermore, the influence of bacterial strains on the plant growth promoting activity and seedling vigor index were assessed using Brassica juncea as a model plant. Inoculation with Bacillus subtilis SRCM 121379 significantly increased the length of B. juncea shoots and roots by 45.6% and 52.0%, respectively. Among the bacterial isolates, Bacillus subtilis SRCM 121379 showed the superior enzyme activities, antagonistic capacity and plant growth promoting effects. Based on the experimental results, Bacillus subtilis SRCM 121379 (GenBank accession no. NR027552) was finally selected as a BCA candidate.

• Journal of Mushroom
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• v.21 no.3
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• pp.140-144
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• 2023

The objective of this study was to achieve biological control of green mold disease in Pyogo mushrooms using antagonistic microorganisms. Bacillus subtilis BSM320 cells inhibited mycelial growth by 48-60% against three Trichodermaisolates including T. hazianumisolated from the substrates of Lentinula edodes, showing their antifungal activity.The bacteria were cultured to a high density of 4.2 × 10⁹±113.7 cfu/mlin aqueous extract of composted spent mushroom substrates of L. edodes containing 1% glucose and showed a higher growth rate than that observed when using the commercial medium, Luria-Bertani broth. The bacterial culture showed a 75% protective effect without damaging the mushroom fruiting bodies. These results suggest that B. subtilis BSM320culture is suitable for biological control of green mold disease during mushroom cultivation.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• Korean Journal of Food Science and Technology
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• v.44 no.3
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• pp.312-316
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• 2012

In order to understand the effect of hydrostatic pressure (HP) on Bacillus subtilis isolated from makgeolli, the survival of B. subtilis after HP treatment (400 MPa for 5 min) in various substrates including phosphate buffer, tryptone soya broth at pH 7 and 4, and makgeolli at pH 4 was evaluated depending on bacterial forms (spores and vegetative cells) and adaptation conditions ($25^{\circ}C$ for 3 h, or $10^{\circ}C$ for 24 h). Spores were generally resistant to HP (<1 log reduction) regardless of conditions. In contrast, vegetative cells were generally susceptible to HP (up to 3 log reduction-except makgeolli) and were more susceptible after 3 h at $25^{\circ}C$ compared to 24 h at $10^{\circ}C$. In vegetative cells inoculated makgeolli (7 log CFU/mL), the colonies were not detected after 24 h at $10^{\circ}C$. Consequently, B. subtilis in makgeolli easily existed as spores and the spores were resistant to HP. Results demonstrate that HP was more promising in the inactivation of vegetative cells.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.439-446
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• 2020

Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.12
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• pp.1576-1582
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• 2008

This study was conducted to investigate the effect of administration of calcium supplementary food containing fermented product of Bacillus subtilis SE4 highly producing poly-$\gamma$-glutamic acid on the growthparameters of adolescent male rats. Four-week old male Sprague-Dawley (SD) rats were fed for 4 weeks and assigned to the following 4 groups: two groups administered orally with new calcium supplementary food (such as 150 mg/kg and 300 mg/kg) containing fermented product of B. subtilis SE4, one group administered with conventional calcium supplementary food product (150 mg/kg) and one saline group as control. Daily weight gain and daily food intake in the two new food product groups were higher than those of conventional food product group and control group. Especially, the content of serum IGF-I in the two new food product groups were significantly higher than those in conventional food product group and in control group (p<0.05). In addition, length and weight of longitudinal bone in the two new food product groups were longer and heavier than those of conventional food product group and control group. Therefore, the addition of fermented food product of B. subtilis SE4 into the conventional calcium supplementary food increased all the parameters examined for the growth of the adolescent male rats.

• Mycobiology
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• v.40 no.1
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• pp.59-65
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• 2012

Antagonistic microorganisms against $Rhizoctonia$ $solani$ were isolated and their antifungal activities were investigated. Two hundred sixteen bacterial isolates were isolated from various soil samples and 19 isolates were found to antagonize the selected plant pathogenic fungi with varying degrees. Among them, isolate C9 was selected as an antagonistic microorganism with potential for use in further studies. Treatment with the selected isolate C9 resulted in significantly reduced incidence of stem-segment colonization by $R.$ $solani$ AG2-2(IV) in Zoysia grass and enhanced growth of grass. Through its biochemical, physiological, and 16S rDNA characteristics, the selected bacterium was identified as $Bacillus$ $subtilis$ subsp. $subtilis$. Mannitol (1%) and soytone (1%) were found to be the best carbon and nitrogen sources, respectively, for use in antibiotic production. An antibiotic compound, designated as DG4, was separated and purified from ethyl acetate extract of the culture broth of isolate C9. On the basis of spectral data, including proton nuclear magneric resonance ($^1H$ NMR), carbon nuclear magneric resonance ($^{13}C$ NMR), and mass analyses, its chemical structure was established as a stereoisomer of acetylbutanediol. Application of the ethyl acetate extract of isolate C9 to several plant pathogens resulted in dose-dependent inhibition. Treatment with the purified compound (an isomer of acetylbuanediol) resulted in significantly inhibited growth of tested pathogens. The cell free culture supernatant of isolate C9 showed a chitinase effect on chitin medium. Results from the present study demonstrated the significant potential of the purified compound from isolate C9 for use as a biocontrol agent as well as a plant growth promoter with the ability to trigger induced systemic resistance of plants.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1720-1728
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• 2020

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.