• The Plant Pathology Journal
• /
• v.25 no.4
• /
• pp.344-351
• /
• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

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

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.29 no.3
• /
• pp.357-363
• /
• 2000

고추장의 품질을 향상시키고 재래식 전통 고추장과 유사한 맛을 지닌 고추장을 만들기 위해 재래식 전통 고추장에서 분리한 B.licheniformis 균주와 공장에서 대량 생산을 위해 개량해온 A. oryzae와, 비교적 호염성이며 알콜 발효 능력이 우수한 S.rouxii를 혼용하여 고추장을 담근 후 180일까지의 각 고추장의 이화학적 특성 변화를 조사한 결과는 다음과 같다. 단도는 모든 구에서 숙성 30일 까지 급격히 증가한 후 180일까지 거의 일정한 수준을 유지하였으며, 곰팡이 단용구에 비해 고초균과 효모 혼용구에서 숙성 기간 동안 높은 적정산도 값을 보였다. 조단백질은 숙성 기간 동안 대조구에서는 숙성 60일까지 약간 증가한 후 숙성 120일까지 급격히 감소하였으며 고초균 혼용구와 효모 첨가구는 숙성 90일 이후 감소한 후 숙성 120일 이후부터는 거의 변화가 없었다. 숙성 90일 이후부터는 B. licheniformis와 효모 혼용구에서 A. oryzae 단용구보다 조단백질 함량이 높았다. 환원당은 세 구간 모두 숙성 60일 경까지는 증가하는 추세를 보였으며, A. oryzae 단용구보다 B.licheniformis 균주와의 혼용구에서 높은 수치를 보여주었다. 비휘발성 유기산은 숙성 기간이 경과하면서 증가하였고 가장 많이 검출된 유기산은 pyroglutamic acid와 citric acid의 순이었다. 따라서 고추장의 숙성 과정 중 이화학적 특성 변화의 측면에서 볼 때 고초균과 효모 혼용구로 제조된 고추장이 구수한 맛의 생성면에서 유용한 담금법인 것으로 사료된다.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.1
• /
• pp.55-64
• /
• 2009

A B cell-specific growth substance (BGS) was isolated from the slime layer of Bacillus licheniformis E1. Unlike LPS, the BGS was not affected by polymixin B, an inhibitor of LPS, or by TLR4, and resulted in the growth of B cells. When BALB/c mice were treated with the BGS, the B cell population was found to increase in both the bone marrow and the spleen, with a marked increase after 24 h in the bone marrow and after 48 h in the spleen. When using antibodies to B cell lineage-restricted surface molecules to analyze the B cell population changes resulting from treatment with the BGS, an increase in immature B cells ($IgM^+$ and $AA4.1^+$) and mature B cells ($IgM^+$ and $IgD^+$) was found in the bone marrow 24 h after treatment with the BGS, whereas a decrease in mature B cells and increase in $IgG^+$ B cells were found in the spleen. When the BGS and OVA antigen were injected into the peritoneal cavity of BALB/c mice, this resulted in a high OVA-specific antibody titer in the sera, similar to that induced by aluminum hydroxide. Therefore, it is anticipated that the mass production of the BGS by B. licheniformis E1 could be used for studies of B cells in immunology, and contribute to the development of a new adjuvant for vaccine manufacture.

• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.2
• /
• pp.171-180
• /
• 2017

Objective: This study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR) silage. Methods: The TMRs were prepared with soybean curd residue, alfalfa hay (ATMR) or Leymus chinensis hay (LTMR), corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties. Results: Both ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens (B. amyloliquefaciens), B. cereus, B. licheniformis, and B. subtilis in ATMR silage and B. flexus, B. licheniformis, and Paenibacillus xylanexedens (P. xylanexedens) in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens, B. licheniformis, and B. subtilis and B. licheniformis, B. pumilus, and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively. Conclusion: The microbial amylase contributes to starch hydrolysis during the ensiling process in both TMR silages, whereas the microbial hemicellulase participates in the hemicellulose degradation only at the early stage of ensiling.

• The Plant Pathology Journal
• /
• v.26 no.3
• /
• pp.238-244
• /
• 2010

Spatial and temporal distribution of Bacillus licheniformis N1 was investigated over time on the leaves, petioles and crowns of the strawberry plants. Bacterial population on the strawberry plants was quantified over time by selective plating. Bacterial population of N1 containing a plasmid pWH43G carrying green fluorescent protein (GFP) declined relatively faster on the plant surface as compared to the Strain N1 itself. However, this result was found to be enough to utilize the strain to visualize bacterial colonization on the plant surface. When B. licheniformis N1 was treated together with Silwet L-77 at 0.03%, the bacterial population on plant surface persisted for up to 7 days. B. licheniformis N1 (pWH43G) containing Silwet L-77 was applied on the strawberry plants and the GFP expressing bacteria were visualized by confocal laser scanning microscopy. Bacterial persistence was also investigated in a growth chamber and in a plastic house after N1 bioformulation treatment on the strawberry plant. The Strain N1 colonized three different tissues well and persisted over 3 to 5 days on the strawberry plants. They formed bacterial aggregates on plant surfaces for at least 3 days, resulting in a biofilm to resist fluctuating plant surface environment. However, the bacterial persistence dramatically declined after 7 days in all tested tissues in a plastic house. This study suggest that B. licheniformis N1 colonizes the strawberry plant surface and persists for a long time in a controlled growth chamber, while it can not persist over 7 days on the plant surface in a plastic house.

• Microbiology and Biotechnology Letters
• /
• v.48 no.2
• /
• pp.185-192
• /
• 2020

The global carbon storage regulator (Csr) system is conserved in bacteria and functions as a regulator in the exponential and stationary phases of growth in batch culture. The Csr system plays a role in the central carbon metabolism, virulence, motility, resistance to oxidative stress, and biofilm formation. Although the Csr was extensively studied in Gram negative bacteria, it has been reported only in the control of motility in Bacillus subtilis among Gram positive bacteria. The goal of this study was to explore the role of the csrA gene of Bacillus licheniformis M2-7 on motility and the bacterial ability to use hydrocarbons as carbon source. We deleted the csrA gene of B. licheniformis M2-7 using the plasmid pCsr-L, harboring the spectinomycin cassette obtained from the plasmid pHP45-omega2. Mutants were grown on culture medium supplemented with 2% glucose or 0.1% gasoline and motility was assessed by electron microscopy. We observed that CsrA negatively regulates motility by controlling the expression of the hag gene and the synthesis of flagellin. Notably, we showed the ability of B. licheniformis to use gasoline as a unique carbon source. Our results demonstrated that CsrA is an indispensable regulator for the growth of B. licheniformis M2-7 on gasoline.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.3
• /
• pp.447-455
• /
• 2021

Strains of four Bacillus spp. were respectively inoculated into sterilized soybeans and the free amino acid profiles of the resulting cultures were analyzed to discern their metabolic traits. After 30 days of culture, B. licheniformis showed the highest production of serine, threonine, and glutamic acid; B. subtilis exhibited the highest production of alanine, asparagine, glycine, leucine, proline, tryptophan, and lysine. B. velezensis increased the γ-aminobutyric acid (GABA) concentration to >200% of that in the control samples. B. sonorensis produced a somewhat similar amino acid profile with B. licheniformis. Comparative genomic analysis of the four Bacillus strains and the genetic profiles of the produced free amino acids revealed that genes involved in glutamate and arginine metabolism were not common to the four strains. The genes gadA/B (encoding a glutamate decarboxylase), rocE (amino acid permease), and puuD (γ-glutamyl-γ-aminobutyrate hydrolase) determined GABA production, and their presence was species-specific. Taken together, B. licheniformis and B. velezensis were respectively shown to have high potential to increase concentrations of glutamic acid and GABA, while B. subtilis has the ability to increase essential amino acid concentrations in fermented soybean foods.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.1
• /
• pp.126-135
• /
• 2006

In a previous study, a biological response modifier (BRM) specifically enhancing the function of B-cells was isolated from Korean fermented soybean paste (Kfsp), but not from non-fermented soybeans. In this study, we attempted to isolate the bacteria producing the BRM from Kfsp (KfspBRM) by ELISA using anti-KfspBRM and by B-cell proliferation. Five bacteria whose culture supernatants showed the BRM activities were isolated, and one of them was identified as Bacillus licheniformis E1. The bacterial BRM (bBRM) originated from a slime layer of B. licheniformis El had a molecular weight of 1,594 kDa, and contained $33\%\;(w/w)$ of reduced sugar and $4.6\%\;(w/w)$ of protein content. The bBRM appeared to be a glycoprotein that is physically, structurally, and functionally similar to the KfspBRM, suggesting that the isolates including B. licheniformis El may produce the KfspBRM in the fermentation process of soybean paste. The mass production of the BRM by the bacterium may help to study B-cells in immunology, and the enrichment of the BRM in Kfsp may help patients in future who are medically in need of potentiation of B-cell proliferation and antibody production.

• Journal of Life Science
• /
• v.20 no.3
• /
• pp.403-410
• /
• 2010

In order to produce high-quality fermenting composts, a microorganism was isolated from the natural world. The bacterium has not only in high enzyme activities but also had good antimicrobial activities against phytopathogenic microorganisms. Its cultivating characteristics were then investigated. Bacterium KJ-9, which contains high CMCase, protease and chitinase activities and excellent antimicrobial activities against phytopathogenic microorganisms, was separated from leaf mold and identified as Bacillus licheniformis by two methods: Bergey's Manual of Systematic Bacteriology and API 50 CHL Carbohydrate Test Kit (Bio Merieux, France) using an ATB (Automated Identification) computer system (Bio Merieux, France). Optimal medium for cultivation of B. licheniformis was 2% soluble starch as a carbon source, 0.5% yeast extract as a nitrogen source and 0.05% $MgSO_4{\cdot}7H_2O$. Optimal growth conditions of pH, temperature and shake speed were pH 7.0, $50^{\circ}C$ and 180 rpm, respectively. Culture broth of B. licheniformis KJ-9 cultured for 36~60 hr was effective in fungicidal activities against plant pathogens including Botrytis cinerea, Corynespora cassicola, Fusarium oxysporum, and Rhizoctonia solani.