稻瘟病拮抗菌株的分离、筛选及鉴定

从水稻病健叶、茎和根组织以及稻田土壤中共分离得到细菌菌株321株。经发酵法初筛,对稻瘟病菌丝生长有抑制作用的有57株,再通过平板对峙法复筛,具有强烈拮抗作用的菌株有5株,其抑菌距离达16mm以上。分别对5个菌株进行形态学观察、生理生化指标进行鉴定,结果有1株(No.156)为Bacillus subtilis,2株(No.171和No.177)为Bacillus pumillus,2株(No.192和No.279)为Bacillus ploymyxa。     

Isolation and characterization of a C12-lipopeptide produced by Bacillus subtilis HSO 121.

A new lipopeptide with C12 fatty acid has been isolated from the cell broth of Bacillus subtilis HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q-TOF MS and 1H-NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q-TOF MS. Comparing the obtained results with surfactin C13 showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso-C12 beta-hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 x 10(-5) mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 degrees C, and the surface tension at CMC (gamma CMC) achieved is as little as 27.71 mN/m. The hemolytic activities of the C12-lipopeptide on 2% human erythrocytes showed a HC50 of 26.5 micromol/l.     

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis

Abstract Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.     

稻瘟菌诱导的水稻 WRKY 基因OsWRKY52 的分离和鉴定

WRKY 蛋白参与植物对生物或非生物胁迫反应和一些发育、代谢过程,在植物中组成一个转录因子大家族 . 从水稻 cDNA 文库中分离到一个新的 WRKY 基因——— OsWRKY52 cDNA ,包括一个 1 719 bp 的开放读码框,推测编码一个由 572 个氨基酸组成的蛋白质,与燕麦 (Avena sativa) AsWRKY1 具有 54 ％的氨基酸一致性 . 该基因被非亲和性稻瘟菌快速诱导 . 凝胶阻滞实验结果表明,原核表达的 OsWRKY52 能与水稻 PR1a 启动子上的 W 盒元件特异结合 . 采用酵母单杂交体系的方法证明了 OsWRKY52 具有转录激活活性 , 其丝氨酸岛、苏氨酸岛和 C 端的富酸性氨基酸区是负责转录激活的区域 . 这些结果提示 OsWRKY52 作为一个转录激活子,可能参与植物对稻瘟菌的应答反应 .     

Isolation and physico-chemical characterization of an antifungal and antibacterial peptide produced by Bacillus licheniformis A12

An antifungal substance named peptide A12-C has been purified to homogeneity from supernatants of sporulated cultures of Bacillus licheniformis A12. It consists of a 0.77-kDa hydrophilic peptide containing two residues of Glu and one of Arg, Ala, Pro, Tyr and Orn. No fatty acids, phosphorus or carbohydrates have been detected. Peptide A12-C is active on several fungi (Microsporum canis CECT 2797, Mucor mucedo CECT 2653, M. plumbeus (CCM F 443, Sporothrix schenckii CECT 2799 and Trichophyton mentagrophytes CECT 2793) and bacteria (Bacillus megaterium, Corynebacterium glutamicum, Sarcina and Mycobacterium), although the latter are less sensitive. Correspondence to: A. Gálvez     

Production and partial characterization of bacteriocin-like pepitdes by Bacillus licheniformis ZJU12

Bacillus licheniformis ZJU12, which was isolated from soil, could produce bacteriocin-like peptides that exhibited a broad spectrum of antagonistic activity against various species of Gram-positive bacteria and fungal pathogens, but not against Gram-negative bacteria tested except Xanthomonas oryzae pv. oryzae, a rice pathogen. The bacteriocin-like peptides were sensitive to proteinase K and trypsin. The activity was stable during temperature exposure up to 100 °C for 30 min, but lost completely at 121 °C for 15 min. The cell-free supernatant of B. licheniformis ZJU12 was shown to retain the activity within the pH range of 2–9, and the optimum pH for the activity was about 6.5. No adverse effect of the antagonistic compound to mice was observed in acute toxicity tests with the dose of 0.8 mg/20 g.     

Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: purification,characterization, effects of surfactants and organic solvents

In this study, the extracellular thermostable alkaline protease out of A10 strain was purified 1.38-fold with 9.44% efficiency through the ammonium sulfate precipitation-dialysis and DE52 anion exchange chromatography methods. The molecular weight of the enzyme in question along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be approximately 40.55?kDa, whereas the optimum pH and temperature ratings were identified as 9.0 and 70?°C, respectively. It was seen that the enzyme had remained stable between pH 7.5–10.5 range, protecting more than 90% of its activity in the wake of 1?h incubation at 60–70?°C. It was also observed that the enzyme enhanced its activity in the presence of Mg²⁺, Mn²⁺, K⁺, while Fe²⁺, Ni²⁺, Zn²⁺, Ag^+?and Co^2+? decreased the activity. Ca²⁺, however, did not cause any change in the activity. The enzyme was seen to have been totally inhibited by phenylmethylsulfonyl fluoride, therefore, proved to be a serine alkaline protease.     

黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1产生的脂肽类抗生素的分离和鉴别

本文旨在对黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1的脂肽类抗生素相关基因进行克隆,对其所产生的脂肽类抗生素进行组分分离,对有抑菌活性的脂肽类抗生素进行结构鉴别。以解淀粉芽孢杆菌B10-6-1基因组DNA为模板,采用PCR方法对ItuA、ItuB、ItuC、ItuD、FenA、FenB、FenD、Sfp、bmyA、bmyB、bmyC合成基因进行DNA扩增。将该菌发酵液经过离心、酸沉淀、甲醇抽提等步骤得到脂肽类抗生素的粗提物,采用高效液相色谱法分离,对收集的洗脱峰组分进行体外抑菌试验,对有抑菌活性的组分进行液质（HPLC-ESI-MS）分析。PCR扩增检测结果显示黄曲霉菌拮抗菌B10-6-1能扩增出ItuA、ItuC、ItuD、FenA、FenD、Sfp、bmyA、bmyB、bmyC合成基因,未能扩增出ItuB、FenB合成基因。高效液相色谱法收集到7种组分,体外抑菌试验证明组分5和7有抑菌活性。经液质测定,组分5和组分7分别为脂肽类抗生素C₁₄Bacillocnycin D和C₁₅Bacillocnycin D。本研究探明了黄曲霉菌拮抗菌B10-6-1所产生的天然抑菌活性成分,为该菌用于黄曲霉菌的生物防治奠定了理论基础。     

地衣芽孢杆菌类细菌素的分离、鉴定及其原核表达

目前,我国饲料中全面禁止添加抗生素,寻找新型的抗生素替代物成为科学研究的热点之一.为获得新的细菌素,本研究以大肠杆菌、沙门氏菌、金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特氏菌为指示菌,通过牛津杯扩散法从羊驼粪便中筛选到高产抑菌物质的芽孢杆菌.基于菌落特征、革兰氏染色和16SrRNA的鉴定结果,将分离到的产抑菌...     

The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain

Shorter lag phases were obtained in cultivations of Bacillus licheniformis using early-compared to late-stationary growth phase inocula and using liquid versus solid propagation medium. Flow cytometry and fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), confirmed that liquid early-stationary growth phase inoculum had a higher vitality and was more homogeneous than solid late-stationary growth phase inoculum. DNA-microarray analyses indicated that liquid early-stationary growth phase inoculum was in a more active state in terms of cell multiplication whereas solid late-stationary growth phase inoculum was induced to some spore formation potentially causing delayed growth initiation.     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.     

地衣芽胞杆菌对白色念珠菌等的拮抗作用

目的了解地衣芽胞杆菌在试管内与阴道正常菌群共生关系的情况。方法将地衣芽胞杆菌菌液分别与葡萄球菌、大肠埃希菌、白色念珠菌、德氏乳杆菌混合培养,定量计数各菌在不同时间内单独培养和混合培养时各菌的活菌数。结果地衣芽胞杆菌生长不受金黄色葡萄球菌、白色念珠菌和大肠埃希菌的影响,金黄色葡萄球菌和白色念珠菌在有地衣芽胞杆菌存在的情况下,其生长受到明显的抑制（P〈0.05）;乳杆菌在12-48 h内,有显著的抑制地衣芽胞杆菌生长的作用,而乳杆菌的生长不受地衣芽胞杆菌的存在与否而正常生长。结论地衣芽胞杆菌对金黄色葡萄球菌及白色念珠菌在体外具有明显的拮抗作用,地衣芽胞杆菌对大肠埃希菌、乳杆菌无明显的体外拮抗作用。     

水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性

【目的】为得到枯草芽孢杆菌(Bacillus subtilis)G87的抗菌蛋白,明确其蛋白理化特性。【方法】采用硫酸铵沉淀和柱层析法进行分离纯化。【结果】获得单一抗菌活性蛋白(峰6-2-1),此抗菌蛋白分子量为50.8 kDa,等电点为5.90。经初步分析,抗菌蛋白不含脂,而含有少量(0.62%)糖;其蛋白部分具有脯氨酸或羟脯氨酸,但不含芳香族氨基酸。抗菌蛋白在高温(≥60℃)和较碱(pH8)环境下活性明显下降,但较抗紫外线、氯仿和胰蛋白酶、蛋白酶K、胃蛋白酶。【结论】枯草芽孢杆菌G87的抗菌蛋白为不含芳烃的糖蛋白,对高温和碱性条件敏感,而对蛋白酶类和紫外线等不敏感。     

Cd胁迫下枯草芽孢杆菌B12产表面活性素及其对生物被膜形成和Cd去除的影响

[背景]枯草芽孢杆菌可通过群集的方式形成生物被膜,而生物被膜在去除重金属方面发挥着重要作用.[目的]探究枯草芽孢杆菌(Bacillus subtilis)B12缓解Cd(CdSO4)胁迫的机制.[方法]考察Cd胁迫对B12生物被膜形成的影响,用酸沉淀法、反相高效液相色谱和液质联用分析(LC-MS)对有无Cd胁迫条件下菌...     

Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain

A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 degrees C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.     

一株产生脂肽的枯草芽孢杆菌的分离鉴定及脂肽对原油的作用

从大庆油田地层水中分离到一组能高效产生生物表面活性剂的菌株,采用sfp基因PCR鉴定的方法从中分离到一株芽孢杆菌ZW-3,该菌株能够产生大量表面活性物质,采用细菌生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株为枯草芽孢杆菌(Bacillus subtilis),通过薄层层析色谱(TLC)、高效液相色谱(HPLC)分析其代谢产物,初步鉴定为脂肽(Lipopeptide);该脂肽生物表面活性剂理化性质显示它能使培养基的表面张力从68.92mN/m降低25.19mN/m、原油/水的界面张力从23.53mN/m降低到4.57mN/m,与1.8%的NaOH溶液复配可以将油水界面张力降低到1.2×10-3 mN/m,其临界胶束浓度为33.3mg/L(3.24×10-5 mol/L),并具有较好的乳化活性和发泡性能,说明该菌株代谢的脂肽生物表面活性剂在提高石油采收率中具有广泛的应用前景.