表型特征结合基因组分析鉴定不同菌落形态Bacillus velezensis ACCC 19742

在中国农业微生物菌种中心(ACCC)菌株的定期转接保藏过程中,发现解淀粉芽孢杆菌ACCC 19742在同一培养基上出现两种不同的菌落形态,将这两个不同形态的菌株编号为19742-1和19742-2。通过形态学、生理生化及基因组分析相结合鉴定不同菌落形态ACCC 19742,并进一步确定该菌株的分类地位。首先将菌株进行分离与纯化,其次将纯化后的菌株进行16S rRNA及gyrB基因扩增及序列分析,通过MEGA 7.0软件构建系统发育树;API 20NE、BIOLOG及脂肪酸等分析菌株的生理生化特性;全基因组分析菌株的ANI和DDH值。两株菌在API 20NE中,仅葡萄糖酸盐同化反应存在差异;脂肪酸检测中主要组成相同,仅是百分含量方面略有差别;两株菌的16S rRNA基因相似性为100%,gyrB基因的相似性为99.4%;全基因组测序表明,两株菌的ANI值为99.95%,DDH值为99.62%。综合遗传学特征和表型特征,证实两者为来源于同一菌株不同的形态变异型,而并非污染所致。同时,19742-1和19742-2与Bacillus velezensis NRRL_B 41580~T的ANI及DDH值最高,分别为97%和77%,且16S rRNA和gyrB系统进化分析也表明,该菌株在分类地位上属于贝莱斯芽孢杆菌(Bacillus velezensis),而非解淀粉芽孢杆菌。这为菌株的保藏提供了一定的参考价值。     

解淀粉芽胞杆菌抗真菌活性研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)具有很强的抑制植物病原真菌的能力。其菌体细胞能产生多种酶类、脂肽类抗生素、生物表面活性素、聚酮类化合物和抑菌蛋白,同时具有诱导植物产生系统抗性(ISR)的能力,因此在工农业、种植业、养殖业、食品加工业、果蔬的采后保鲜和饲料业等行业具有重要价值。本文对解淀粉芽胞杆菌抗真菌作用、抗真菌能力提高策略、抗菌化合物合成调节、抑制真菌机制及其引发的ISR等问题进行了深入探讨和综述。     

解淀粉芽孢杆菌生防菌BS-3全基因组测序及生物信息分析

【背景】解淀粉芽孢杆菌BS-3是从健康橡胶树树根中分离获得的一株对真菌具有较强抗菌活性的内生细菌,有作为生物农药的潜力。【目的】解析菌株BS-3的基因组序列信息,以深入研究该菌株防病促生机制及挖掘次级代谢产物基因资源。【方法】采用第二代BGISEQ与第三代Pac Bio平台相结合的测序技术,对生防菌BS-3进行全基因组测序,并对测序数据进行基因组组装、基因预测与功能注释、共线性分析及次级代谢产物合成基因簇预测等。【结果】BS-3全基因组大小为3 870 130 bp,平均GC含量为46.88%,共编码4 161个基因;含有92个t RNA基因、28个r RNA、10个sRNA;含有122个串联重复序列、98个小卫星DNA、2个微卫星。在COG、GO、KEGG、NR和Swiss-Prot数据库分别注释到基因2 875、2 620、1 885、4 040和3 328个。同时,预测到BS-3中有10个次级代谢产物合成基因簇,编码表面活性素、丰原素、多烯类、儿茶酚型嗜铁素等抑菌物质。基因组测序数据提交至NCBI获得GenBank登录号为CP060384。【结论】为基因组层面上解析菌株BS-3具有良好防病效果的内在原因提供基础数据,为深入了解解淀粉芽孢杆菌次级代谢合成途径提供参考信息,对菌株BS-3后续相关研究具有重要意义。     

Endophytic bacterium,Bacillus amyloliquefaciens,enhances ornamental hosta resistance to diseases and insect pests

A total of 84 bacterial endophytes were isolated from seeds of 6 cultivars of ornamental hostas, and they were identified to 5 species based on morphological characteristics and 16S rDNA sequence analysis. Among them, the strain ‘Blu-v2’, which was isolated from the seeds of cultivar ‘Blue Umbrella’ and identified to be Bacillus amyloliquefaciens, showed highest antifungal activity and capacity to deter feeding by Fall armyworms (Spodoptera fruigiperda). Lipopeptides in cultures of Blu-v2 were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its antifungal activities were verified. However, the lipopeptide preparation did not show toxicity to larvae of Fall armyworms. In a greenhouse experiment, Blu-v2 was inoculated into small plantlets of hosta (cultivar ‘Rainforest Sunrise’). The leaves of plants with bacteria (endophyte-infected?=?E+) and without bacteria (endophyte-free?=?E?) were used in seven-day feeding experiments employing fourth-instar larvae of Fall armyworms. We found that there was a significant decrease in the weights of larvae fed with E+ compared to E? plants; and the mortality rate of larvae fed with E? leaves was lower (3.33%) compared to that of larvae fed with E+ leaves (30%). Based on our studies, we suggest that endophytic B. amyloliquefaciens strain Blu-v2 has potential value as a biocontrol agent to reduce damage from fungal diseases and insect pests of hosta cultivars.     

解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K⁺对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds

AIMS: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean. METHODS AND RESULTS: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were resistant over a wide pH range and to hydrolytic enzymes. By (1)H-NMR and thin-layer chromatography analyses surfactin and iturin-like compounds were partially identified. Moreover, soya bean seeds bacterization with BNM 122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene. According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis. PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic fingerprinting revealed a close genetic relationship to B. amyloliquefaciens. However, by physiological characterization using API tests, this strain resembled more B. subtilis. CONCLUSIONS: This is the first report describing the co-production of surfactin and iturin-like compounds by a putative strain of B. amyloliquefaciens. The synergistic effect of both lipopetides is a remarkable trait for a candidate biocontrol agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This kind of research has relevance in order to minimize the use of synthetic fungicides and surfactants, contributing to the preservation of the environment.     

解淀粉芽孢杆菌高效合成胞苷的代谢调控机制及育种策略

胞苷是合成抗病毒、抗肿瘤药物的良好中间体,也是核苷酸类保健食品和功能性食品的重要原料。主要论述了解淀粉芽孢杆菌高效合成胞苷的代谢调控机制和构建胞苷高产菌株的育种策略。重点阐述了通过提高解淀粉芽孢杆菌嘧啶操纵子转录水平,增强胞苷合成代谢途径、阻断胞苷降解途径、增大磷酸戊糖途径向胞苷合成途径的分流量、提高胞苷合成前端代谢物PRPP的合成量、增强中心碳代谢流向胞苷合成途径、减少旁路代谢途径及促进胞苷的分泌等方法,选育出胞苷高产菌株,不仅为胞苷高产菌株的选育提供参考,也为解决目前胞苷工业化生产中存在的问题提供思路。