基于气相色谱-飞行时间质谱联用技术分析枯草芽孢杆菌对变异链球菌的抑制作用

【背景】龋病危害着人类健康,变异链球菌(Streptococcusmutans)是公认的主要致龋菌。我们前期的研究从口腔中分离出一株枯草芽孢杆菌(Bacillussubtilis),其能抑制S.mutans的生长,但尚未明确其抑菌作用的组分。【目的】利用非靶向代谢组学技术检测分析B. subtilis zh78抑制S. mutans生长可能的小分子代谢物,以评估其在龋病防治研究及应用中的前景。【方法】取B. subtilis zh78经生长0 h、7 h、12 h、5 d时的菌液,利用牛津杯法将冷甲醇萃取后的代谢物对S. mutans进行抑菌实验;采用气相色谱-飞行时间质谱联用技术(GC-TOF-MS)对代谢物进行检测;利用主成分分析(Principalcomponentanalysis,PCA)、正交偏最小二乘判别分析(Orthogonalpartialleastsquaresdiscriminantanalysis,OPLS-DA)和皮尔森相关分析(Pearson correlationanalysis)等方法进行数据分析。【结果】B.subtiliszh78生长5d时的代谢产物对S.mutans具有明显抑菌作用,其代谢物中木糖醇、氨基酸类及有机酸类等36种物质与其对S.mutans的抑制作用显著相关。【结论】B. subtilis zh78可能产生木糖醇、氨基酸类及有机酸类等可能促进B. subtilis zh78抑制S. mutans生长的小分子代谢产物,具有一定的防龋应用研究前景。     

枯草芽孢杆菌J-15抗大丽轮枝菌次生代谢产物对棉田土壤真菌多样性的影响

【背景】目前利用拮抗菌进行作物病害防治的研究较多,但拮抗菌次生代谢产物如何影响棉花根际土壤微生物群落相关的研究较少。【目的】探讨枯草芽孢杆菌J-15抗大丽轮枝菌次生代谢产物对棉田土壤真菌多样性的影响,为利用枯草芽孢杆菌J-15及其次生代谢产物防治棉花黄萎病的土壤微生物生态安全进行评估。【方法】以新疆北部玛纳斯地区棉田为土壤采样点,随机选取10个点进行采样后混合,经枯草芽孢杆菌J-15抗大丽轮枝菌次生代谢产物处理一定时间后,提取土样总DNA,利用Illumina HiSeq高通量技术,对样品中真菌ITS1-ITS2区进行高通量测序,分析J-15抗大丽轮枝菌次生代谢产物处理对土样真菌多样性的影响。【结果】在97%相似度水平下,处理10、30d后,样品中真菌的OTU数量、Chao1和ACE丰度指数均分别高于相同时间放置的未处理的对照组,而Simpson指数低于其对照组。从群落组成分析来看,与对照组相比,受J-15次生代谢产物处理的土壤样品,子囊菌门(Ascomycota)的盘菌属(Tricharina)和被孢霉门(Mortierellomycota)的被孢霉属(Mortierella)等优势真菌相对丰度提高,而丰度高于1%的2类病原真菌轮枝孢属(Verticillium)、镰孢霉属(Fusarmm)的丰度显著降低。【结论】J-15抗大丽轮枝菌次生代谢产物对棉田土壤真菌群落及丰度有显著影响,但不改变影响农业生产的土壤真菌群落的结构。     

枯草芽孢杆菌fmb60菌株非核糖体肽类化合物对铜绿微囊藻生长的抑制作用

铜绿微囊藻是一种分布广泛的水华微藻,可对人类健康和生态环境造成严重危害,而枯草芽孢杆菌作为一种生防微生物可通过非核糖体肽合成酶合成多种生物活性物质。因此,枯草芽孢杆菌fmb60非核糖体肽(Non-ribosomal peptide,NRP)类产物对铜绿微囊藻生长抑制作用的研究在水华治理方面具有重要的意义。利用基因组挖掘技术从枯草芽孢杆菌fmb60中分离鉴定出bacillibactin、表面活性素(Surfactin)和芬芥素(Fengycin)3种NRP类产物,进而通过添加不同浓度的NRP类产物研究其对铜绿微囊藻生长的影响,结果显示其对铜绿微囊藻4 d的半效应浓度值EC50.4 d为26.5 mg/L,且随着样品浓度的增加,枯草芽孢杆菌fmb60的NRP类产物对铜绿微囊藻的抑制作用增强。当向其分别加入50 mg/L的样品时,培养4 d的铜绿微囊藻Fv/Fm、Fv/Fo、Yield参数分别比对照组降低了2.8%、1.7%、2.0%。表明枯草芽孢杆菌fmb60 NRP类产物能够显著抑制铜绿微囊藻的光合作用强度及代谢合成,从而为枯草芽孢杆菌抑藻剂的开发奠定理论基础。     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria.

Bacteria subjected to a hypotonic osmotic shock lose internal ions and also metabolites, without lysis of the cells. We show that the presence in the shock medium, at submillimolar concentrations, of the ion gadolinium, recently shown to block stretch-activated channels in Xenopus oocytes [Yang, X.-C. & Sachs, F. (1989) Science 243, 1068-1071], was sufficient to inhibit shock-induced release of metabolites such as lactose and ATP in Escherichia coli and ATP in Streptococcus faecalis. Moreover, gadolinium was observed, in patch-clamp experiments, to inhibit the giant stretch-activated channels of E. coli, S. faecalis. and Bacillus subtilis. Taken together, these data suggest that stretch-activated channels are localized in the cytoplasmic membrane of Gram-negative and Gram-positive bacteria, where they control the efflux of osmotic solutes, thus probably playing a major role in the response to hypotonic osmotic shock.     

Use of antagonistic Bacillus subtilis bacteria for treatment of nosocomial urinary tract infections

Effect of Bactisporin--a probiotic, containing spores of aerobic Bacillus subtilis 3H bacterium--for complex treatment of patients with nosocomial urinary tract infections was studied. 68 Cultures of different species of conditionally pathogenic bacteria were isolated from urine of the patients. Susceptibility of the isolated cultures to antibiotics before and after application of B. subtilis 3H metabolites was determined. The metabolites were accumulated on potato-glucose agar (PGA) while bacterium was cultivated on kapron membranes placed on surface of the medium. Influence of obtained metabolites on isolated strains was assessed by cultivation of each strain in metabolites-rich PGA during 24 h. Metabolites of B. subtilis led to decrease in resistance of isolated uropathogenic microflora to antibiotics. Use of Bactisporin in complex treatment of nosocomial urinary tract infections resulted in accelerated elimination of causative microorganism.     

Quantitative metabolome analysis using capillary electrophoresis mass spectrometry

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.     

Specific toxic effects of 2,4,6-trinitrotoluene on Bacillus subtilis SK1

Using Bacillus subtilis SK1 as an example, it was demonstrated for the first time that 2,4,6-trinitrotoluene (TNT) transformation pathways change with TNT concentration. The growth of cultured B. subtilis SK1, delayed at 20 mg/l TNT (minimum toxic concentration), was resumed following TNT transformation. Aromatic amines were predominant metabolites detected in the culture medium at early stages of TNT transformation. The culture growth was completely inhibited by 200 mg/l TNT. As this took place, nitrites accumulated in the culture medium.     

A rapid method for detecting interconnections between functionally and/or evolutionary close biological sequences]

It is now common practice to retrieve, by key words, highly specialized selections of sequences from general-purpose databases such as EMBL, GenBank, etc. The sequences included in a selection are often interconnected, which means that there are duplications, embeddings, intersections, homology, common structural elements. Knowledge of these interconnections is necessary for further processing of the sequences. We propose a rapid (single scan) method for identification of such interconnections by means of complexity analysis that generalizes the Lempel-Ziv approach. Analysis of a selection of 5'-flanking regions of vertebrate growth hormone genes from EMBL is presented as an example.     

一株海洋链霉菌MMHS020的抗菌活性代谢产物

【背景】海洋微生物因其生存环境的多样性与独特性,已成为天然产物研究的重要来源。【目的】以一株太平洋海泥来源链霉菌MMHS020为出发菌株,筛选可促进其产生丰富代谢产物的发酵条件,挖掘菌株在抗菌抗肿瘤方面的潜力。【方法】采用单菌株多次级代谢产物策略对MMHS020菌株进行培养诱导,使其产生更丰富的活性代谢产物。双层平板法测定发酵产物对6种指示菌的抑菌活性。以硅胶柱层析、葡聚糖凝胶层析和制备层析等方法对代谢产物进行分离纯化,再通过质谱技术和~1H-NMR和~(13)C-NMR对化合物进行结构解析。【结果】链霉菌属MMHS020菌株可在较高浓度盐离子环境中产生丰富的抑菌活性代谢产物,显示出对枯草芽孢杆菌、结核分枝杆菌和藤黄微球菌等多种指示菌的抑制活性。从发酵产物中分离鉴定了3个化合物,分别是诺卡胺素(1)、麦角甾醇(2)和星形孢菌素(3)。其中星形孢菌素表现出白色念珠菌的抑制活性,而诺卡胺素则对其他几个指示菌表现出较强的抑制活性。【结论】海洋链霉菌MMHS020菌株可代谢产生丰富多样的生物活性物质,具有开发成为新型抑菌生物制剂的潜力。     

Characterization and regulation of new secondary metabolites from Aspergillus ochraceus M18 obtained by UV mutagenesis

UV irradiation of Aspergillus ochraceus NRRL 3174 conidia led to stable mutations in ochratoxin and penicillic-acid pathways. These mutants, especially M18, produced an unexpectedly large number of new metabolites. Two new compounds were purified by TLC and HPLC and their chemical structures were determined. They are 2,10-dimethyl 4-hydroxy-6-oxo-4-undecen-7-yne (1) and 4-(3-methyl-2- butenyl) oxy 1-phenyl acetic acid (2). Compound 1 is very active against Gram-positive bacteria, such as Staphylococcus aureus and Bacillus subtilis, but inactive against Gram-negative bacteria, fungi, and yeasts. However, compound 2 has no antibiotic activity. The production of 1 was generally associated with growth, whereas that of compound 2 was dissociated from growth. The biosynthesis of these 2 metabolites was influenced by the sources of carbon and nitrogen.     

Changes in fecal microflora induced by intubation of mice with Bacillus subtilis (natto) spores are dependent upon dietary components.

We examined changes in mouse fecal microflora after various dietary components and Bacillus subtilis (natto) spores were delivered by intubation. The administration of intact spores of Bacillus subtilis (natto) did not affect fecal Enterobacteriaceae and Enterococcus spp. in all three diet groups; on the other hand, it did affect fecal Bacteroidaceae and Lactobacillus spp., depending upon the diets fed. The administration of autoclaved spores did not alter fecal microflora. In vitro cultures of Lactobacillus murinus obtained from mouse feces, together with Bacillus subtilis (natto) under aerobic conditions as a mixed culture, revealed that the growth of L. murinus was enhanced by the addition of intact spores of Bacillus subtilis (natto). This enhancement of growth was displayed only in media containing either sucrose, glucose, maltose, or fructose but not in media containing cornstarch, soluble starch, or microcrystalline cellulose. From these results it was evident that some metabolites of Bacillus subtilis (natto) produced during germination and (or) outgrowth of spores of this strain, requiring monosaccharides or oligosaccharides, participated in the enhancement of the growth of L. murinus.     

Biological control of Tiarosporella phaseolina the causal agent of charcoal rot of soybean

Charcoal rot caused by Tiarosporella phaseolina (Tassi) Van der Aa is an important disease of soybean in Gorgan province of Iran. Experiments were carried out with 95 bactenal isolates that were collected from the rhizosphere of soybean plant. Among these bacteria only 50 isolates showed antagonistic effect on Tiarosporella phaseolina using dual culture test. Six highly effective bacteria were selected for subsequent studies. Based on biochemical physiological and morphological tests, isolates Pf-12 and Pf-63 were identified as Pseudomonas fluorescens, isolates B-13, B-42,B-126 and B-84 as Bacillus subtilis. The isolates of P. fluorescens produced antibiotics as well as volatile metabolites that inhibited mycelial growth of fungus. Bacillus subtilis isolates inhibited the fungal growth through volatile and non-volatile metabolites production. Only P. fluorescens isolates produced hydrogen cyanide. In greenhouse studies, the isolates B-13 and B-126 reduced 59% and 66% the intensity of charcoal rot of soybean respectively. The combinations of isolates B-13 and B-126 were also effective on reducing the intensity of disease.     

Rapid Method for Identification of Interconnections between Functionally and/or Evolutionarily Related Biological Sequences

It is now common practice to retrieve, by key words, highly specialized selections of sequences from general-purpose databases such as EMBL, GenBank, etc. The sequences included in a selection are often interconnected, which means that there are duplications, embeddings, intersections, homology, common structural elements. Knowledge of these interconnections is necessary for further processing of the sequences. We propose a rapid (single scan) method for identification of such interconnections by means of complexity analysis that generalizes the Lempel–Ziv approach. Analysis of a selection of 5"-flanking regions of vertebrate growth hormone genes from EMBL is presented as an example.     

Transport Alterations in a Phosphatidylethanolamine-Deficient Mutant of Bacillus subtilis

A mutant of Bacillus subtilis ATCC 6051, demonstrated to be deficient in phosphatidylethanolamine, was found to differ from the parent strain in the rate of uptake of a variety of metabolites.     

细微短足软珊瑚的化学成分研究

本文对采自广西涠洲岛海域细微短足软珊瑚（Cladiellasubtilis）的化学成分进行研究,经理化常数和波谱数据分析,分别鉴定为（20R,24S）-5-烯-21羧基-麦角甾-3β-醇（1）、柳珊瑚甾醇（2）、鲨肝醇（3）及麦角甾-5-烯-3β-醇（4）。对细微短足软珊瑚（Cladiella subtilis）化学成分的研究尚属首次。     

杜仲内生真菌的抑菌活性筛选

以金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌为测试菌种。对杜仲(Eucommia ulmoides Oliv.)根、茎、叶中分离出的20株内生真菌及其次生代谢物进行抗菌活性筛选。结果表明：有15个菌株至少对1种实验细菌具有抑菌活性,19株的代谢产物至少对1种实验细菌具有抑菌活性,其中有3株内生真菌及其次生代谢产物对测试病原细菌均有较强抑制作用。