链霉菌IMS002的分类鉴定及其抗真菌活性物质解析

【目的】对一株分离自植物根际土壤的具有抗真菌活性的链霉菌IMS002进行菌株分类鉴定,通过活性追踪分离纯化并鉴定有机相中的活性物质。【方法】通过16S rDNA和5个不同基因(atpD,gyrB,recA,rpoB,trpB)串联聚类分析以及生理生化实验分析,对链霉菌IMS002进行菌株分类鉴定,用扫描电子显微镜观察该株链霉菌的菌丝及孢子形态,以尖孢镰刀菌(Fusarium oxysporum)为指示菌进行生物活性追踪,通过硅胶柱层析、凝胶柱层析及高压液相色谱(HPLC)对活性物质进行分离和纯化,使用液质联用高分辨质谱仪、500 MHz核磁共振波谱仪以及圆二色光谱仪确定该物质的化学结构。【结果】IMS002经初步鉴定与产二素链霉菌(Streptomycesambofaciens)具有较近的亲缘关系,其发酵液对尖孢镰刀菌具有良好的抑菌效果,经分离和纯化以及现代波谱技术分析,确定有机相中的抑菌活性组分为Borrelidin。【结论】链霉菌IMS002能够产生化合物Borrelidin,该化合物对尖孢镰刀菌具有抑制活性。     

药用植物内生放线菌的分离、筛选及活性菌株YIM 61470鉴定

从云南西双版纳热带雨林多种药用植物中分离到272株内生放线菌,活性筛选表明 146株菌的发酵产物具有抗菌活性,其中94株菌具有拮抗病原细菌活性,127株菌具有抑制病原真菌的功能.分离菌株YIM 61470具有广谱抗菌活性,通过形态特征、培养特征、生理生化特征、细胞化学分类特征和基于16S rRNA基因序列的相似性分析等研究,菌株YIM 61470被鉴定为链霉菌属(Streptomyces)氢化链霉菌(S.llydrogenans)的一个菌株.     

抗鱼类病原菌的放线菌分离鉴定及其活性研究

为了研究鱼类疖疮病的生物防治方法,以引起鱼类疖疮病的病原菌杀鲑气单胞菌(Aeromonas salmonicida)为指示菌,从土壤中筛选到对该致病菌有较强拮抗活性的放线菌I6,经16S rRNA分析鉴定为链霉菌(Strptomyces sp.)。链霉菌I6的次级代谢产物对杀鲑气单胞菌具有很强的抗菌活性且对鱼类无毒力,在鱼类疖疮病的治疗和防控中具有应用潜力。为了提高链霉菌I6抗菌活性物质的产量,对该I6菌株进行发酵条件优化,确定其最佳发酵培养基为AM3-1,最适初始pH为7～8,最佳培养时间为5 d,种子液最适菌龄为36 h。所有这些研究结果表明,链霉菌I6对鱼类疖疮病病原菌杀鲑气单胞菌有显著抑制作用,在渔业生物防治中有潜在的开发应用价值。     

一株皱叶南蛇藤内生放线菌的分离、鉴定及活性研究

目的:分离具有抗肿瘤作用与抗菌作用的内生放线菌,并对其进行分子鉴定和系统发育分析.方法:从皱叶南蛇藤中分离内生放线菌,通过滤纸片法和SRB法对其进行抗菌活性和抗肿瘤活性筛选,然后利用菌株形态特征、培养特征、生理生化特性和16S rRNA基因序列分析对活性菌株进行鉴定结果:从皱叶南蛇藤中分离到10株内生放线菌,其中内生放线菌LCB369具有较好的抗菌活性和抗肿瘤活性,该菌对5种致病菌和肝癌细胞株HepG2均有抑制作用.经分子分类学分析鉴定,该菌与Streptomyces microflavus 在同一个分支上,同源性为99%.结论:皱叶南蛇藤内生菌LCB369具有明显抗菌和抗肿瘤作用,经鉴定为链霉菌Streptomyces microflavus.     

拉鲁湿地自然保护区放线菌组成分析及生物活性测定

探究拉鲁湿地自然保护区的放线菌组成及其抑菌和酶活性,为放线菌新药物先导化合物和高活性酶的筛选提供资源。从拉鲁湿地自然保护区不同土壤类型、不同优势植被采集25份土样。用分散差速离心法分离了拉鲁湿地中温放线菌和低温放线菌。从中温放线菌中选择15株代表菌株进行了初步分类鉴定。采用打孔法检测了其对2株细菌和4株病原真菌的抑菌活性。结果表明：（1）拉鲁湿地放线菌数量从水生环境向陆地生态系统递增,中温放线菌数量显著多于低温放线菌;（2）拉鲁湿地土壤中分离到链霉菌属、小单孢菌属、诺卡氏菌属、马杜拉菌属、小链孢菌属5个放线菌属。其中以链霉菌属和小单孢菌属为优势属。链霉菌属以金色类群、白孢类群和粉红孢类群为主,小单孢菌分离到黄橙类群和黑褐类群;（3）供试菌株分解纤维素能力较强,分解蛋白质活性较低,具有抗菌活性的菌株很少,且抗菌活性较弱;（4）供试菌株耐毒性物质的能力较强。这些菌可用于毒害有机物污染物的处理。     

大连海域沉积物中可培养海洋链霉菌活性及其多样性

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。     

百部内生放线菌的分离、分类及次级代谢潜力

【目的】以对叶百部块根为材料分离内生放线菌,并对分离菌株进行分类、抗菌活性和次级代谢产物合成基因研究。【方法】样品经过严格的表面消毒,选用4种培养基分离百部内生放线菌;分离菌株通过形态观察和16S rRNA序列分析进行分类鉴定;采用琼脂移块法测试分离菌株的抗菌活性;通过PCR检测分离菌株的PKS/NPRS和卤化酶基因;使用HPLC-UV/VIS-ESI-MS/MS分析发酵产物。【结果】从6个样品中获得18株内生放线菌,分属链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、假诺卡氏菌属(Pseudonocardia)和甲基杆菌属(Methylobacterium)。分离菌株绝大部分具有抗菌活性和次级代谢产物合成基因,其中13株对耐药金黄色葡萄球菌和/或绿脓杆菌有拮抗活性,17株具有PKS/NRPS基因,8株菌具有卤化酶基因,且卤化酶阳性代表菌株的发酵产物具有抗细菌活性和卤代化合物特征。【结论】百部作为一种传统中药,其内生放线菌以链霉菌和小单孢菌为主,在次级代谢产物合成方面具有很好的潜力,可作为一类重要微生物资源进行活性产物开发。     

一株拮抗可可球二孢菌放线菌的分离及鉴定

目的:筛选具有拮抗植物病原真菌可可球二孢菌活性的放线菌.方法:选择高氏一号培养基分离放线菌,以可可球二孢菌为指示菌进行抑菌活性筛选,通过形态学特征、生理生化特征、培养特征以及16S rRNA基因序列分析等研究对筛选得到的高活性菌株进行菌种鉴定.结果:从南方红豆杉根际土壤中筛选得到一株高活性菌株KLBMP 2284,16S rRNA基因序列比对结果显示其与弗吉尼亚链霉菌(Streptomyces virginiae)相似性98.963％,发酵液稀释300倍后抑制率为41.64％.结论:KLBMP 2284为弗吉尼亚链霉菌的一个菌株.     

链霉菌H03发酵液中具有抗菌活性多糖的分离纯化及其单糖组成分析

时链霉菌H03发酵产物进行了分离纯化,并对其进行了初步鉴定．对链霉菌H03发酵产物离心,采用减压蒸馏,乙醇沉淀,沉淀组分用Sevage法、盐析法去蛋白,用透析法除去小分子物质以及用Sephadex-100柱层析等技术进行纯化,纯化物质仍具有较强的抗菌活性、利用化学方法、紫外光谱、红外光谱、气质联用等方法分析该抗菌活性物质的理化性质．结果表明：从链霉菌的发酵产物中分离纯化得到的具有抗菌活性物质是多糖,这种多糖是由甘露糖、葡萄糖、半乳糖3种单糖组成的,其组成比例约为2：1：1．     

内生拮抗放线菌FRo2的鉴定及抑菌活性物质的分离

【目的】鉴定从东乡野生稻根部分离得到的对多种农作物病原真菌具有拮抗活性的内生放线菌株FRo2,并对其抑菌活性物质进行分离。【方法】根据FRo2形态特征观察、生理生化特性、细胞壁组分和16S rRNA基因序列对其进行鉴定。采用管碟法和菌丝生长速率法测定了该菌株的抗菌活性,活性追踪法结合正相硅胶柱层析及凝胶(Sephadex LH-20)柱层析等技术对抑菌组分进行分离,并通过NMR对抑菌活性物质进行解析。【结果】菌株FRo2属于链霉菌属,与娄彻氏链霉菌(Streptomyces rochei)极为相似。该菌株发酵液对小麦赤霉菌、立枯丝核菌等7种主要农作物致病菌具有较好的抑制活性;从菌株FRo2发酵液中分离得到抑菌活性化合物AW2,结构鉴定为邻苯二甲酸二丁酯。【结论】研究阐明了内生放线菌FRo2抑菌活性物质,也为该菌今后的农业生防应用提供物质基础。     

A neutral lipase applicable in biodiesel production from a newly isolated Streptomyces sp. CS326

In an attempt to isolate a biocatalyst able to catalyze biodiesel production from microbial source, Streptomyces sp. CS326 was screened from hundreds of soil isolates collected from various parts of Korea. In 16S rRNA sequence analysis, the strain showed high degree of similarity with Streptomyces xanthocidicus (99.79%); therefore, it is classified as Streptomyces sp. CS326. An extracellular lipase produced by the strain (LP326) was purified using a single step gel permeation chromatography on Sepharose CL-6B. Molecular weight of LP326 was estimated to be 17,000 Da by SDS-PAGE. The activity was optimum at 40 °C and pH 7.0 and was stable at pH 5.0-8.0 and below 50 °C. It preferred p-nitrophenyl palmitate (C16), a long chain substrate; and K (m) and V (max) for the substrate were determined to be 0.24 mM and 4.6 mM/min mg, respectively. First 10 N-terminal amino acid sequences were APDLVALQSE, which are different from so far reported lipases. LP326 catalyzed biodiesel production using methanol and various oils; therefore, the enzyme can be applicable in the field of biofuel.     

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%–5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.     

Oxidation of methyl halides by the facultative methylotroph strain IMB-1.

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [(14)C]formaldehyde to (14)CO(2) but had only a small capacity for oxidation of [(14)C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [(14)C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2). The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

Streptomyces pharetrae sp. nov., isolated from soil from the semi-arid Karoo region

An actinomycete, strain CZA14T, was isolated from Worcester in the Western Cape province of South Africa. Based on rapid genus identification, 16S-rDNA sequence similarities and chemotaxonomy, strain CZA14T was identified as a member of the genus Streptomyces. It exhibited weak antibiosis against Bacillus coagulans ATCC 7050T, Mycobacterium aurum A+ and Acinetobacter calcoaceticus C91. The results of physiological tests and analysis of the 16S-rDNA sequence allowed for the differentiation of strain CZA14T from other species of the genus Streptomyces. Strain CZA14T therefore represents a new species for which the name Streptomyces pharetrae is proposed, with the type strain CZA14T (= DSM 41856T = NRRL 24333T).     

Studies on an Enzyme Capable of Splitting β-d-1,6-Glucosidic Linkage

Examination was made on the morphological and cultural characteristics of the lutease-producing Streptomyces strain No. OP-4-5 isolated from a dust. The strain was identified as Streptomyces griseus. In addition, it was proved that 2 strains of Streptomyces griseus produce lutease in a test for lutease production in Streptomyces species. Streptomyces parvus and Streptomyces niveoruber also produce the same enzyme. However, production of the lutease by these 4 strains was less than that of produced by Streptomyces griseus strain No. OP-4-5 which was isolated by the authors.     

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

APOBEC3G (hA3G) is a host inhibitor for human immunodeficiency virus, type 1 (HIV-1). However, HIV-1 Vif binds hA3G and induces its degradation. We have established a screening system to discover inhibitors that protect hA3G from Vif-mediated degradation. Through screening, compounds IMB-26 and IMB-35 were identified to be specific inhibitors for the degradation of hA3G by Vif. The inhibitors suppressed HIV-1 replication in hA3G-containing cells but not in those without hA3G. The anti-HIV effect correlated with the endogenous hA3G level. HIV-1 particles from hA3G(+) cells treated with IMB-26/35 contained a hA3G level higher than that from those without IMB-26/35 treatment and showed decreased infectivity. IMB-26/35 bound directly to the hA3G protein, suppressed Vif/hA3G interaction, and therefore protected hA3G from Vif-mediated degradation. The compounds were safe with an anti-HIV therapeutic index >200 in vitro. LD₅₀ of IMB-26 in mice was >1000 mg/kg (intraperitoneally). Therefore, IMB-26 and IMB-35 are novel anti-HIV leads working through specific stabilization of hA3G.     

Identification of methyl halide-utilizing genes in the methyl bromide-utilizing bacterial strain IMB-1 suggests a high degree of conservation of methyl halide-specific genes in gram-negative bacteria

Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.     

Oxidation of Methyl Halides by the Facultative Methylotroph Strain IMB-1

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 μM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [¹⁴C]formaldehyde to ¹⁴CO₂ but had only a small capacity for oxidation of [¹⁴C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [¹⁴C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K_s values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO₂. The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

1株土壤放线菌的分类鉴定及其抗菌活性的研究

对从土壤微生物中筛选到的放线菌菌株1356进行分类学和抗菌活性的研究。采用多相分类法,对菌株的形态特征、培养特征、生理生化特性及16 SrRNA基因序列进行了研究。结果表明:该菌株的形态特征、培养特征、生理生化特性为链霉菌属的特征;16S rDNA序列分析及系统进化树分析表明其序列与灰色产色链霉菌的同源性最高;该菌株的发酵产物对番茄叶霉、白色念珠菌、小麦根腐菌等17种真菌均有不同程度的抑制作用。放线菌1356菌株具有广谱抗真菌活性而对细菌无作用;初步确定其为链霉菌属灰色产色链霉菌的一个亚种。