八门湾红树林土壤小单孢菌的分离及其多样性

采用平板稀释法从海南八门湾红树林潮间带、木果榄和海莲红树根际土壤样品中分离和筛选小单孢菌科放线菌,对其分离方法进行了评价和比较,并通过16S rRNA基因测序探索了八门湾红树林小单孢菌科放线菌的多样性。采用4种预处理方法和5种分离培养基,经过排除重复菌株后共得到115株放线菌。16S rRNA基因测序结果显示,这些放线菌分布在5个科、9个属。其中小单孢菌科的菌株约占全部菌株的90%(105株),主要分布于3个属:Micromonospora(85%),Jishengella(9.5%)和Verrucosispora(5.5%),与其亲缘关系最近的菌株的相似性分布于98.5%~100%之间。     

Studies on the microbial populations of the rhizosphere of big sagebrush (<Emphasis Type="Italic">Artemisia tridentata</Emphasis>)

Prolonged use of broad-spectrum antibiotics has led to the emergence of drug-resistant pathogens, both in medicine and in agriculture. New threats such as biological warfare have increased the need for novel and efficacious antimicrobial agents. Natural habitats not previously examined as sources of novel antibiotic-producing microorganisms still exist. One such habitat is the rhizosphere of desert shrubs. Here, we show that one desert shrub habitat, the rhizosphere of desert big sagebrush (Artemisia tridentata) is a source of actinomycetes capable of producing an extensive array of antifungal metabolites. Culturable microbial populations from both the sagebrush rhizosphere and nearby bulk soils from three different sites were enumerated and compared, using traditional plate-count techniques and antibiotic activity bioassays. There were no statistical differences between the relative numbers of culturable non-actinomycete eubacteria, actinomycetes and fungi in the rhizosphere versus bulk soils, but PCR amplification of the 16S rRNA gene sequences of the total soil DNA and denaturing gradient gel electrophoresis showed that the community structure was different between the rhizosphere and the bulk soils. A high percentage of actinomycetes produced antimicrobials; and the percentage of active producers was significantly higher among the rhizosphere isolates, as compared with the bulk soil isolates. Also, the rhizosphere strains were more active in the production of antifungal compounds than antibacterial compounds. 16S rRNA gene sequence analysis showed that sagebrush rhizospheres contained a variety of Streptomyces species possessing broad spectrum antifungal activity. Scanning electron microscopy studies of sagebrush root colonization by one of the novel sagebrush rhizosphere isolates, Streptomyces sp. strain RG, showed that it aggressively colonized young sagebrush roots, whereas another plant rhizosphere-colonizing strain, S. lydicus WYEC108, not originally isolated from sagebrush, was a poor colonizer of the roots of this plant, as were two other Streptomyces isolates from forest soil. These results support the hypothesis that the rhizosphere of desert big sagebrush is a promising source of habitat-adapted actinomycetes, producing antifungal antibiotics.     

辐射污染区土壤中放线菌的分离及多样性

从辐射污染区采集42份土样, 分别采用6种分离培养基进行放线菌的分离, 共获得152株放线菌。经形态、核糖体DNA扩增片段限制性内切酶(Amplifed ribosomal DNA restriction analysis, ARDRA)分析比较, 选取其中的60株进行16S rRNA基因测序。通过序列比对、聚类分析, 60株菌分布在放线菌纲中的12个属, 链霉菌属占大多数, 有大量稀有放线菌, 这表明辐射污染区具有较丰富的放线菌属种多样性。     

Characterization of Ni-resistant bacteria in the rhizosphere of the hyperaccumulator <Emphasis Type="Italic">Alyssum murale</Emphasis> by 16S rRNA gene sequence analysis

The diversity of 184 isolates from rhizosphere and bulk soil samples taken from the Ni hyperaccumulator Alyssum murale, grown in a Ni-rich serpentine soil, was determined by 16S rRNA gene analysis. Restriction digestion of the 16S rRNA gene was used to identify 44 groups. Representatives of each of these groups were placed within the phyla Proteobacteria, Firmicutes and Actinobacteria by 16S rRNA gene sequence analysis. By combining the 16S rRNA gene restriction data with the gene sequence analysis it was concluded that 44.6% (82/184) of the isolates were placed within the phylum Proteobacteria, among these 35.9% (66/184) were placed within the class α-Proteobacteria, and 20.7% (38/184) had 16S rRNA gene sequences indicative of bacteria within genera that form symbioses with legumes (rhizobia). Of the remaining isolates, 44.6% (82/184) and 5.4% (10/184) were placed within the phyla Actinobacteria and Firmicutes, respectively. No placement was obtained for a small number (10/184) of the isolates. Bacteria of the phyla Proteobacteria and Actinobacteria were the most numerous within the rhizosphere of A. murale and represented 32.1% (59/184) and 42.9% (79/184) of all isolates, respectively. The approach of using 16S rRNA gene sequence analysis in this study has enabled a comprehensive characterization of bacteria that predominate in the rhizosphere of A. murale growing in Ni-contaminated soil.     

西沙海藻共附生放线菌的分离鉴定及其抗菌活性评价

【目的】为了探究南海海藻共附生放线菌资源的多样性及潜在的应用价值,对中国西沙群岛来源的海藻进行共附生放线菌的分离鉴定与抗菌活性筛选。【方法】利用稀释涂布平板法,采用2种不同分离培养基对不同采样位点的6种海藻进行放线菌分离;通过16S rRNA基因序列分析、构建系统发育树对分离的放线菌进行鉴定;用打孔法对无乳链球菌(Streptococcus agalactiae)等10种敏感细菌进行抗菌活性筛选;对筛选得到的目标活性菌株HZ014进行全基因组测序,通过AntiSMASH在线工具分析其次级代谢产物生物合成基因簇,预测其产生新型活性物质的潜力。【结果】从6种海藻中分离得到36株共附生放线菌,基于16S rRNA基因序列比对和系统发育分析,鉴定结果为链霉菌属(Streptomyces) 2株、红球菌属(Rhodococcus) 2株、诺卡氏菌属(Nocardia)3株、小单孢菌属(Micromonospora) 5株和盐孢菌属(Salinispora) 24株;抗菌活性筛选结果表明,36株共附生放线菌发酵粗提物对至少1种敏感细菌表现出一定的抑制作用,不同菌株发酵粗提物的抗菌活性存在明显差异,...     

Biodiversity of Actinomycetes Associated with Caribbean Sponges and Their Potential for Natural Product Discovery

Marine actinomycetes provide a rich source of structurally unique and bioactive secondary metabolites. Numerous genera of marine actinomycetes have been isolated from marine sediments as well as several sponge species. In this study, 16 different species of Caribbean sponges were collected from four different locations in the coastal waters off Puerto Rico in order to examine diversity and bioactive metabolite production of marine actinomycetes in Caribbean sponges. Sediments were also collected from each location, in order to compare actinomycete communities between these two types of samples. A total of 180 actinomycetes were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 14 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora, Microbacterium and Cellulosimicrobium. Seventy-eight of the isolates (19 from sediments and 59 from sponges) shared 100 % sequence identity with Micromonospora sp. R1. Despite having identical 16S rRNA sequences, the bioactivity of extracts and subsequent fractions generated from the fermentation of both sponge- and sediment-derived isolates identical to Micromonospora sp. R1 varied greatly, with a marked increase in antibiotic metabolite production in those isolates derived from sponges. These results indicate that the chemical profiles of isolates with high 16S rRNA sequence homology to known strains can be diverse and dependent on the source of isolation. In addition, seven previously reported dihydroquinones produced by five different Streptomyces strains have been purified and characterized from one Streptomyces sp. strain isolated in this study from the Caribbean sponge Agelas sceptrum.     

Identification of Streptomyces sp. nov. WH26 producing cytotoxic compounds isolated from marine solar saltern in China

A moderately halophilic actinomycetes strain, designated as WH26, was isolated from Weihai Solar Saltern in China. The identification of the strain WH26 was performed by its morphological characteristics, physiological and biochemical tests as well as phylogenetic analysis based on 16S rRNA sequence comparison. The results showed that the nucleotide sequence of the 16S rRNA gene (1,677 bp) of the strain WH26 exhibited close similarity (97–99 %) with other Streptomyces 16S rRNA genes and the strain WH26 was identified to belong to the genus Streptomyces. An ethyl acetate extraction of Streptomyces sp. nov. WH26 demonstrated significant cellular toxicity. Two compounds, 8-O-methyltetrangulol and naphthomycin A were isolated from the extract via silica gel column chromatography and HPLC. These two compounds showed potent cytotoxic activity against several human tumor cell lines including A549, HeLa, BEL-7402 and HT-29. The present studies suggest that moderately halophilic actinomycetes may be a novel biological source for the discovery of anticancer agents.     

西沙石珊瑚共附生放线菌的分离培养及抗菌活性评价

【背景】珊瑚礁生态系统是海洋中一类极其重要的生态系统,健康珊瑚礁中丰富的共附生放线菌群体是珊瑚抵御各种致病菌的重要防线,因此,这类放线菌是寻找抗菌活性分子的重要资源,其药用潜力巨大。【目的】从西沙石珊瑚样品中分离共附生放线菌,并从中筛选具有良好抗菌活性的菌株。【方法】通过稀释涂布法分离珊瑚共附生放线菌,并根据16S rRNA基因序列构建系统发育树进行菌种鉴定;通过平板对峙法对放线菌进行抗菌活性筛选并确定目标菌株;将目标菌株涂布于不同氯化钠浓度的ISP2固体培养基上培养,测试其盐度耐受能力;通过平板对峙法对该菌株发酵产物的热稳定性和光稳定性进行测试;采用NanoPore和Illumina方法完成目标活性放线菌全基因组测序,并通过antiSMASH在线分析预测其次级代谢产物生物合成基因簇及其结构类型。【结果】从6份西沙石珊瑚样品中分离得到104株可培养放线菌,根据菌落形态和分离来源去重后对其中27株放线菌进行16S rRNA基因序列测序,通过序列比对和系统发育树分析将菌株初步鉴定为盐孢菌属(Salinispora)(25株)、链霉菌属(Streptomyces)(1株)和戈登菌属(Gord...     

佳西热带雨林土壤芽胞杆菌分离与多样性分析

采用热处理法从海南省佳西热带雨林土壤中分离到147株芽胞杆菌,并利用16S rDNA PCR-RFLP与序列分析技术对其遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析结果表明,在100%的相似性水平上,这些芽胞杆菌分属13个遗传类群。不同遗传类型代表菌株的16S rRNA基因序列分析结果显示,它们分布在Bacillaceae、Planococcaceae和Paenibacillaceae科的Bacillus、Lysinibacillus、Paucisalibacillus、Bhargavaea和Paenibacillus五个属,其中Bacillus为优势属(占50%);有3株芽胞杆菌的16S rRNA基因序列与数据库中相应模式菌株的最大相似性在98.3%~98.9%之间。结果表明,佳西热带雨林土壤中芽胞杆菌有着较为丰富的遗传多样性。     

南方红豆杉内生及根际放线菌多样性及其生物活性

【目的】研究药用植物南方红豆杉内生及根际土壤放线菌的多样性及其抑菌、抗肿瘤等重要生物活性并获得一些具有强抑制植物病原真菌以及抗肿瘤等重要生物活性的菌株。【方法】选择7种培养基从南方红豆杉及其根际土壤中分离放线菌,对链霉菌进行形态学分类,去重复后对其进行抑制植物病原真菌以及抗肿瘤活性的筛选并对高活性菌株进行初步鉴定。对部分菌株进行16S rRNA基因测序分析研究其多样性。【结果】研究共分离得到277株放线菌,经去重复后剩余111株放线菌,可归类到6个亚目、7个科、8个属。其中链霉菌可分为10个类群。生物活性研究结果显示:30.9%的菌株具有抑制植物病原真菌活性,其中6株放线菌对多种植物病原真菌显示了强的抑菌活性。分别有44.1%和33.3%的菌株对胃癌肿瘤细胞株SGC-7901和肺癌肿瘤细胞株NCI-H460的抑制率在40%以上。【结论】药用植物南方红豆杉及其根际土壤蕴含种类丰富的放线菌资源,具有良好的生物学活性。菌株KLBMP 2170具有显著的抑菌以及抗肿瘤活性,值得我们去进一步研究。     

Non-Frankia Actinomycetes Isolated from Surface-Sterilized Roots of Casuarina equisetifolia Fix Nitrogen

Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from ¹⁵N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.     

Molecular evidence for the presence of novel actinomycete lineages in a temperate forest soil

PCR primers were designed to selectively recover partial (1100 bp) actinomycete 16S ribosomal DNA sequences from a temperate forest soil. A gene library was made and colony PCR was used to identify clones containing inserts. Unique clones were identified and partial or complete insert sequences were determined for 53 clones. Phylogenetic analyses revealed that 46 (87%) of the clones sampled contained 16S rDNA sequences which fell within the actinomycete radiation. The largest group of 34 sequences formed two closely related monophyletic groups in the 16S rRNA tree, which in turn formed a weakly supported sister group with the sequence fromActinomadura madurae. Four novel 16S rDNA lineages were detected inMycobacterium, one inPropionibacterium and one inCorynebacterium. Three novel sequences weakly grouped withSporichthya polymorpha. Two sequences formed an isolated lineage not closely related to any of the reference actinomycetes. Our results lend strong support to the hypothesis that cultured (and sequenced) actinomycetes do not adequately describe the diversity of this group in the environment.     

高望界土壤放线菌抗菌活性筛选及生物学特征

为了从土壤环境中筛选产生抗菌活性物质的放线菌,采用琼脂扩散法对分离自湖南省高望界自然保护区森林土壤的169株放线菌进行抗菌活性检测,采用16S RNA基因序列分析法进行系统发育分析,采用常规方法进行生物学特性研究。结果表明,有47株受试菌株的发酵产物显示抗菌活性阳性(27.8%),其中6株具有较强抗菌活性。形态观察结果表明,这6株菌株均为典型的产丝产孢放线菌,在多数培养基上生长良好,气生菌丝和孢子丝丰富。系统发育分析表明,6株菌株均属于链霉菌属(Streptomyces),归为5个物种。菌株JSM 147612、JSM 147786、JSM 147831和JSM 147846分别与链霉菌属的已知物种S.violascens(序列相似性为99.93%)、S.malachitospinus(99.65%)、S.anulatus(99.72%)和S.aureus(99.38%)系统发育关系最为密切;菌株JSM 147823和JSM 147842之间的序列相似性为99.86%,应该属于同一物种,与它们系统发育关系最为密切的是S.albidoflavus(相似性分别为99.72%和99.58%)。     

八角炭疽病拮抗放线菌RX2-2的分离鉴定及其生物活性评价

[背景]八角炭疽病主要是由哈锐炭疽菌(Colletotrichum horii)引起的真菌性病害,给八角产业带来严重的经济损失.[目的]从八角根系土壤中分离筛选对哈锐炭疽菌具有拮抗作用的放线菌菌株,并对其进行种属鉴定及抗菌活性评价.[方法]采用稀释涂布平板法分离放线菌,并以哈锐炭疽菌作为指示菌,利用平板对峙法筛选具有高...     

Biodegradation of 2-Nitrotoluene by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain SMN-1

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.     

Evaluation of the taxonomic diversity of prosthecate bacteria belonging to the genera <Emphasis Type="Italic">Brevundimonas</Emphasis> and <Emphasis Type="Italic">Caulobacter</Emphasis> isolated from various eurasian ecosystems by analysis of the 16S rRNA genes

A taxonomic study of 35 cultures of prosthecate bacteria of the genera Brevundimonas and Caulobacter isolated from various soil and aquatic ecosystems of Eurasia was performed by amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequencing. The most widespread groups of prosthecate bacteria belonging to these genera were revealed; at least two new species belonging to the genus Brevundimonas were found. The genus Brevundimonas includes both prosthecate and non-prosthecate species; however, it is quite possible that some Brevundimonas species may exhibit heterogeneity in such an important taxonomic characteristics as the ability to form prosthecae.     

Repression of alkaline protease in salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1 under the influence of amino acids in minimal medium

A salt-tolerant alkaliphilic actinomycete (strain Mit-1) was isolated from Mithapur (Western Coast, Gujarat, India) and identified as Streptomyces clavuligerus. Based on 16S rRNA gene sequence (EU146061) homology, it was found to be related to Streptomyces sp. (AY641538.1). The strain secreted alkaline protease optimally at 5% NaCl and pH 9 during the early stationary phase and could utilize the amino acids methionine, alanine, leucine, phenylalanine, tyrosine, tryptophan, arginine, asparagine, histidine, and glutamic acid as the sole source of nitrogen. Above their threshold levels, these amino acids caused repression of alkaline protease production. Protease production with methionine (120 U/mL), histidine (140 U/mL), and aspartic acid (118 U/mL) was comparable to that with complex medium (130 U/mL). However, the production increased with an increasing number of different amino acids in the growth medium. Repression of protease production as influenced by the amino acids generated valuable information on enzyme synthesis in actinomycetes, as such data is scarce. Optimization of the conditions for enzyme production by actinomycetes in general, and in haloalkaliphilic actinomycetes in particular, appears to be an attractive proposition for biocatalysis.     

Efficient Transformation Procedure of a Newly Isolated <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TN58 Strain Producing Antibacterial Activities

A new aerobic Gram-positive bacterium designated TN58 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. The nucleotide sequence of the 16S rRNA gene (1516 bp) of the TN58 strain showed high similarity (96–98%) to the Streptomyces 16S rRNA genes, especially with that of Streptomyces lavendulae which produces the anti-tumor compound mitomycin C, and the cyclic peptide antibiotic, complestatin. Cultural characteristic studies, alignment data of the 16S rRNA gene, and analysis of the nucleotide sequence of a 2.2 kb genomic DNA fragment from TN58 strongly suggested that this strain could be an actinomycete and most probably belongs to the genus Streptomyces. Study of the influence of different nutritional compounds on antibiotic production showed that the highest antibacterial activities were obtained when glycerol at 1% (w/v) was used as sole carbon source in the presence of potassium. In analytical conditions, the application to supernatant culture of the TN58 strain of various extraction and purification steps led to the isolation of two pure active molecules having a retention time of 38.6 and 50.2 min, respectively. TN58 strain was untransformable with the Streptomyces cloning vector pIJ702 via classical polyethylene glycol (PEG) protoplast transformation and previously described Streptomyces electroporation procedures. Transformation was rendered possible by the electroporation technique only after utilization of a preculture medium without sucrose and a regeneration plate containing a low sucrose concentration.     

硝尔库勒湖可培养放线菌多样性及其功能酶和抗细菌活性

【目的】认识和了解硝尔库勒湖可培养放线菌的多样性、功能酶和抗细菌活性特点,为今后的开发和利用奠定基础。【方法】应用可培养技术和基于16S r RNA基因序列的系统发育分析硝尔库勒盐湖沉积物中放线菌的多样性。常规方法检测样品成分因子,并筛选了嗜盐放线菌的蛋白酶、淀粉酶和酯酶活性;抑菌圈法检测放线菌新种的抗细菌活性。【结果】分离获得了51个OTUs,分属于24个不同的属,其中15个OTUs代表了放线菌新种;链霉菌属是优势菌属,占全部分离菌株数量的16.25%。硝尔库勒湖放线菌类群数量一定程度上受样品成分因子的协同影响。代表新种的菌株展示了良好的功能酶活性和抗细菌活性,其中代表链霉菌新种的菌株XHU5011不仅具有多种酶活性,而且具有强大的抗金黄葡萄球菌、耻垢分枝杆菌和荧光假单胞菌的能力,具有很好的开发潜能。【结论】硝尔库勒盐湖中存在丰富的可培养放线菌多样性,潜藏着大量的放线菌新资源,并且具有很好的功能酶和天然产物挖掘潜力。     

Isolation and Characterization of Novel Marine-Derived Actinomycete Taxa Rich in Bioactive Metabolites

A unique selective enrichment procedure has resulted in the isolation and identification of two new genera of marine-derived actinobacteria. Approximately 90% of the microorganisms cultured by using the presented method were from the prospective new genera, a result indicative of its high selectivity. In this study, 102 actinomycetes were isolated from subtidal marine sediments collected from the Bismarck Sea and the Solomon Sea off the coast of Papua New Guinea. A combination of physiological parameters, chemotaxonomic characteristics, distinguishing 16S rRNA gene sequences, and phylogenetic analysis based on 16S rRNA genes provided strong evidence for the two new genera (represented by strains of the PNG1 clade and strain UMM518) within the family Micromonosporaceae. Biological activity testing of fermentation products from the new marine-derived actinomycetes revealed that several had activities against multidrug-resistant gram-positive pathogens, malignant cells, and vaccinia virus replication.