繁茂膜海绵中可培养稀有放线菌的多样性

摘要：【目的】本文旨在尝试改进分离培养方法从大连海域繁茂膜海绵中筛选稀有放线菌,并对其多样性进行研究。【方法】根据繁茂膜海绵元素组成配制微量元素溶液,加入到放线菌分离培养基中,同时将部分培养基稀释成寡营养培养基,结合富集培养法,对繁茂膜海绵中放线菌进行分离培养。采用16S rDNA的限制性片断长度多态性(Restriction Fragment Length Polymorphism, RFLP)分析和序列分析,揭示其多样性。【结果】共获得可培养放线菌59株,通过形态、颜色观察,将其归为27个类群。RFLP分析表现为15种不同的图谱类型。16S rDNA序列分析表明：它们分别属于放线菌的10个属,其中布劳氏菌属（Prauseria）和糖单胞菌属（Saccharomonospora）是首次报道从海绵中分离培养。【结论】改进的分离培养基适合于繁茂膜海绵中稀有放线菌的分离培养,进一步揭示了该海绵中丰富的稀有放线菌,同样的方法有可能应用于其他海绵放线菌的分离培养。     

不同红树林地区老鼠簕内生放线菌的分离及其环境适应性

【目的】比较不同红树林地区的老鼠簕内生放线菌的地理分布,了解内生放线菌与其所处环境的相关性。【方法】分别从5个不同地点的红树林采集老鼠簕全株植物,采用9种分离培养基,从植株不同部位分离内生放线菌,用16S rRNA基因序列分析鉴定到属,用添加不同NaCl浓度的ISP 2液体培养基进行耐盐度测试,用无氮基础培养基进行固氮活性测试。【结果】共分离得到内生放线菌52株,其中从叶、茎和根部分别获得5株、2株和45株,花和果中未分离到。52株内生放线菌分别属于小单孢菌属(47株),链霉菌属(3株),疣孢菌属(1株)和继生菌属(1株)。48株菌表现出耐盐或嗜盐特征,其中18株最高耐盐度20%,4株不能在无盐条件下生长,12株菌可在含有3.3%NaCl的培养基上生长良好。4株菌可在无氮培养基下生长。【结论】对47株内生小单孢菌的地理分布分析表明,老鼠簕内生小单孢菌的类群因不同地理位置有很大差异。耐盐和固氮活性测试结果表明了老鼠簕内生放线菌对环境的适应性。     

Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord, Norway: isolation, diversity and biological activity

Actinomycete bacteria produce a wide variety of secondary metabolites with diverse biological activities, some of which have been developed for human medicine. Rare actinomycetes are promising sources in search for new drugs, and their potential for producing biologically active molecules is poorly studied. In this work, we have investigated the diversity of actinomycetes in the shallow water sediments of the Trondheim fjord (Norway). Due to the use of different selective isolation methods, an unexpected variety of actinomycete genera was isolated. Although the predominant genera were clearly Streptomyces and Micromonospora, representatives of Actinocorallia, Actinomadura, Knoellia, Glycomyces, Nocardia, Nocardiopsis, Nonomuraea, Pseudonocardia, Rhodococcus and Streptosporangium genera were isolated as well. To our knowledge, this is the first report describing isolation of Knoellia and Glycomyces species from the marine environment. 35 selected actinomycete isolates were characterized by 16S rDNA sequencing, and were shown to represent strains from 11 different genera. In addition, these isolates were tested for antimicrobial activity and the presence of polyketide synthase and non-ribosomal peptide synthetase genes. This study confirms the significant biodiversity of actinobacteria in the Norwegian marine habitats, and their potential for producing biologically active compounds.     

Isolation and identification of actinobacteria from surface-sterilized wheat roots

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.     

16S rDNA_RFLP分析繁茂膜海绵可培养放线菌的多样性

海绵是迄今为止已知海洋天然产物的最大来源。由于海绵的底栖过滤性摄食和消化选择性等生理特性,其体内蕴藏了丰富的微生物种群。近几年来,发现越来越多的海绵微生物产生很强的生物活性物质,有些并被证明是海绵天然产物的真正生产者。对大连海域繁茂膜海绵中的放线菌进行分离,对于得到的菌株进行16SrDNA扩增和RFLP及测序分析。研究表明海绵中蕴含着丰富的放线菌资源。其中包含链霉菌(Streptomycetes),拟诺卡氏菌(Nocardiopsis),假诺卡氏菌(Pseudonocardia),诺卡氏菌(Nocardia),小单孢菌(Micromonospora),红球菌(Rhodococcus),异壁放线菌(Actinoalloteichus)等属。利用限制性内切酶HhaⅠ对16SrDNA进行的RFLP分析能够对海绵中放线菌的16SrDNA多样性进行有效的分析,结果达到属,部分到种的级别。揭示了繁茂膜海绵中蕴藏着丰富的放线菌资源。     

嗜酸丝状放线菌的选择性分离与多样性

摘要:【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16S rRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为：土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸（各50 mg/L）的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属：链霉菌属(Streptomyces)、小单孢菌属(Micromonospora) 、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea) 、韩国生工属(Kribbella) 、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora) 和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。     

Functional Gene-Guided Discovery of Type II Polyketides from Culturable Actinomycetes Associated with Soft Coral Scleronephthya sp

Compared with the actinomycetes in stone corals, the phylogenetic diversity of soft coral-associated culturable actinomycetes is essentially unexplored. Meanwhile, the knowledge of the natural products from coral-associated actinomycetes is very limited. In this study, thirty-two strains were isolated from the tissue of the soft coral Scleronephthya sp. in the East China Sea, which were grouped into eight genera by 16S rDNA phylogenetic analysis: Micromonospora, Gordonia, Mycobacterium, Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. 6 Micromonospora strains and 4 Streptomyces strains were found to be with the potential for producing aromatic polyketides based on the analysis of KS(α) (ketoacyl-synthase) gene in the PKS II (type II polyketides synthase) gene cluster. Among the 6 Micromonospora strains, angucycline cyclase gene was amplified in 2 strains (A5-1 and A6-2), suggesting their potential in synthesizing angucyclines e.g. jadomycin. Under the guidance of functional gene prediction, one jadomycin B analogue (7b, 13-dihydro-7-O-methyl jadomycin B) was detected in the fermentation broth of Micromonospora sp. strain A5-1. This study highlights the phylogenetically diverse culturable actinomycetes associated with the tissue of soft coral Scleronephthya sp. and the potential of coral-derived actinomycetes especially Micromonospora in producing aromatic polyketides.     

The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.     

海南近海30株抗B16细胞活性放线菌的16S rDNA多样性分析

从海南近海 ,包括文昌红树林、海口红树林以及洋浦港等地采集样品 ,经苯酚、SDS、加热等预处理 ,稀释涂布麦芽汁_酵母膏琼脂 (YE)、淀粉酪素琼脂 (SC)、葡萄糖天冬氨酸琼脂 (GA) ,或者直接将样品稀释涂布加有重铬酸钾的高氏一号琼脂 (Gause)和麦芽汁_酵母膏琼脂等进行平板分离。共获得 35 4株放线菌 ,其中有 76株具有不同程度的抗B16细胞毒活性。比较发现加热预处理法和重铬酸钾选择培养法对于广泛分离筛选抗肿瘤活性放线菌不失为一种快速、简便、行之有效的方法。YE、Gause培养基无论在分离到的放线菌总数 ,还是细胞毒活性菌株的比例上都显示了良好的效果。对 30株具有较强抗B16细胞活性的链霉菌进行了扩增性 16SrDNA限制性酶切片段多样性分析 (16SARDRA) ,表明这 30株链霉菌之间有较大的基因差异性。 0 5 0 6 4 2、0 6 0 386和 0 6 0 5 2 4等 3株菌序列分析进一步证明这 3株菌属于链霉菌属 ,其中菌株 0 5 0 6 4 2与其亲缘关系最近的Streptomycescattleya的相似性仅为 95 % ,因此可能是一个新种。     

Phylogenetic diversity of actinomycetes cultured from coastal multipond solar saltern in Tuticorin, India

ABSTRACT: BACKGROUND: Hypersaline solar salterns are extreme environments in many tropical and subtropical regions throughout the world. In India, there are several coastal solar salterns along with the coastal line of the Bay of Bengal and Arabian Sea and inland solar salterns around Sambhar saltlake, from which sodium chloride is obtained for human consumption and industrial needs. Studies on characterization of such coastal and inland solar salterns are scarce and both the bacterial and archaeal diversity of these extreme saline environment remains poorly understood. Moreover, there are no reports on exclusive diversity of actinomycetes inhabiting Indian solar salterns. RESULTS: Soil sediments were collected from both concentrator and crystallizer ponds of solar salterns and subjected to detailed physico-chemical analysis. Actinomycetes were selectively isolated by employing selective processing methods and agar media. A total of 12 representatives were selected from the 69 actinomycete isolates obtained from the saltern soil samples, using Amplified Ribosomal DNA Restriction Analysis. Sequencing and analysis of 16S rDNA from chosen representative isolates displayed the presence of members affiliated to actinobacterial genera: Streptomyces, Micromonospora, Nocardia, Nocardiopsis, Saccharopolyspora and Nonomuraea. The genus Streptomyces was found to be the dominant among the isolates. Furthermore, rare actinomycete genus Nonomuraea was isolated for the first time from Indian solar salterns. CONCLUSIONS: To the best of our knowledge, this study constitutes the first characterization of actinomycete diversity centred on solar salterns located in the eastern coastal region of India. Furthermore, this is the very first report of isolation of Nonomuraea species from solar salterns and also from India. As actinomycetes encompass recurrently foremost sources of biotechnologically important member of the microbial communities, the actinomycetes retrieved from the Indian saltern soil samples laid the platform to search for novel biotechnologically significant bioactive substances.     

Diversity of Streptomyces spp. in Eastern Himalayan region - computational RNomics approach to phylogeny

Isolation and characterization of actinomycetes from soil samples from altitudinal gradient of North-East India were investigated for computational RNomics based phylogeny. A total of 52 diverse isolates of Streptomyces from the soil samples were isolated on four different media and from these 6 isolates were selected on the basis of cultural characteristics, microscopic and biochemical studies. Sequencing of 16S rDNA of the selected isolates identified them to belong to six different species of Streptomyces. The molecular morphometric and physico-kinetic analysis of 16S rRNA sequences were performed to predict the diversity of the genus. The computational RNomics study revealed the significance of the structural RNA based phylogenetic analysis in a relatively diverse group of Streptomyces.     

Bioprospecting Micromonospora from Kaziranga National Park of India and their anti-infective potential

Large number of strains was isolated from soils of Kaziranga National Park of North-East India using selective isolation procedure. They were assigned to the genus Micromonospora on the basis of their typical colonial and pigmentation features. The taxonomic identities of the isolates were confirmed on the basis of their molecular characters (16SrDNA). A total of one hundred Micromonospora strains were isolated during the present investigation. The diagnostic cell wall sugar and amino acids were determined from these Micromonospora strains. After preliminary screening most of the isolates exhibited excellent anti-infective activity against human bacterial pathogens Staphylococcus aureas, Bacillus subtilis, Proteus vulgaris, Echerichia coli, Pseudomonas aeroginosa and fungal pathogens Aspergillus niger, Fusarium oxysporum and Candida albicans. Among these isolates one strain designated as HK-10 showed promising activity against human pathogens S. aureas, B. subtilis, P. vulgaris and P. aeroginosa.     

Cultivatable microbial biodiversity: gnawing at the Gordian knot

Rapid and inexpensive sorting of bacterial isolates may be achieved using Fourier transform infrared spectroscopy (FT-IR), a method that has hitherto been applied to identification and classification. The comprehensive characterization of environmental samples requires the isolation of large numbers of isolates using different growth media and growth conditions. In such cases, sorting the isolates is critical before isolates are subjected to more detailed studies. Using FT-IR, isolates are grown under standardized conditions, and 100 strains can be tested within less than 8 h. Chemotaxonomic and molecular characterization of members of clusters emerging from FT-IR analysis either at a level of spectral distance values below 20–30 (analysis of region 600–800 cm⁻¹, average linkage algorithm) or at spectral heterogeneity values below 75 (regions 1200–900, 3000–2798 and 901–698, scaling to first region, Ward's algorithm) reveals great similarities in fatty acids and 16S rDNA sequences. As judged from riboprinting analyses and fatty acid analyses, FT-IR analysis is able to unravel intraspecific subclustering. The example used in this study of 100 isolates from a mat system, Lake Fryxell, Dry Valleys, Antarctica, selected from a larger number of isolates, picked mainly on the basis of colony pigmentation and form, reveals the utility of the method for identifying the number of putative species quickly. The method described is able to select strains rapidly that represent clusters at the specific and intraspecific level for subsequent characterization.     

Humic acid-vitamin agar,a new medium for the selective isolation of soil actinomycetes

A new medium, designated HV agar, containing soil humic acid as the sole source of carbon and nitrogen was developed.The HV agar was superior to other currently used media, including colloidal chitin agar, glycerol-arginine agar and starch-casein-nitrate agar, for the isolation and enumeration of soil actinomycetes: It allowed the growth of the largest numbers of actinomycete colonies belonging to each genus of Streptomyces, Micromonospora, Microbispora, Streptosporangium, Nocardia, Dactylosporangium, Microtetraspora and Thermomonospora on the plate, while restricting the development of true bacteria. The HV agar supported adequate growth and good sporulation for these actinomycetes.Even when spore suspensions were used as the inoculum, the HV agar produced remarkably larger numbers of actinomycetes, especially strains of the genera Micromonospora, Microbispora, Streptosporangium, Dactylosporangium and Saccharomonospora, than did glycerol-arginine agar. It was found that the spores of these actinomycetes were activated upon germination by treatment at 20°C for 30 min with a O.2% solution of humic acid prior to incubation.     

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。     

Phylogenetic Diversity and Population Densities of Culturable Cellulolytic Soil Bacteria across an Agricultural Encatchment

Abstract A typical, small encatchment (catena B?lkendorf) in the moraine, northeast German agricultural landscape Schorfheide-Chorin was studied with respect to summit, midslope, and foot-slope positions at northern and southern slope exposure, respectively, including a central noncultivated kettle hole position (pot hole). Across the sequence of seven distinct sampling positions, soil organic carbon and total nitrogen contents, soil gravimetric water content, and soil microbial biomass displayed maxima at the kettle hole position. Soil pH revealed a decreasing trend at the northern exposed slope and a minimum at the kettle hole position. Against this background, the population density of total culturable bacteria clearly displayed a minimum at the kettle hole position, whereas the population density of carboxymethylcellulose decomposing bacteria was not clearly differentiated in relation to sampling positions. To study the phylogenetic diversity of culturable cellulolytic bacteria, 311 isolates were obtained from the sampling positions across the entire encatchment and examined by restriction analysis of PCR-amplified 16S rDNA. Using the restriction enzyme ScrFI, isolates were classified into 31 pattern groups. Although the ratio of actinomycetes within total isolates ranged from 0.73 to 0.94, only 16 pattern groups originated from actinomycetes, but 15 from other bacteria. At all sampling positions, a dominant pattern group was identified, containing 38 to 65% of total isolates. Two site-specific pattern groups could be identified, representing significant parts of the total population, which were highly specific for the kettle hole (19% of total isolates) and for foot- and midslope positions (15–18% of total isolates), respectively. In general, the composition of cellulolytic isolates across the encatchment displayed differences with respect to slope positions, but was not significantly affected by soil properties. Based on 16S rDNA sequence analysis, isolates of the dominant as well as the specific pattern groups could be assigned to the genus Streptomyces. Furthermore, sequencing of 16S rDNA of isolates of another three pattern groups revealed a high phylogenetic diversity among these isolates, including cellulomonads and bacilli. Received: 12 August 1998; Accepted: 1 February 1999     

Culturability and In situ abundance of pelagic bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

热带药用植物根际放线菌的分离、鉴定及生物活性分析

从海南热带植物园采集12种药用植物的根际土样,采用选择性分离方法,分离得到400株根际放线菌。使用5种活性筛选模型对分离菌株进行生物活性评价,154株放线菌在一个或多个活性筛选模型中显示为阳性,菌株初筛阳性率达38.5%;根据菌株形态特征并结合代谢产物的生物活性,从中挑选出28株菌进行16S rRNA基因序列分析,发现其分属于链霉菌属、诺卡氏菌属、小单孢菌属和野野村菌属。     

水霉拮抗放线菌的分离、筛选与鉴定

目的:以珍珠健康养殖水体底泥为材料分离放线菌,筛选对水产动物水霉病病原菌有抗菌活性的放线菌。方法:采用稀释涂布法,选用萘啶酮酸和放线菌酮双抗平板分离获得放线菌;以黄颡鱼和湘云鲫鱼卵水霉病原菌为靶标菌,采用琼脂块法测试所分离菌株抗水霉菌活性及其稳定性;对拮抗活性强的放线菌采用形态观察和16S r DNA序列分析进行分类鉴定。结果:从分离获得的27株放线菌中筛选出3株对水霉病原菌有拮抗活性的菌株QF1、DNC17和QHV2,其中QHV2抗菌活性与稳定性最好;形态学观察与16S r DNA序列分析结果表明QF1、DNC17和QHV2均属于链霉菌属(Streptomycete sp.),分别鉴定为Streptomyces diastatochromogenes、Streptomyces variabilis和Streptomyces collinus。结论:3株放线菌对水霉病原菌具有较好的拮抗活性,具有开发成抗水霉药物的潜在价值。