专性海洋放线菌盐孢菌的研究进展

盐孢菌属(Salinispora)作为首个被报道的专性海洋放线菌,主要分布于热带和亚热带海洋沉积环境中,在海绵、海鞘中也有发现。与其他大多数放线菌一样,盐孢菌属的菌株可以产生大量具有抗细菌、抗病毒、抗肿瘤细胞活性、结构新颖的次级代谢产物且表现出物种特异性。全基因组序列分析显示,盐孢菌属菌株基因组中超过10%的基因序列与次级代谢产物合成相关,但绝大多数生物合成基因簇编码的产物未被发现,表明盐孢菌属还存在巨大的生物合成潜能,有待深入发掘。目前新的培养方法、测序技术及生物信息学、基因组发掘技术、合成生物学技术的发展对提升盐孢菌属菌株新型药物的生产潜力发挥重要作用。本文对盐孢菌属的物种多样性、系统分类与化合物发现等方面的研究进行了系统综述。     

永兴岛白穗软珊瑚共附生放线菌筛选及部分活性次级代谢产物的鉴定

【目的】从白穗软珊瑚中分离和鉴定共附生放线菌,运用PCR技术对所分离的放线菌进行I型聚酮合酶(PKS)筛选,研究其次级代谢产物。【方法】使用11种培养基对白穗软珊瑚共附生放线菌进行分离、鉴定,构建16S rRNA基因系统发育进化树,以基于I型PKS的KS基因设计的简并引物对所分离放线菌进行基因筛选,对阳性菌株用3种培养基发酵检测,对目标菌株进行放大规模发酵分离鉴定次级代谢产物。【结果】从白穗软珊瑚中分离到20株共附生放线菌,包括链霉菌属10株、迪茨氏菌属2株和盐水孢菌属8株,筛选获得18株I型PKS阳性菌株,并从菌株Salinospora arenicola SH04中分离到化合物rifamycin S和rifamycin W。【结论】首次从珊瑚共附生环境中分离得到海洋专属性稀有放线菌盐水孢菌属,并以I型PKS基因筛选为指导,分离鉴定了聚酮类化合物rifamycins,为研究软珊瑚共附生可培养放线菌的多样性和基于基因筛选指导分离次级代谢产物提供了可靠依据。     

大连海域海洋放线菌多样性及分类鉴定初探

海洋孕育着丰富的微生物资源,其中海洋放线菌能够产生各种次级代谢产物及生理活性物质,对海洋放线菌的研究具有重要的实际意义。实验对筛选自大连海域的30株放线菌进行了形态及生理生化鉴定、16S rDNA序列的分析及系统发育树构建,结果表明分离到的海洋放线菌中13株分属于放线菌目链霉菌不同种或亚种,说明大连海域的海洋放线菌具有一定的多样性。     

深海放线菌可望成为新抗癌药物的来源

ScienceNews 2 0 0 3年 16 3卷 5期 78页报道 :很多种药物包括抗生素类的链霉素 ,均来自土壤微生物放线菌。无独有偶 ,最近美国加利福尼亚州拉霍亚市Scripps海洋研究所的科学家WilliamFenical及其同事也发现 ,先前未知的放线菌菌株可制造具有抗生或抗癌特性的化学物质。Fenical把他在深海沉积中采集到的这种新的放线菌称之为盐孢菌 (Salinospora)根据Fenical的初步检测 ,在其所鉴定的 2 5 0 0个盐孢菌株中 ,有很多菌株可产生具有潜在治疗效果的化学物质。进一步研究中 ,Fenical及其同事检测了盐孢菌中产生的盐孢菌酰胺的分子结构。还发…     

微波处理对嗜碱和嗜盐海洋放线菌分离效果的影响

【目的】研究微波处理对于分离嗜碱和嗜盐海洋放线菌的效果。【方法】用微波处理7份海泥样品,梯度稀释后涂布于3种分离培养基,分离具有嗜碱和嗜盐特性的海洋放线菌。【结果】微波处理后的7份样品中,4份样品中嗜碱海洋稀有放线菌和3份样品的嗜盐海洋稀有放线菌数量极显著提高;7份样品中的嗜碱、嗜盐海洋小单孢菌属、游动放线菌属、诺卡氏菌属等稀有放线菌数量均有显著增加,不同样品中新分离到链孢菌属、小双孢菌属、链孢囊菌属及其他未鉴定的海洋稀有放线菌,分离到属的数量提高了1-4个。【结论】微波处理不仅显著提高嗜碱和嗜盐海洋放线菌的分离数量,而且明显增加了海洋稀有放线菌的分离种类。     

拟孢囊菌属放线菌的研究进展

拟孢囊菌属(Kibdelosporangium)是一个经典的丝状放线菌类群。自Shearer等于1986年建立该属以来,不断有新的菌株从不同生境中被纯培养分离得到,目前已有效发表8个种、2个亚种。拟孢囊菌属作为稀有放线菌属,是获得新型抗生素类次生代谢产物的重要资源。本文结合我们分离到的一株该属菌株的功能研究、基因组分析和相关文献资料,系统综述了拟孢囊菌属放线菌的建立、分类学特征、属内种的分布、活性次级代谢产物的发现以及其他功能应用和开发前景,以期为该属其他新分离菌株的分类鉴定、新颖次级代谢产物的发现、功能基因资源的挖掘与开发提供借鉴。     

湛江高桥红树林沉积物放线菌多样性及其中一株链霉菌产生的germicidins类化合物的鉴定

【目的】从3种红树林植物根际沉积物中分离和鉴定放线菌,进行抑菌活性初筛获得目标菌株并研究其次级代谢产物。【方法】使用5种培养基对红树林植物根际沉积物放线菌进行分离,采用16S rRNA基因序列比对的方法研究沉积物中的放线菌多样性,结合抑菌活性筛选获得目标菌株后进行放大规模发酵和分离鉴定次级代谢产物,根据生物合成基因簇定位和分析对化合物的生物合成途径进行推导。【结果】从3种红树林植物根际沉积物中分离得到放线菌49株,包括链霉菌属(Streptomyces)31株、小单孢菌属(Micromonospora)14株、小双孢菌属(Microbispora)、链孢子囊菌属(Streptosporangium)、野野村氏菌属(Nonomuraea)和糖单孢菌属(Saccharomonospora)各1株。获得粗浸膏抑菌活性较好的菌株Streptomyces sp. SCSIO 40067,从中分离鉴定了6个α-吡喃酮类化合物：germicidin A–C、germicidin I和isogermicidin A–B,并首次报道了germicidin A的晶体结构。从菌株SCSIO 40067基因组...     

贵州兴义喀斯特洞穴可培养放线菌多样性及抗菌活性初筛

【目的】进一步了解兴义喀斯特洞穴可培养放线菌资源及产活性代谢产物的能力。【方法】选取多种分离培养基,利用稀释直接涂布平板法对贵州黔西南兴义市多个喀斯特洞穴的土壤和岩石进行可培养放线菌资源分离;利用三种发酵培养基对相关放线菌进行生物产物初筛。【结果】根据16S rRNA基因序列的比对分析,将分离得到的251株放线菌分别归类到44个属,其中链霉菌属(Streptomyces)占分离菌株的比例为24.30%,小单孢菌属(Micromonospora)占比11.95%,红球菌属(Rhodococcus)占比9.16%,微杆菌属(Microbacterium)占比7.17%,诺卡氏菌属(Nocardia)占比6.37%,该五类放线菌为该地区可培养放线菌的优势菌群。对70株细菌进行活性次级代谢产物筛选,其中35株放线菌对指示菌具有抑制活性,且主要类群为链霉菌属和小单孢菌属。【结论】贵州兴义喀斯特洞穴中存在丰富多样的放线菌类群,且蕴藏大量具有产生活性次级代谢产物能力的菌株,为医药产业提供潜力菌株资源,极具进一步发掘和研究的价值。     

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。     

智利海洋沉积物中放线菌多样性

【目的】认识智利海洋沉积物中放线菌的多样性。【方法】分别采用选择性分离培养和非培养的基于16SrRNA基因序列系统发育分析方法,对来自智利南部海域海洋沉积物中放线菌多样性进行研究。采用6种选择性分离培养基分离放线菌;利用放线菌特异性引物对样品总DNA进行16SrRNA基因序列扩增并构建了16SrRNA基因克隆文库。分别挑选不同培养特征的22株放线菌和59个基因克隆进行16SrRNA基因序列的系统进化分析;测定分离的放线菌对海水的依赖性及产生抗菌活性化合物的能力。【结果】共分离到328株放线菌;挑选的22株放线菌分别属于小单孢菌属、多形孢菌属、链霉菌属、迪茨氏菌属、气微菌属和短状杆菌属;挑取的59个克隆属于40个分类单元,其中60%分类单元属于放线菌门放线菌亚纲、酸微菌亚纲和红色杆菌亚纲,另外40%的分类单元在放线菌内形成几个独立的进化分支,有可能代表放线菌新类群。22株放线菌有19株具有抗菌活性,50%的生长依赖海水的放线菌也具有抗菌活性。【结论】智利海域沉积物存在丰富的放线菌系统发育多样性并能产生活性次级代谢产物,而且还蕴藏丰富的新类型的放线菌资源。     

Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora

Salinispora is the first obligate marine genus within the order Actinomycetales and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two Salinispora species have thus far been cultivated, suggesting limited species-level diversity. To further explore Salinispora diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a Salinispora-specific seminested PCR technique, was used to detect Salinispora from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (S. arenicola). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from Taq polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly Salinispora spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.     

Marine actinomycetes as an emerging resource for the drug development pipelines

Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinomycetes from terrestrial sources have been studied and screened since the 1950s, yielding many important anti-infective and anti-cancer drugs. However, frequent re-discovery of the same compounds in terrestrial actinomycetes have made them less attractive for screening programs in the recent years. At the same time, actinomycetes isolated from the marine environment currently receive considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. This review highlights achievements and challenges in the isolation of marine actinomycetes, some examples of bioactive metabolites identified by conventional screening, and presents new developments in the field of genome mining and heterologous expression of biosynthetic gene clusters leading to the discovery of novel compounds.     

Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments

A major taxon of obligate marine bacteria within the order Actinomycetales has been discovered from ocean sediments. Populations of these bacteria (designated MAR 1) are persistent and widespread, spanning at least three distinct ocean systems. In this study, 212 actinomycete isolates possessing MAR 1 morphologies were examined and all but two displayed an obligate requirement of seawater for growth. Forty-five of these isolates, representing all observed seawater-requiring morphotypes, were partially sequenced and found to share characteristic small-subunit rRNA signature nucleotides between positions 207 and 468 (Escherichia coli numbering). Phylogenetic characterization of seven representative isolates based on almost complete sequences of genes encoding 16S rRNA (16S ribosomal DNA) yielded a monophyletic clade within the family Micromonosporaceae and suggests novelty at the genus level. This is the first evidence for the existence of widespread populations of obligate marine actinomycetes. Organic extracts from cultured members of this new group exhibit remarkable biological activity, suggesting that they represent a prolific resource for biotechnological applications.     

链霉菌基因组及次生代谢研究进展

链霉菌属革兰氏阳性放线菌,具有复杂的生活周期和次生代谢途径,并产生大量具有重要价值的天然代谢物。本文概述了链霉菌基因组染色体的独特结构与次生代谢途径的研究进展,重点论述了利用基因组信息改造和调控链霉菌次生代谢途径的研究成果。后基因组时代的功能基因组研究使人类能深入了解链霉菌家族,对链霉菌进行更加合理高效的遗传操作,为提高具有重要价值的天然代谢物的产量和获得新代谢物创造更有利的条件。     

Evolution of secondary metabolite genes in three closely related marine actinomycete species

The marine actinomycete genus Salinispora is composed of three closely related species. These bacteria are a rich source of secondary metabolites, which are produced in species-specific patterns. This study examines the distribution and phylogenetic relationships of genes involved in the biosynthesis of secondary metabolites in the salinosporamide and staurosporine classes, which have been reported for S. tropica and S. arenicola, respectively. The focus is on "Salinispora pacifica," the most recently discovered and phylogenetically diverse member of the genus. Of 61 S. pacifica strains examined, 15 tested positive for a ketosynthase (KS) domain linked to the biosynthesis of salinosporamide K, a new compound in the salinosporamide series. Compound production was confirmed in two strains, and the domain phylogeny supports vertical inheritance from a common ancestor shared with S. tropica, which produces related compounds in the salinosporamide series. There was no evidence for interspecies recombination among salA KS sequences, providing further support for the geographic isolation of these two salinosporamide-producing lineages. In addition, staurosporine production is reported for the first time for S. pacifica, with 24 of 61 strains testing positive for staD, a key gene involved in the biosynthesis of this compound. High levels of recombination were observed between staD alleles in S. pacifica and the cooccurring yet more distantly related S. arenicola, which produces a similar series of staurosporines. The distributions and phylogenies of the biosynthetic genes examined provide insight into the complex processes driving the evolution of secondary metabolism among closely related bacterial species.     

Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.     

<Emphasis Type="Italic">Salinispora</Emphasis><Emphasis Type="Italic">arenicola</Emphasis> from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes

The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.     

Actinomycete integrative and conjugative pMEA-like elements of Amycolatopsis and Saccharopolyspora decoded

Actinomycete integrative and conjugative elements (AICEs) are present in diverse genera of the actinomycetes, the most important bacterial producers of bioactive secondary metabolites. Comparison of pMEA100 of Amycolatopsis mediterranei, pMEA300 of Amycolatopsis methanolica and pSE211 of Saccharopolyspora erythraea, and other AICEs, revealed a highly conserved structural organisation, consisting of four functional modules (replication, excision/integration, regulation, and conjugative transfer). Features conserved in all elements, or specific for a single element, are discussed and analysed. This study also revealed two novel putative AICEs (named pSE222 and pSE102) in the Sac. erythraea genome, related to the previously described pSE211 and pSE101 elements. Interestingly, pSE102 encodes a putative aminoglycoside phosphotransferase which may confer antibiotic resistance to the host. Furthermore, two of the six pSAM2-like insertions in the Streptomyces coelicolor genome described by Bentley et al. [Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., et al., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141-147] could be functional AICEs. Homologues of various AICE proteins were found in other actinomycetes, in Frankia species and in the obligate marine genus Salinispora and may be part of novel AICEs as well. The data presented provide a better understanding of the origin and evolution of these elements, and their functional properties. Several AICEs are able to mobilise chromosomal markers, suggesting that they play an important role in horizontal gene transfer and spread of antibiotic resistance, but also in evolution of genome plasticity.