海绵及其共附生微生物的活性物质与化学防御

海绵及其共附生微生物具有多种化学防御途径,具体表现在能够抵御潜在病原微生物、抗鱼类捕食、阻止污浊生物和降解表面活性剂等方面,这可能与海绵及其共附生微生物产生的活性物质有关。本文对海绵及其共附生微生物的化学防御进行讨论,希望对相关机制的揭示有所帮助。     

海绵共附生真菌多样性及其次级代谢产物的研究进展

海绵由于其独特的生理结构、进食方式使其体内部聚集了大量的微生物,这些微生物产生了多种结构新颖的生物活性物质,因此海绵及其共附生微生物的研究成为了海洋药物研发的热点。就海绵中共附生真菌的分布情况,新技术的应用及其生物次级代谢产物的生物活性展开综述。     

北部湾海洋植物及其共附生微生物次级代谢产物研究进展(综述)

海洋植物及其共附生微生物是海洋生物的重要组成部分,能够产生许多结构新颖、活性独特的次级代谢产物,承担多种生理生态功能。北部湾海洋植物物种资源丰富,据统计,海洋植物有3门43种。该文综述了从2002年起北部湾海洋植物及其共附生微生物次级代谢产物的研究进展,从11种红树植物和7种共附生微生物中获得59个新化合物和35个已知活性化合物,从3种海草植物中获得3个新化合物和7个已知活性化合物,从6种海藻植物和1种共附生微生物中获得25个新化合物和8个已知活性化合物,主要涉及结构类型有萜类、生物碱、黄酮类、甾醇,多数具有良好的抗菌、抗氧化、抗肿瘤、抗炎、增强机体免疫力等功效。在此基础上,进一步提出了北部湾海洋植物研究方向及后续的研究建议。该综述为深入研究和开发利用北部湾海洋植物及其共附生微生物提供了参考。     

基于PCR-DGGE指纹的南海海绵共附生细菌优势种群的揭示与系统发育分析

采用PCR-DGGE指纹、克隆测序和系统发育分析技术较系统地对我国南海贪婪倔海绵(Dysidea avara)和澳大利亚厚皮海绵(Craniella australiensis)共附生的优势细菌进行了研究。研究发现变形菌门(Proteobacteria)细菌是这两种海绵中的主要优势细菌,贪婪倔海绵中的变形菌包含了α、β、γ三种类型,而澳大利亚厚皮海绵中仅有γ一种类型。两种海绵都有拟杆菌(Bacteroidetes),但是具体的种类不同。这些细菌都是第一次在海绵中被发现。澳大利亚厚皮海绵共附生的优势细菌还包括放线菌属(Actinobacterium)及厚壁菌门(Firmicutes)细菌,菌群多样性要比贪婪倔海绵的丰富。两种海绵尽管来自于同一海域但其共附生优势细菌的组成明显不同,这说明海绵共附生微生物具有宿主特异性。     

海绵内/共生稀有放线菌Dermacoccus sp. X4次级代谢产物分离纯化及结构解析

从中国南海西沙群岛10 m深海水中采集得到一株未鉴定海绵样品,对其进行共附生微生物的分离,得到真菌及细菌共16株。通过分子生物学鉴定及菌株发酵液抑菌活性测定,发现其中一株稀有放线菌皮生球属菌株Dermacoccus sp.X4对金黄色葡萄球菌具有较好抑菌活性。大量发酵制备该菌株发酵液,通过硅胶分配层析、ODS反相层析、SephadexTMLH-20凝胶过滤层析及C18反相层析等分离方法对发酵液成分进行分离,并使用液相质谱连用、一维核磁及二维核磁分析对分离得到的单一化合物进行鉴定,确定二酮哌嗪类化合物1个,吲哚酸酯类化合物2个。本研究中吲哚酸酯类化合物为首次从微生物次级代谢产物中得到,为丰富海洋微生物药用资源作出贡献。     

海洋共附生微生物天然产物生物合成基因研究进展

对海洋无脊椎动物天然产物的研究表明,很多种活性物质的真正生产者是与其共生或附生的未培养微生物.克隆这些未培养微生物中特定活性物质的生物合成基因,不仅为活性物质的来源提供遗传学证据,也使通过异源表达相关生物合成基因来大量获取目标化合物成为可能.本文综述了来源于海绵、海鞘、苔藓虫、深海管状蠕虫和深海沉积物中共附生微生物天然产物生物合成基因簇的研究进展.     

海绵共生微生物研究中的分子技术

海绵共生微生物与海绵活性物质有紧密的关系,由于其大多不可培养,分子生物学技术成为海绵微生物研究的有效方法。本文重点介绍了海绵共生微生物研究中经常应用到的FISH、PCR、16S rRNA克隆测序、DGGE、RFLP等各种分子技术,对它们的特点及缺陷进行了分析与小结,希望有助于海绵共生微生物分子研究的进一步发展。     

海绵微生物生物活性物质的研究进展

海绵独特的摄食、滤食系统使其体内体表富集了大量的微生物,这些微生物能够产生多种结构新颖的生物活性物质,对海绵微生物的研究正在成为开发海洋药物资源的重要内容之一。本文概述了近十年来对海绵微生物生物活性物质的化学成分和生物活性的研究进展。     

可培养海绵共附生微生物的PKS基因筛选

利用PCR技术对21株分离自我国南海澳大利亚厚皮海绵的放线菌及9株分离自贪婪倔海绵的芽孢杆菌进行了聚酮合酶(PKS)基因筛选。从芽孢杆菌C89中获得了一条669bp片段,BLAST比对结果表明该基因对应的氨基酸序列和枯草芽孢杆菌I型聚酮合成酶基因(PKS)KS域的相似性达96%。通过系统发育分析推测芽孢杆菌C89PKS基因属于trans-AT型。首次证明了贪婪倔海绵共附生微生物中存在PKS基因,这为海绵活性物质的微生物来源假说提供了证据;同时也为可以产生聚酮类化合物的微生物筛选以及聚酮类化合物的发酵制备奠定了基础。     

海绵共附生细菌种群组成的PCR-DGGE基因指纹分析

采用不依赖于分离培养的16S rDNA的PCR-DGGE基因指纹技术对我国南海的细薄星芒海绵、皱皮软海绵、贪婪倔海绵、澳大利亚厚皮海绵体内的优势细菌的种群组成进行了比较分析。结果显示:每种海绵体内都含有丰富多样的细菌;通过DGGE指纹图的聚类分析发现来自同一海域的不同海绵的共附生细菌的种群组成具有明显不同,即共附生细菌具海绵宿主特异性;同时也发现有相同的细菌存在于不同的海绵体内。     

Diversity and biotechnological potential of the sponge-associated microbial consortia

Sponges are well known to harbor diverse microbes and represent a significant source of bioactive natural compounds derived from the marine environment. Recent studies of the microbial communities of marine sponges have uncovered previously undescribed species and an array of new chemical compounds. In contrast to natural compounds, studies on enzymes with biotechnological potential from microbes associated with sponges are rare although enzymes with novel activities that have potential medical and biotechnological applications have been identified from sponges and microbes associated with sponges. Both bacteria and fungi have been isolated from a wide range of marine sponge, but the diversity and symbiotic relationship of bacteria has been studied to a greater extent than that of fungi isolated from sponges. Molecular methods (e.g., rDNA, DGGE, and FISH) have revealed a great diversity of the unculturable bacteria and archaea. Metagenomic approaches have identified interesting metabolic pathways responsible for the production of natural compounds and may provide a new avenue to explore the microbial diversity and biotechnological potential of marine sponges. In addition, other eukaryotic organisms such as diatoms and unicellular algae from marine sponges are also being described using these molecular techniques. Many natural compounds derived from sponges are suspected to be of bacterial origin, but only a few studies have provided convincing evidence for symbiotic producers in sponges. Microbes in sponges exist in different associations with sponges including the true symbiosis. Fungi derived from marine sponges represent the single most prolific source of diverse bioactive marine fungal compounds found to date. There is a developing interest in determining the true diversity of fungi present in marine sponges and the nature of the association. Molecular methods will allow scientists to more accurately identify fungal species and determine actual diversity of sponge-associated fungi. This is especially important as greater cooperation between bacteriologists, mycologists, natural product chemists, and bioengineers is needed to provide a well-coordinated effort in studying the diversity, ecology, physiology, and association between bacteria, fungi, and other organisms present in marine sponges.     

Identification,classification, and functional characterization of novel sponge-associated acidimicrobiial species

Sponges are known to harbour an exceptional diversity of uncultured microorganisms, including members of the phylum Actinobacteriota. While members of the actinobacteriotal class Actinomycetia have been studied intensively due to their potential for secondary metabolite production, the sister class of Acidimicrobiia is often more abundant in sponges. However, the taxonomy, functions, and ecological roles of sponge-associated Acidimicrobiia are largely unknown. Here, we reconstructed and characterized 22 metagenome-assembled genomes (MAGs) of Acidimicrobiia from three sponge species. These MAGs represented six novel species, belonging to five genera, four families, and two orders, which are all uncharacterized (except the order Acidimicrobiales) and for which we propose nomenclature. These six uncultured species have either only been found in sponges and/or corals and have varying degrees of specificity to their host species. Functional gene profiling indicated that these six species shared a similar potential to non-symbiotic Acidimicrobiia with respect to amino acid biosynthesis and utilization of sulfur compounds. However, sponge-associated Acidimicrobiia differed from their non-symbiotic counterparts by relying predominantly on organic rather than inorganic sources of energy, and their predicted capacity to synthesise bioactive compounds or their precursors implicated in host defence. Additionally, the species possess the genetic capacity to degrade aromatic compounds that are frequently found in sponges. The novel Acidimicrobiia may also potentially mediate host development by modulating Hedgehog signalling and by the production of serotonin, which can affect host body contractions and digestion. These results highlight unique genomic and metabolic features of six new acidimicrobiial species that potentially support a sponge-associated lifestyle.     

Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the delta subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored.     

Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.     

Abundance and Bioactivity of Cultured Sponge-Associated Bacteria from the Mediterranean Sea

In this study, the search for new antibiotics was combined with quantitative ecological studies. The cultured fraction of the associated bacterial communities from ten different Mediterranean sponge species was investigated. To obtain quantitative and qualitative data of sponge-associated bacterial communities and to expand the cultured diversity, different media were used. The largest morphological diversity and highest yield of isolates was obtained by using oligotrophic media, which consisted of natural habitat seawater amended with (1% additional carbon sources. The dominant bacterial morphotypes were determined and bacterial isolates were tested for antimicrobial activity and identified using 16S rDNA sequencing. The sponge-associated most abundant morphotypes were all affiliated to the Alphaproteobacteria and showed antimicrobial activity against at least one of the tested strains. In contrast, the ambient seawater was dominated by Gammaproteobacteria. One single alphaproteobacterium, which was related to Pseudovibrio denitrificans, was shown to dominate the cultured community of at least six of the sponges. This designated MBIC3368-like alphaproteobacterium has been isolated from sponges before and seems to be restricted to associations with members of the phylum Porifera. It displays a weak and unstable antimicrobial activity, which gets easily lost during cultivation. However, this bioactive bacterium was present in the sponges by up to 10⁶ cells per gram wet-weight sponge tissue and dominated the cultured fraction with up to 74%. The association of this alphaproteobacterium with sponges is probably evolutionary young and facultative and possibly involves biologically active secondary metabolites. Besides a demonstrated vertical transfer, additional horizontal transfer between the sponges is assumed. Members of the genus Bacillus displaying antimicrobial activity were found regularly, too. However, actinomycetes, which are known for their production of bioactive substances, were present in very low abundance.     

极地微生物 —— 新天然药物的潜在来源

微生物是极地生态系统的重要组成部分.由于极地独特的地理、气候及环境特点,极地微生物所具有的新颖性与多样性,在科学研究、应用开发等方面都具有重要价值,并逐渐受到人们的广泛关注.有关极地微生物的资源勘探与代谢活性产物研究,已成为国际微生物学研究的热点之一.结合笔者的工作实践,对近年来极地微生物资源的勘探与收集情况进行了介绍,并着重对以医药与生物农药等为目标的极地微生物产活性化合物的研究现状进行了概述,同时还对目前一些影响研究工作开展的限制因素进行了讨论.可以预见,针对极地微生物资源的研究开发工作,将会给新型天然药物的筛选与发现带来新的机遇与突破.