【作者】 秦启龙；

【导师】 张玉忠；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 海洋是地球上最重要的生态系统之一,其中蕴藏着种类繁多的微生物类群,是获取新药和新酶的宝贵资源,具有很好的开发前景。地球表面的60%被深度超过1000米的海水所覆盖,并且深海区地球表面覆盖着生物和非生物来源的深海沉积物。由于巨大的广度和深度,海底沉积物和上部洋壳是地球上最大的生态系统之一,蕴藏着巨大的生物特别是微生物资源。海洋底部是一个极端环境,是嗜冷、嗜热、嗜压、嗜盐和嗜低营养等嗜极端环境微生物的理想来源。对这些极端微生物的研究开发将会在工业应用、医药应用、环境保护等方面具有潜在的、巨大的应用价值。但是由于采样和培养困难,对深海沉积物微生物资源的开发还远远不够。因此,对深海沉积物中微生物资源的发现和开发就具有重要的意义。本论文研究内容首先是对一株分离自南冲绳海槽深海沉积物的细菌SM-A87进行了菌种鉴定。然后对菌株SM-A87进行了全基因组测序,以期通过基因组信息揭示其在深海沉积物PON降解中的作用。Pseudoalteromonas sp. SM9913是从冲绳槽附近海域1855米深的海底沉积物中分离的深海适冷菌。能分泌大量的胞外多糖和适冷蛋白酶。本论文对其全基因组序列进行了测序和分析,并与南极浅海适冷菌Pseudoalteromonas haloplanktis TAC125进行了比较基因组学的研究。以期发现假交替单胞菌属内深海细菌对深海环境适应的独特机制。Trichodermapseudokoningii SMF2是分离自海滩土壤的一株丝状真菌。其对线虫有杀伤作用,并且其分泌的次级代谢物哌珀霉素有抗菌和抗肿瘤活性。本论文对其基因组进行了测序和分析。希望能够发现哌珀霉素的产生途径并为以后的研究做好基础。菌株SM-A87的菌种鉴定：SM-A87分离自南冲绳海槽深海沉积物海底表层下2米,海水深度为1245米。SM-A87革兰氏染色为阴性,无游动和滑动能力。在海水LB平板上28℃培养48 h后,菌落呈黄色到橘红色,菌落圆形且紧贴平板,不透明,直径2-3 mm,高1-2 mm。菌体细胞呈短杆状,长约1.5-3.3μm,宽约0.3-0.6μm。长期培养可形成球状菌体。SM-A87是严格好氧菌,氧化酶和接触酶阳性。产黄色色素,不形成芽孢。能在4-38℃范围内生长,最适生长温度范围为25-30℃。生长的pH范围是pH5.0-8.5,能在0-12%的NaCl浓度下生长,最适生长NaCl浓度为3%。SM-A87含有黄杆菌科主要的呼吸醌MK-6。含有Flexirubin色素,主要合成末端分支的脂肪酸。SM-A87的DNA G+C含量为35.8 mol%。通过16S rRNA基因系统进化树分析发现,与SM-A87最相近的菌株为黄杆菌科的Salegentibacter holothuriorum,两者的16S rRNA基因序列相似性为92.9%。另外,SM-A87 16S rRNA基因序列与同属于黄杆菌科的Mesonia algae和Gramella portivictoriae相似性分别为91.8%和91.5%。综合传统分类学、化学分类学和分子系统学分类的结果,将SM-A87归类为黄杆菌科的一个新属新种,定名为Wangia profunda。最后属名在International Journal of Systematic and Evolutionary Microbiology (IJSEM) Validation List no.116上更改为Zunongwangia。Zunongwangia profunda SM-A87基因组学研究：SM-A87的全基因组只有一条染色体,长度为5128187 bp,含有4653个预测的ORF,ORF平均长度为960 bp。有47个tRNA基因和3个rRNA操纵子。是目前为止Bacteroidetes第一个被测序的深海沉积物细菌。基因组中含有糖酵解,戊糖磷酸途径和三羧酸循环的全部基因以及ED代谢途径的关键酶。这反应了SM-A87代谢途径的多能性。SM-A87可以产生大量的荚膜多糖,基因组分析发现其基因组有两个多糖合成和输出的基因簇。SM-A87可以编码大量分泌型的水解酶。其中在130个肽酶中,61个带有信号肽。并且带信号肽的肽酶比不带信号肽的肽酶更有嗜盐特性,这有利于SM-A87在海水环境中对胞外蛋白质进行更好的水解。其胞外肽酶主要以金属肽酶和丝氨酸肽酶为主。SM-A87中与糖类分解和转运相关的酶也比较多。因此,深海来源的Bacteroidetes的菌株具有很强的对胞外有机碳和有机氮降解的能力。SM-A87中有两个CRISPR位点。第一个位点为5595 bp,第二个位点为2115bp。CRISPR被认为与抗病毒有关,但是SM-A87的两个CRISPR位点的间隔区序列在现有病毒库中搜不到同源序列。这可能是由于现有病毒数据库中仅包含了海洋病毒的很少一部分序列的原因。SM-A87能耐受0-12%的NaCl,是中度嗜盐菌。嗜盐蛋白具有比较多酸性氨基酸残基从而使自身等电点比较低。通过预测SM-A87基因组全蛋白的等电点来预测其耐盐特性。SM-A87胞外蛋白的等电点明显低于胞内蛋白的等电点,说明其胞外蛋白比胞内蛋白更具有嗜盐性。同时也说明SM-A87胞内的盐离子浓度不是很高,很可能是通过富集有机物来维持其细胞内外渗透压平衡。其基因组能编码甜菜碱转运子也验证了这个推论。Pseudoalteromonas sp. SM9913基因组及比较基因组学研究：P. sp. SM9913(简称SM9913)是一株深海适冷菌,最适生长温度为15℃。P. haloplanktisTAC125是分离自南极表层海水的适冷菌,并且基因组已经测序完成。SM9913基因组由两条染色体组成,大小分别为3.3 Mb(染色体Ⅰ)和700 kb(染色体Ⅱ),与P. haloplanktis TAC125(简称TAC125)两条染色体的大小和结构都很相似。SM9913的两个染色体共含有3711个ORF,其中66.9%可以注释上已有的或预测的功能。62个tRNA基因和8个rRNA操纵子以及一个额外的5S rRNA基因都位于染色体Ⅰ上。根据16S rRNA基因构建的进化树表明SM9913和TAC125在进化关系上是非常接近的。SM9913和TAC125的平均核苷酸相似度为85%,说明SM9913和TAC125是假交替单胞菌属不同的种但是具有很高的相似性。两者共有2698个直系同源基因,分别占SM9913和TAC125全部基因的72.7%和77.4%。SM9913中共鉴定出了12个长度大于15 kb的基因岛(gene island,GI)。其中11个位于染色体Ⅰ,一个位于染色体Ⅱ。基因岛中的基因大部分是在TAC125中没有直系同源基因的SM9913特有基因,所以这些基因岛的特有基因会赋予SM9913一些区别于TAC125的特性。与TAC125相比,SM9913中双加氧酶个数比较少并且缺少TAC125中与H2O2抗性相关的脂肪酸代谢基因簇。因此推测SM9913对H2O2比较敏感。实验结果也证实了这一点,SM9913只能在5 mM H2O2浓度下生长,而TAC125可以在10-15 mM H2O2浓度下生长。这可能是因为深海沉积物中氧气浓度比较低,导致ROS (reactive oxygen species)的产生量比较少,从而使SM9913只能耐受低浓度的H2O2。SM9913基因岛2编码与抗生素抗性相关基因。实验结果也表明SM9913对某些抗生素有抗性,如青霉素、阿莫西林,而TAC125比较敏感。这可能是因为冲绳海槽位于中国东海大陆架边缘,与南极海水相比,冲绳海槽海底沉积物会有大量的来自于陆地的物质,其中包括抗生素。从而导致SM9913对许多抗生素有抗性。SM9913的基因岛8和9中有许多重金属离子抗性基因。实验结果证明SM9913对锌离子比TAC125有更高的抗性。我们推测可能是因为SM9913可以分泌大量的带负电的胞外多糖,这些胞外多糖有时可能会吸附过量的重金属离子,这会导致SM9913面临高浓度的重金属离子。所以SM9913对一些重金属离子具有抗性。SM9913拥有极生鞭毛与侧生鞭毛两套鞭毛合成基因簇,并且侧生鞭毛合成基因簇在TAC125基因组是没有的。拥有两套鞭毛系统,SM9913就既可以在海水中游动又可以在固体表面滑动。这对于其在深海沉积物中获取颗粒状的营养物质非常有帮助。极生鞭毛可以在透射电镜下观察到,但是没有观察到侧生鞭毛的存在,可能是由于侧生鞭毛合成基因在常压下不表达。另外SM9913还能编码糖原合成基因,这可以使其在营养丰富时合成糖原来储存能量以度过营养匮乏时期。这有利于其在深海环境中生存。Trichoderma pseudokoningii SMF2基因组学研究：通过Solexa对Trichoderma pseudokoningii SMF2基因组进行测序和组装,共得到114个contig和93个scaffold,基因组序列大小约为31 Mb。其胞外蛋白酶以金属蛋白酶和丝氨酸蛋白酶为主。与同属内其他测序菌株有一定的相似性,但是在GO功能分类上有一定的不同。基因组中有22个与次级代谢产物合成有关的基因簇,这些基因簇可能与哌珀霉素及其他次生代谢产物的合成相关。本文对Trichoderma pseudokoningii SMF2基因组的初步分析为其基因组、转录组和蛋白质组的深入研究奠定了基础。更多还原

【Abstract】 Marine is one of the most important ecosystems on the Earth. It harbors a variety of bacteria which are valuable resources for discovery of new drugs and enzymes. More than 60% of the Earth’s surface is covered by sea water with depth more than 1000 m. The deep-sea floor is covered with fine-grained sediments. Deep-sea sediment is a dynamic geo- and biosphere that hosts rich microbial communities because of its great width and depth. The deep-sea is an extreme environment and harbors a variety of extremophiles adapted to low or high temperature, high pressure, high salty and low nutrient. These extremophiles will have great potential in industry, medicine and environment protection. Though deep-sea sediments contain huge microbial resources, it is still less exploited because of the sample and culture difficulty. So it is important to discover and exploit the bacterial resources in deep-sea sediments.In this dissertation, we identified a bacterium, SM-A87, isolated from southern Okinawa Trough. Then the complete genome of SM-A87 was sequenced and analyzed to find its role in deep-sea particulate organic material (POM) degradation. Pseudoalteromonas sp. SM9913 is a cold-adapted bacterium isolated from Okinawa Trough with water depth of 1855 m. It can secret a large quantity of exopolysaccharides and cold-adapted proteases. In order to find the special features of deep-sea species of Pseudoalteromonas, the complete genome of P. sp. SM9913 was sequenced and compared to Pseudoalteromonas haloplanktis TAC125 which is a cold-adapted bacterium isolated from the Antarctic surface water. Trichoderma pseudokoningii SMF2 was a filamentous fungus isolated from beach soil. It has nematicidal activity and can kill nematode Meloidogyne incognita. The secondary metabolite peptaibols T. pseudokoningii SMF2 secreted have antimicrobial activity and can induce programmed cell death of tumor. In this dissertation, we sequenced and analyzed the genome of SMF2 in order to find the pathway of peptaibols synthesis and provide foundation for further research.Identification of strain SM-A87:Strain SM-A87 was isolated from southern Okinawa Trough sediments at 2 meters below seafloor with water depth of 1245 m. It is a Gram-negative, non-motile bacterium. After 48 h cultivation at 28℃on marine agar, the colonies were yellow to orange and circular, about 1-3mm in diameter, and were adherent to the agar. Cells were rod-shaped and ranged from 0.3 to 0.6 mm in width and from 1.5 to 3.3 mm in length and were non-motile. Cells in old cultures might form coccoid bodies.Cells are strictly aerobic, oxidase-and catalase-positive. Flexirubin-type pigments are absent. MK-6 is the predominant respiratory quinone. SM-A87 synthesized mainly terminally branched iso-and anteiso-fatty acids. The strain can grow at 4-38℃(25-30℃optimum), at pH 5.0-8.5 and in the presence of 0-12% NaCl (3%, optimum).The DNA G+C content of SM-A87 is 35.8%. The phylogenetic tree revealed that strain SM-A87 can form a distinct lineage within the family Flavobacteriaceae. Strain SM-A87 had 92.9% 16S rRNA gene sequence similarity to its nearest neighbor Salegentibacter holothuriorum, and 91.8% and 91.5% to Mesonia algae and Gramella portivictoriae, respectively.According to the phenotypic characteristics, chemotaxonomy and phylogeny analysis, SM-A87 was classified as a new genus and species in the family Flavobacteriaceae for which the name Wangia profunda is proposed. The type strain is SM-A87T. In the International Journal of Systematic and Evolutionary Microbiology (IJSEM) Validation List no.116, it was renamed to Zunongwangia profunda.Genomic study of Zunongwangia profunda SM-A87:SM-A87 is a new genus and species in the family Flavobacteriaceae of the phylum Bacteroidetes that was isolated from deep-sea sediment. Its genome is composed of only one circular chromosome with 5128187 bp. It has 4653 predicted ORFs of which the average length is 960 bp. The genome contains 47 tRNA genes and 3 rRNA operons. This is the first sequenced genome of a deep-sea bacterium from the phylum Bacteroidetes. The genome harbors all the genes of glycolysis, the pentose phosphate pathway and the tricarboxylic/citric acid cycle as well as the key enzyme of ED metabolic pathway. All this reflects the metabolic versatility of SM-A87.SM-A87 can produce a large quantity of capsular polysaccharide, and the genome contains two gene clusters for polysaccharide synthesis and export. SM-A87 contains 130 predicted peptidases,61 of which have signal peptides. The extracellular peptidases are more halophilic than the intracellular peptidases. The halophilicity of the extracellular peptidases helps them function in saline environments and decompose extracellular organic nitrogen matter in the marine salty condition. The extracellular peptidases mainly belong to families of metallopeptidases and serine peptidases. SM-A87 has many genes related to carbohydrate transport and metabolism. This shows that the deep-sea bacterium of the phylum Bacteroidetes is also good at decomposing extracelluar materials.SM-A87 has two CRISPR loci. The first one is 5595 bp and the second one is 2115 bp. CRISPR is related to phage defense., But the spacers of these two CRISPR loci do not have similar sequences in the virus databases, this may because the updated virus databases just have a small section sequences of all marine virus.SM-A87 is a moderate halophile and can tolerate 0-12% NaCl. Halophilic proteins usually contain more acidic residues and have lower pIs (predicted isoelectric point) than nonhalophilic proteins. We predicted the pIs of all the proteins of SM-A87 and found that the pIs of extracelluar proteins are lower than that of intracellular proteins. This shows that its extracellular proteins are more halophilic than intracelluar proteins. This also indicated that SM-A87 may concentrate organic compatible solutes in the cell so the intracellular ion concentration is not high. The presence of glycine betaine transporter in SM-A87 confirms this conclusion.Genomic and comparative genomic study of Pseudoalteromonas sp. SM9913: P. sp. SM9913 (SM9913 for short) is a deep-sea cold adapted bacterium with the optimum growth temperature of 15℃. The genome of SM9913 is composed of two chromosomes. One is 3.3 Mb (chrⅠ) and the other is 700 kb (chrⅡ). The genome size and structure are all similar to the two chromosomes of P. haloplanktis TAC125 (TAC125 for short). The two chromosomes of SM9913 have 3711 ORFs in total, of which 66.9% can be annotated with known or predicted function. Sixty-two tRNA genes and eight rRNA operons as well as one extra 5S rRNA gene are all located in chr I.Phylogenetic tree based on the 16S rRNA gene indicates that SM9913 and TAC125 are with high similarity. The average nucleotide identity between SM9913 and TAC125 is 85%, which indicates that the two strains are not the same species, but have a high identity. The two strains share 2698 orthologous genes, accounting for 72.7%and 77.4%of all the genes of SM9913 and TAC125, respectively. Twelve genomic islands (GIs) larger than 15 kb can be identified. Eleven GIs are located in chr 1 and one GI is located in chr 2. Most genes in the GIs of SM9913 are specific genes that do not have orthologs in TAC125. These specific genes may confer some features of SM9913 that differentiate it from TAC125SM9913 has fewer dioxygenase genes than TAC125 and lacks the fatty acid metabolism gene cluster that is related to ROS (reactive oxygen species) resistance, indicating a possible sensitivity to reactive oxygen species. Accordingly, experimental results showed that SM9913 was less tolerant of H2O2 than TAC125. SM9913 was only able to grow at concentrations of up to 5 mM H2O2, while TAC125 grew well even at 10-15 mM H2O2. This may be because the oxygen concentration in deep sea sediment is very low, so the production of ROS in deep-sea sediment is less than that in surface sea water.GI-2 of SM9913 contains some genes that are related to drug resistance. SM9913 can resist some antibiotics, such as ampicillin, penicillin and amoxicillin, while TAC125 is susceptible to them. The Okinawa Trough is at the edge of the continental shelf of the East China Sea. Compared to Antarctic sea water, the water and sediments in the Okinawa Trough are likely to contain more materials from the continent, including antibiotics. So SM9913 can resist many antibiotics.There are some heavy metal resistance and efflux genes in GI-8 and GI-9 of SM9913. Experimental results proved that SM9913 is more resistant to zinc than TAC125. It is still unclear why deep-sea bacteria are more resistant to heavy metals than surface sea bacteria. However, SM9913 may be adapted to high metal concentrations because the negatively-charged EPS that is secreted by deep-sea bacteria sometimes can adsorb more cations around the cell than are neededWith the polar and lateral flagellar systems, SM9913 can swim in the sea water and swarm on the sediment particle surface, which is advantageous in the acquisition of nutrients from particle materials in deep-sea sediment. The polar flagellum can be seen under SEM. But the lateral flagella were not observed. This may be because lateral flagella biosynthesis genes are not expressed under normal pressure.The genome contains a glycogen production operon, SM9913 can accumulate glycogen with this operon when nutrients are widely available and use the stored glycogen when nutrients are absent from the environment, which would improve its ability to survive in the deep-sea environment.Genomic study of Trichoderma pseudokoningii SMF2:The genomic sequence of T. pseudokoningii SMF2 was sequenced and assembled by Solexa Genome Analyzer. The genome composed of 114 contigs and 93 scaffolds is about 31 Mb. Its extracelluar proteases are mainly metallo and serine peptidases. Genomic sequence has some similarity to other sequenced strains in the same genus but has differences in GO function categories. The genome has 22 gene clusters related to synthesis of secondary metabolite. These clusters may be responsible for the synthesis of peptabols, which needs further research. Our prelimitary study on the genome of SMF2 will be beneficial for further research on its genome, transcripsome and proteinsome.更多还原