【作者】 王怡倩；

【导师】 章晓波；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 海洋占整个地球表面积的71％，多样性的生态环境蕴藏着丰富的生物资源。深海热液口是地球上一个极其恶劣的生存环境-高温、高压、缺氧、含有大量有毒的化学物质，但是其中及其周围却存在着一个完整的生态系统。噬菌体被认为是地球上最丰富的生命体，几乎可以在地球上的每个生态环境中找到，同样也暗示着它们在微生物多样性和生态平衡上发挥着重要作用。嗜热菌作为深海热液口生态群落的初级生产者，是推动营养和能量循环的主要动力。噬菌体是一类特殊群体，它们寄生在细菌体内，根据环境变化发生溶原或者裂解，从而引起宿主菌在整个生态群落中的分布。由于深海热液口在地理分布上具有一定的独立性，所以噬菌体与宿主的关系直接影响着整个热液口生态群落的存在和结构。随着基因组测序技术的发展，越来越多的噬菌体基因组信息添加到基因组数据库中，这为噬菌体生物信息学研究提供了大量的数据，不仅包括噬菌体序列之间的分析，还有利于研究噬菌体群体之间的结构组成。噬菌体的基因组虽然很小，但是却编码了自身复制所需的蛋白，如DNA包装蛋白、头尾结构蛋白、DNA复制相关蛋白、转录调控蛋白、裂解相关蛋白等。通过比较基因组学，可以得到大量的基因多样性数据，同时发现基因组之间存在着非常明显的序列相似性。这些序列可以通过同源或者点特异性重组促进噬菌体基因模块发生互换；或者，通过非定向的遍及整个基因组发生非同源重组。在本论文中，通过各种筛选培养基对深海热液区样品中的嗜热菌和高温噬菌体进行了分离、纯化，并得到了4株高温噬菌体，对其中的高温噬菌体D6E进行了进一步研究。结果表明，高温噬菌体D6E为肌尾科病毒，有二十面体的头部，长长的尾巴和尾丝。经测序，D6E基因组有49335 bp，为双链环状DNA，可以编码49个预测有功能的蛋白。和其它噬菌体一样，D6E的基因排列也是成簇分布的，按功能可以分为四个部分：DNA包装和头部装配、尾部组成、溶原与裂解、DNA复制和转录。通过在NCBI数据库中进行比对分析发现，D6E的大部分结构相关蛋白与已知噬菌体蛋白的同源性非常低，但是溶原和裂解相关基因以及DNA复制和转录相关基因的核苷酸序列与本实验室另外一株已测序的高温噬菌体GVE2不管在基因排列、还是这些基因编码的氨基酸序列上都具有非常高的相似性。采用蛋白质组学技术，对D6E病毒粒子的蛋白质组进行了分析，结果得到了10个与结构相关的蛋白，包括核衣壳蛋白、入口蛋白、支架蛋白等，其中质谱匹配率非常高的两个未知蛋白ORF3(第3条带)和ORF16(第9条带)的功能有待进一步研究。根据本实验室分离获得的深海嗜热菌噬菌体GVE2基因组测序结果和预测阅读框分析，重组表达并纯化了20个GVE2蛋白，制备相应的多克隆抗体，采用Western Blot对噬菌体的基因表达进行了分析。通过免疫共沉淀(Co-IP)和GST下拉(GST pull-down)试验寻找在感染过程中GVE2与宿主可能发生相互作用的蛋白。通过实验，得到了两种蛋白复合体：与GVE2 ORF5(核衣壳蛋白，VP371蛋白)相互作用的分子伴侣蛋白；与GV2 ORF36相互作用的未知蛋白。本实验室前期工作中发现宿主的天冬氨酸转氨酶和分子伴侣蛋白参与了噬菌体的感染过程，因此，本试验进一步研究噬菌体GVE2 VP371蛋白与宿主天冬氨酸转氨酶和分子伴侣形成的蛋白复合体在噬菌体感染中的作用。Western blot和细菌双杂交试验结果显示，VP371蛋白和分子伴侣蛋白之间、分子伴侣蛋白和天冬氨酸转氨酶之间存在相互作用，而VP371蛋白和天冬氨酸转氨酶之间没有相互作用，复合体中3个蛋白呈串联结构结合在一起。在噬菌体感染实验中我们发现，随着感染时间的增加，VP371的转录表达量逐渐增大，同时分子伴侣蛋白和天冬氨酸转氨酶都比未感染时有了明显上调表达。因此，在噬菌体感染过程中，GVE2核衣壳蛋白VP371和宿主的分子伴侣蛋白发生相互作用，并依次递进促进了分子伴侣蛋白和天冬氨酸转氨酶的上调表达，从而进一步调控了宿主细胞的代谢活动。本论文对对高温噬菌体GVE2的ORF3(入口蛋白)和ORF56(胸腺嘧啶合成酶)进行了功能鉴定。ORF3预测为噬菌体入口蛋白，虽然在氨基酸序列上它与其他噬菌体具有很低的同源性，但与SPP1、HK97的入口蛋白都具有相似的螺旋／折叠排列结构，属于HK97入口蛋白家族，在DNA包装过程中发挥着相同的功能。预测二级结构分析发现，GVE2同SPP1和Phi29都具有非常保守的α-Helices，可能是在基因组包装过程中保留下来的一种古老结构域。通过免疫电镜定位，结果表明入口蛋白位于头尾的连接处。Orf56编码原核型的胸腺嘧啶合成酶，与已知的不同物种的胸腺嘧啶合成酶有很高的相似性。ORF56中可以找到符合胸腺嘧啶合成酶的活性中心的保守序列，还具有五个非常保守的motif，包括叶酸结合位点、催化活性中心、dUMP-结合位点、质子转运位点和功能待定区域。通过体外结合试验，结果发现ORF56编码的蛋白可以与其自身mRNA结合。更多还原

【Abstract】 Bacteriophages can be detected in almost every biological niche and represent ahuge source of biodiversity and possibly the largest part of the biomass on the planet.Therefore,phages are thought to play major roles in the ecological balance ofmicrobial life and in microbial diversity.This view has gained strong support from thework in the past 30 years or so on viruses in extreme ecosystems.With the discoveryof deep-sea hydrothermal vents and their attendant communities,researches onhydrothermal vent communities have become attractive interests in the field ofoceanography and biology.The isolation and characterization of viruses often lead tonew insights into virus relationships and to a more detailed understanding of thebiochemical environment of their host cells.The increasing number of viral (andcellular) genomes that have been sequenced has directed our attention to utilize thisinformation to rationalize the organization of viral life forms.In general,ourunderstanding of the deep-sea thermophilic bacteriophages is far behind ourknowledge of the terrestrial,mesophilic bacteriophages.The recent discovery of manynovel extremely thermophilic bacteriophage,especially among members isolatedfrom deep-sea hydrothermal vents,is likely to lead to a more complete understandingof not only thermophiles,but also the biochemical adaptations required for the life inextreme environments,and to new insights into both host and virus evolution.Genome sequencing has revealed the important role of horizontal gene transfer inprokaryotes.Comparative viral genomics has created a wealth of information that hasmade it possible to construct gene and genome phylogenies as well as to observecomplex relationships among virus genomes.However,phages that infectthermophilic eubacteria have remained mostly unexplored.Genomes of only a few ofsuch phages have been sequenced completely.The only phage infecting thermophilicbacteria which identified from deep-sea hydrothermal vent that has been characterizedat the molecular level is GVE2,a siphovirus that infects Gebacillus sp.The functionalanalysis of the GVE2 genome and its gene expression strategy has revealed a wealthof new data about transcription and replication regulation,indicating that furtherstudies of phages infecting thermophilic bacteria are warranted.Our studies focus on the molecular biology of host-phage interactions especially at protein level using biochemistry,molecular microbiology and molecular biologyapproaches.In this investigation,several thermophilic bacteriophages were isolatedfrom deep-sea hydrothermal vents in east pacific.Among them,the thermophilicbacteriophage D6E was characterized.D6E was an atypical myovirus with anicosahedral capsid (60 nm in diameter),a tail (16 nm in width,60 in length) and a tailfiber (4 nm in width,60 nm in length).An accurate mode of DNA pyrosequencingwas used to sequence the genome and mass spectrometry was used to accomplish thecomprehensive virion protein survey.Based on sequencing,the phage contained a49335-bp double-stranded genomic DNA.The genome encoded 49 putative openreading frames (ORFs).Functions for D6E gene products were predicted on the basisof similarity to proteins of known functions from diverse phages and bacteria.D6Eencoded four clusters of proteins involved in DNA packaging and headmorphogenesis,lysis and lysogeny,DNA replication and transcription.Advancedbioinformatics techniques were used to identify classical morphogenesis genes.Thestructural genes of D6E,most of which had no similarity to sequences in publicdatabases,were identified by mass spectrometric analysis of purified virions.Therewas also evidence that the capsid protein and portal protein could generate tertiaryand quaternary structures similar to corresponding proteins of other bacteriophages,despite the lack of significant sequence similarity.A comparative analysis of D6Esequence with GVE2,isolated from east pacific,was conducted.The comparisonsconfirmed that these phages were genetic mosaics,with mosaic segments separated bysharp transition in the sequence.The mosaicism provided clear evidence thathorizontal exchange of genetic material myoviruse and siphovirus was a resource forfuture studies of vertical gene transmission.Based on proteomic analysis of thepurified D6E virions,10 structural proteins were revealed,including capsid protein,portal protein,etc.To reveal the protein-protein interaction between bacteriophage and its host,thethermophilic bacteriophage GVE2 was characterized.Twenty of the predicted 62GVE2 ORFs were expressed as recombinant proteins,and the purified recombinantproteins were used for antibody preparations.The expression profiles of these geneswere analyzed by Western blots.Based on GST pull down,Co-IP and nativeSDS-PAGE,several protein complexes associated with host-phage interaction wereobtained.Subsequently the interactions between ORF5 encoding capsid protein (VP371),chaperonin protein (CHG) and aspartate aminotransferase (AST) werecharacterized by Western blot and bacteria two-hybrid system.The results showedthat the VP371 was bound with CHG and CHG bound with AST.However no bindingwas observed between VP371 and AST.Northern blots and Western blots indicatedthat chg and ast genes were up-regulated after phage infection.It is well known thatCHG and AST were involved in energy metabolism,matter and energy transportation,suggesting that they might play very important roles in host immune response againstbacteriophage infection.In this paper,novel portal protein and thymidylate synthase were identifiedfrom the deep-sea thermophilic bacteriophage GVE2 for the first time.Portal protein,located asymmetrically at one of the twelve vertices of the capsid,play very importantroles in viral DNA packaging.The GVE2 portal protein (designated as VP411 protein)shared low similarity to known portal proteins from other species,but they showedhigh similarities in the predicted secondary structures,suggesting that they had thesame function in viral DNA packaging.The Northern blot and Western blot resultsdemonstrated that the vp411 gene was expressed in the late stage of GVE2 infection,implying that it might be a viral late gene.As revealed by immuno-electronmicroscopy,the gold particles were observed in the junction between the phage headand the phage tail when the anti-VP411 IgG was used as the primary antibody,indicating that it had the location in the virion expected of a portal protein.Thymidylate synthase (TS) is essential for de novo synthesis of dTMP and is a keyenzyme involved in DNA synthesis and transcriptional regulation of organisms.Dueto their biologic importance,TSs have been intensively studied.In this investigation,a thermostable TS was identified from the deep-sea thermophilic bacteriophageGeobacillus virus E2 (GVE2).It was demonstrated that the GVE2-TS was highlyhomologous to known TSs and contained five characteristic conserved domains.Thetemporal analyses by Northern and Western blots revealed that the GVE2-TS wastranscribed and expressed early after Geobacillus virus E2 infection,identifying it asan early viral gene.As shown by gel mobility shift assays,the recombinant GVE2-TSprotein had the capacity to bind its own mRNA.Our study presented the first reporton thymidylate synthase from deep-sea thermophilic bacteriophage.更多还原