【作者】 张永雨；

【导师】 焦念志；

【作者基本信息】 厦门大学 ， 环境科学， 2009， 博士

【摘要】 海洋噬菌体是海洋生态系统中丰度最高的一种生物类群，它在控制原核生物的死亡率，调节微生物群落结构、影响微食物网过程以及参与海洋中碳、氮等元素的生物地球化学循环中发挥着重要作用。由于噬菌体感染对细菌的生理生化过程具有十分重要的影响，因此对海洋中某一重要细菌类群的生态动力学过程及其生态意义进行综合考察与评价时，其周围环境中大量存在的噬菌体与它们之间的相互作用也是一个不可或缺的重要考察因素。对海洋细菌与噬菌体之间的相互关系进行研究具有十分重要的生态学意义。本文选择了两株具有重要生态地位的海洋细菌，即好氧不产氧光合异养细菌Roseobacter denitrificans OCh114和玫瑰杆菌属细菌Silicibacter pomeroyi DSS-3作为宿主菌，对其敏感性噬菌体进行了分离与鉴定，并就它们与噬菌体之间的相互关系进行了研究。首先，我们以R.denitrificans OCh114作为宿主菌，自中国南海分离到一株烈性噬菌体RDJLΦ1，分别从电镜形态、感染周期、宿主范围、核酸和蛋白质组成等方面对该噬菌体进行了分析和鉴定。电镜观察显示，该噬菌体具有一个等面体的头部(直径约69 nm)和一个长约170 nm、宽约9 nm的尾部，属于病毒分类学中的长尾病毒科(Siphoviridae)噬菌体。RDJLΦ1对氯仿具有很强的抗性，表明该噬菌体的衣壳内不含脂类物质。噬菌体RDJLΦ1具有较强的裂解能力，短时N间内即可引起宿主细菌的大量死亡。它感染宿主细菌时，在宿主细胞内的潜伏期约为80min，宿主菌被裂解时，平均每个细胞约释放203个子代噬菌体粒子。该噬菌体具有较强的感染专一性，在受试的24株细菌中，仅R.denitrificansOCh114能被其感染。对噬菌体RDJLΦ1的核酸进行限制性酶切分析后，证实RDJLΦ1的基因组是一种双链DNA分子，并且其基因组中存在着某种形式的修饰化碱基，使得其对一些常见的限制性内切酶具有抗性作用。对该噬菌体的基因组进行RAPD-PCR扩增，并对扩增产物进行克隆测序后，得到了一段1，651 bp的噬菌体基因组片段，对该基因组片段序列进行聚类分析后，发现噬菌体RDJLΦ1与另一株海洋噬菌体JL001具有较近的亲源关系。最后，使用SDS-PAGE结合MALDI-TOF MS／MS质谱鉴定技术对噬菌体RDJLΦ1的蛋白质组成进行了分析与鉴定，揭示RDJLΦ1至少由12种不同的蛋白质所组成，其中4个蛋白为其宿主同源蛋白，另外的8个蛋白由于在现有的数据库中找不到其同源匹配物，被认为是目前未知的新蛋白。以我们所知，本文中RDJLΦ1代表了第一株分离到的好氧不产氧光合异养菌(Aerobic anoxygenic phototrophic bacteria，AAPB)噬菌体，为将来人们研究AAPB与其噬菌体之间的相互关系拉开了序幕。其次，结合使用原子力显微镜成像技术(Atomic Force Microscopy，AFM)与比较蛋白质组学的方法，我们对噬菌体RDJLΦ1感染R．denitrificans OCh114的整个过程中，宿主菌的动态响应过程及机制进行了研究与探讨。在原位状态下，对受感染宿主菌的实时AFM成像揭示了R．denitrificans OCh114在受噬菌体感染的整个过程中其细胞表面形态的动态变化过程，尤为值得一提的是，噬菌体裂解细菌这一极其短暂的动态过程被以AFM图片的形式追踪记录了下来，这也是原子力显微镜技术在原位实时地观察噬菌体裂解细菌这一短暂过程中得以成功应用的首次报道，为人们直观的了解噬菌体感染细菌的动态过程提供了珍贵的图片资料。另外，利用基于双向凝胶电泳(2D-PAGE)与MALDI-OF MS／MS质谱鉴定技术的蛋白质组学方法，对R．denitrificans OCh114受RDJLΦ1感染后不同时间点(分别是受感染后的0、0．5、1．5、3和4．5 h)的蛋白质组表达谱进行了比较与分析，发现R.denitrificans OCh114受噬菌体感染后，在很短的时间(约30 min)内即能够完成全部的蛋白质响应，所需时间大大短于噬菌体RDJLΦ1在其宿主细胞内的潜伏时间(约80 min)，对宿主细菌蛋白质的双向电泳图谱进行比较分析后，发现约有91种蛋白质点的表达发生了显著变化。为了对这一响应机制进行初步的了解，我们对部分重要的响应蛋白进行了质谱鉴定，并对其生理功能进行了探讨。从一个新的视角出发，该研究将有助于我们对细菌与噬菌体(尤其是海洋细菌与其噬菌体)之间的相互关系进行更加全面的了解。在噬菌体感染原核生物细胞的过程中，一些原核生物细胞有时可以通过突变获得对某些噬菌体的抗性，从而得以存活下来。M1是由海洋玫瑰杆菌Silicibacterpomeroyi DSS-3在噬菌体Φ1感染的强胁迫诱导作用下产生的一株突变体，它获得了一种能抵抗噬菌体Φ1感染的能力。在本论文最后部分，我们对突变株M1抵抗噬菌体Φ1感染的作用机制进行了研究，逐一对以下几种机制发生的可能性进行了排除，它们分别是流产感染、吸附抑制、以及基于CRISPRs(clusteredregularly interspaced short palindromic repeats)的抗感染机制。最后，利用比较蛋白质组学的方法，对DSS-3与其突变株M1的蛋白表达图谱进行比较分析后，发现胞内4种高丰度蛋白的某种翻译后修饰可能赋予了突变株M1对噬菌体RDJLΦ1感染的抗性作用，其中的一种蛋白质经MALDI-FOF MS分析后，被成功鉴定为一种基于S.pomeroyi DSS-3基因组进行注释得到的假定蛋白(SPOA0343)。该部分的研究结果暗示了像SPOA0343这类蛋白的某种翻译后修饰参与了细菌抵抗噬菌体感染的过程。以我们所知，这是将蛋白质组学技术引入到细菌对噬菌体感染的抗性机制研究中的首次报道，丰富了人们对细菌抵抗噬菌体感染的方式和机制的了解。更多还原

【Abstract】 Phages are the most abundant microbial group in the ocean.They play importantroles in dominating the mortality of prokaryotic cells,structuring the microbialcommunity,influencing the microbial food web process and promoting thebiogeochemical (such as C and N etc.) cycles in the ocean.Since phage infection hasan important impact on the bacterial physiological and biochemical process,for acomprehensively investigation and evaluation of the kinetic process and ecologicalsignificance of one microbial group,the substantial existence of phages in thesurrounding environment and their interactions with these bacteria would be anindispensable factor to be considered.Studies on the interactions between bacteria andphages are of great ecological meanings.In this study,the two bacteria,Roseobacterdenitrificans OCh114 and Silicibacterpomeroyi DSS-3,who both have an importantecological status in the ocean,were served as the host bacteria to isolate theirsusceptive phages and to set up their specific host-phage systems,then using thesehost-phage systems,the interactions of host bacteria and their phages wereinvestigated.Firstly,by using R.denitrificans OCh114 as the host to challenge viralconcentrate collected from the South China Sea,we isolated the roseophage RDJLΦ1and then characterized this phage from several aspects,such as phage morphology,growth characteristics,host range,nucleic acids and proteomic compostion,etc.Transmission electric microscopy revealed that RDJLΦ1 has an isometric head (ca.69 nm in diameter) and a long,flexible,non-contractile tail (ca.170 nm long and ca.9nm wide),morphologically belonging to the Siphoviridae family.The strongresistance to chloroform indicated that RDJLΦ1 is a lipid-free phage.It is capable ofrapid lysis of host cells with a burst size of ca.200 phages per cell and a latent periodof ca.80 min.Host-range test revealed a highly specific infectivity of RDJLΦ1 whichcan only infect R.denitrificans OCh114 among 24 tested bacterial strains.Its double-stranded genomic DNA is refractory to several commonly used restrictionenzymes,suggesting that it contains some modified nucleic acid bases.Sequencealignment analysis of a 1,651-bp phage DNA fragment,which was revealed byrandomly amplified polymorphic DNA PCR coupled with cloning and sequencing ofthe amplicons,indicated a close relatedness of RDJLΦ1 with another marinesiphophageΦJL001.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ofthe virion proteins revealed that RDJLΦ1 is composed of at least 12 proteins.Surprisingly,4 of them were identifed as the host cellular proteins via matrix assistedlaser desorption/ionization time-of-flight mass spectrometry/mass spectrometry(MALDI-TOF MS/MS) method.While since no homologues of the other 8 proteinswere found in the NCBI database,they were considered to be novel proteins.R.denitrificans OCh114 is the first discovered aerobic anoxygenic phototrophicbacterium (AAPB),and RDJLΦ1 isthe firstphage isolated from the AAPBfunctional group.They represent the first host-phage model for studying theinteractions between AAPB and its susceptive phages.Secondly,we employed atomic force microscopy and proteomic techniques toinvestigate the host responses of R.denitrificans OCh114 during the whole course ofinfection by RDJLΦ1.In situ real-time atomic force microscopic imaging of theinfected R.denitrificans OCh114 cells revealed the morphological dynamic changesin the host surface following phage infection.Notably,several valuable atomic forcemicroscopic images revealing the highly dynamic phage lysis process were obtainedfor the first time.It showed that several large hollows with increasing diameter anddepth were abruptly and successively formed on the host surface,finally leading tothe collapse of the host cell.Then,using the two-dimensional polyacrylamide gelelectrophoresis method,at a global scale,we investigated the dynamic proteinexpression profiles of the host ceils at different timepoints (0,0.5,1.5,3 and 4.5 hpostinfection) following phage infection.Interestingly,the host protein responseswere found to be almost completed in only 30 min,which is much shorter than thelatent period ofRDJLΦ1(ca.80 min).For a preliminary investigation of the responsemechanism,several response proteins with significant changes in expression were identified using MALDI-TOF MS/MS method and are discussed separately in thisarticle.From a new perspective,this study contributes to our knowledge about theinteractions of bacteria and their phages,especially those which exist in the marinesystem.Under phage infection,sometimes some prokaryotic cells can survive byobtaining a mutation conferring upon themselfthe anti-phage ability.M1 is just such aphageresistant mutant of the marine roseobacter Silicibacter pomeroyi DSS-3.In thelast part of this paper,the phage resistance mechanisms of the mutant strain M1 wereinvestigated.Upon establishment of phage resistant strain M1 of S.pomeroyi DSS-3,our results excluded the possibilities of several phage resistance mechanisms,including abortive infection,adsorption inhibition,and the clustered regularlyinterspaced short palindromic repeats (CRISPRs).The comparison of proteinexpression profiles between the wild type strain (DSS-3) and mutant strain (M1)identified a potential phage resistance response due to the modification of four highlyexpressed proteins.One of these four proteins was successfully identified via massspectrometry,and matched a hypothetical protein (SPOA0343) annotated from the S.pomeroyi DSS-3 genome.Our results suggest that the modification of these proteinslike the protein SPOA0343 involves in the development of resistance to phageinfection.To the best of our knowledge,this work constitutes the first application ofcomparative proteomics to the study of phage resistance.As a result,it expands ourunderstanding of the anti-phage mechanisms of bacteria.更多还原