- 學位論文
噬菌體EmPhiS感染表孔珊瑚內生桿菌之轉錄體時序變化
Transcriptome analysis of Endozoicomonas montiporae under EmPhiS infection
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摘要
病毒能夠主導珊瑚礁生態系碳循環、增加珊瑚礁內微生物基因多樣性，並控制病原菌數量使珊瑚免於疾病侵擾。然而當珊瑚礁生態系面臨環境壓力，會使珊瑚病毒多樣性組成改變造成疾病。隨著對於珊瑚礁微生物研究增加，學者也發現細菌對珊瑚礁生態系有諸多益處。其中內生桿菌屬(Endozoicomonas)備受矚目，他們能減少營養源自珊瑚礁生物體流失，或是提供幫助對抗熱逆境物質。表孔珊瑚內生桿菌(Endozoicomonas montiporae CL-33T, CL-33)，是第一個自珊瑚礁中分離出內生桿菌物種(Yang et al., 2010)，其基因體帶有數個噬菌體相關基因，且具有能對抗噬菌體的回文重複序列叢集關聯蛋白系統(Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR)。這些發現人們好奇是否有能感染CL-33噬菌體，從而發現了噬菌體EmPhiS。噬菌體EmPhiS來自墾丁出水口，萼形柱珊瑚(Stylophora pistillata)周圍海水。本研究透過形態、基因註解、生理及轉錄體學研究，分析噬菌體EmPhiS。實驗結果發現，以形態上來看，EmPhiS屬於肌尾噬菌體科(Myoviridae)，並具有巨型噬菌體(Jumbo phage)特徵。基因註解結果發現EmPhiS具有大量核苷酸代謝相關基因，其餘功能如感染與裂解、能量調控及結構相關基因數量則相互接近。生理實驗結果顯示EmPhiS為裂解性噬菌體，在不同感染比例下會顯現出不同病毒動態。轉錄體實驗結果發現噬菌體基因表現能夠分為前、中、後等不同時期，同時宿主在前期受影響基因數量也為最多。感染前期，噬菌體表現許多感染相關基因，並啟動核苷酸複製與調控宿主代謝，宿主則產生壓力表現基因應對入侵活動；感染中期，噬菌體抑制宿主胺基酸代謝及壓力反應，避免噬菌體表現蛋白質被降解，並逐漸開始製造結構蛋白；感染後期，噬菌體著重於結構組裝，進一步增加能量需求，細菌內部環境逐漸無法維持穩定，隨著宿主裂解完成感染循環。本研究為第一個與珊瑚有益細菌噬菌體轉錄體分析，透過此研究，可增加對珊瑚礁共生體中病毒所扮演角色及生態意義認知。
並列摘要
Viruses play a crucial role in coral reef ecosystems by regulating carbon cycling, enhancing microbial genetic diversity, and controlling pathogen populations to protect corals from diseases. However, environmental stress can alter the diversity and composition of coral-associated viruses, leading to disease outbreaks. As research on coral reef microbiomes progresses, scientists have discovered numerous benefits of bacteria to coral reef ecosystems. Among these, the genus Endozoicomonas has gained significant attention for its ability to reduce nutrient loss from coral organisms and provide substances that help corals cope with thermal stress. Endozoicomonas montiporae CL-33T, the first species of Endozoicomonas isolated from coral reefs (Yang et al., 2010), possesses a genome with numerous phage-related genes and a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system that provides resistance against phages. This discovery led researchers to investigate whether phages capable of infecting CL-33 exist, resulting in the identification of the phage EmPhiS. EmPhiS was isolated from the seawater surrounding Stylophora pistillata corals in Kenting. This study analyzed EmPhiS through morphological, genomic, physiological, and transcriptomic approaches. Morphologically, EmPhiS belongs to the family Myoviridae and exhibits characteristics of jumbo phages. Genomic annotation revealed that EmPhiS contains numerous genes related to nucleotide metabolism, with similar numbers of genes involved in infection and lysis, energy regulation, and structural functions. Physiological experiments demonstrated that EmPhiS is a lytic phage, displaying different viral dynamics at varying infection ratios. Transcriptomic analysis showed that phage gene expression occurs in distinct early, middle, and late stages of infection, with the highest number of host genes affected during the early stage. During the early stage, EmPhiS expresses many infection-related genes, initiates nucleotide replication, and regulates host metabolism, while the host responds with stress-related genes. In the middle stage, EmPhiS suppresses host amino acid metabolism and stress responses to prevent degradation of phage proteins and begins producing structural proteins. In the late stage, EmPhiS focuses on structural assembly, increasing energy demands, and leading to the eventual lysis of the host cell, completing the infection cycle. This study represents the first transcriptomic analysis of a phage infecting beneficial coral-associated bacteria, providing insights into the role and ecological significance of viruses within coral holobiont.
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