兩株台灣軸孔珊瑚內生桿菌新種之特性與基因體分析

Abstract

珊瑚擁有不同的微生物夥伴，比如真菌、藻類及細菌等，且彼此有著複雜的交互作用。珊瑚的不同部位居住著不同的微生物，其中有一屬的細菌被稱為內生桿菌，常被發現在珊瑚的黏液及組織中。內生桿菌群集的豐度會隨著珊瑚的健康程度而有所不同，相比於生病的或受環境壓迫的珊瑚，健康的珊瑚體具有較高豐度的內生桿菌，因此內生桿菌被認為與珊瑚健康有關係，並且被認為是一群珊瑚健康的益生菌。近年來，環境快速變遷導致珊瑚大量死亡，益生菌成為其中一個拯救或復育珊瑚的手段。內生桿菌為珊瑚益生菌的重要候選菌種之一，但目前由石珊瑚分離的可培養內生桿菌只有兩株，其中Endozoicomonas montiporae CL-33T為第一株具有基因體序列的內生桿菌，並且在基因體分析中發現E. montiporae CL-33T具有與宿主互動的基因而且可能在珊瑚遇到逆境時保護宿主的粒線體，來防止其失去功能，並且當珊瑚沒有足夠的葡萄糖時可以促使宿主轉而利用脂質，促進糖質新生。今年另一個研究從基因體方面證實了內生桿菌藉由代謝二甲基巯基丙酸(dimethylsulfoniopropionate, DMSP )，產生氣候調節化合物，二甲基醚(dimethyl suldife, DMS)參與珊瑚的硫循環。 鑑於目前只有兩株由珊瑚分離的內生桿菌，分離更多可培養的內生桿菌可以幫助我們更暸解其與珊瑚可能的互動關係。在這個研究中，我們分別由澎湖及墾丁分離兩株新的內生桿菌，E. penghunesis 4G與E. ruthgatesii 8E ，兩株菌與最近的內生桿菌E. euniceicola 16S rRNA的親緣關係相似性分別為96.68%及96.99%。兩株菌在最適生長溫度、鹽度及酸鹼值上展現了不同的特性，E. ruthgatesii 8E的最適生長溫度為25°C、pH值7、鹽度1-2%，而E. penghunesis 4G則最適合生長於20至25°C，pH值8、鹽度1-2%。除此之外，我們也將兩株新菌進行基因體定序，並且獲得低污染(污染<1%)，接近完整(>97%)的基因體。在基因體大小上，E. penghunesis 4G為5.73 Mb，E. ruthgatesii 8E則具有目前內生桿菌中最大的基因體，7.1Mb，也具有假定可以將DMSP當成碳源並代謝成DMS的基因操縱組。進一步我們量化從珊瑚分離出的這四株內生桿菌消耗DMSP與產生DMS的濃度，並發現E. ruthgatesii 8E 相比於E. acroporae Acr-14T可以消耗更多的DMSP。最後本論文提出兩株內生桿菌屬新種，並首度量化珊瑚優勢共棲菌的DMSP消耗量及DMS產生量。

Corals harbor diverse microbial partners (e.g., fungi, algae, and bacteria), leading to complex yet intriguing mesh of interactions among and within coral holobiont. Different organs of coral harbor diverse microbial communities, such as members of genus Endozoicomonas are abundant in coral mucus and epidermal tissues. Interestingly, Endozoicomonas are often abundant in healthy corals and their abundance declines in diseased or stressed corals, consequently, members of this genus are hypothesized to be potential probiotics of coral health. Recently, the identification of beneficial microorganisms for coral has been at the center of developing probiotics for coral reefs to mitigate stress from heat-induced coral bleaching. Endozoicomonas species which are potential candidates for coral probiotics lack cultured isolates with only two species isolated from scleractinian corals. Endozoicomonas montiporae CL-33T the first cultured and genome sequenced isolate has been shown to have the potential to internalize and interact with host cells and harbor genes to protect mitochondrial dysfunction and promote gluconeogenesis in the host. A recent study provided first genomic and functional evidence of the role of Endozoicomonas in the coral sulfur cycle by metabolizing dimethylsulfoniopropionate (DMSP) to climate active gas dimethylsulfide (DMS). With only two cultured isolates to date, it becomes important to identify and culture new species from this diverse genus in order to ascertain its role in coral reefs. In this study, we isolated and cultured two novel bacteria species, E. penghuensis 4G and E. ruthgatesii 8E from dominant coral Acroporae sp. of the coast of Penghu Archipelago and Kenting, Taiwan, respectively. The nearest neighbor of these new species is E. eunicicola (16S rRNA gene identity: 96.68%, 96.99% respectively). The two isolates exhibit different physiological and biochemical characteristics (optimal pH, temperature and salinity). E. ruthgatesii 8E grows in on optimal temperature of 25 °C, with an optimal pH of 7 and salinity 1-2 %, whereas E. penghuensis 4G grows at a temperature of 20-25 °C, a pH, and salinity of 8 and 1-2%, respectively. Furthermore, we sequenced and assembled high-quality (contamination <1%) near-complete (>97%) draft genomes of the two species. E. ruthgatesii 8E has the largest genome (7.1 Mb) in cultured species of Endozoicomonas to date, but E. penghuensis 4G has a genome size (5.73 Mb) comparable other species. E. ruthgatesii 8E harbors a putative operon to metabolize DMSP to DMS and use it as a carbon source. Quantification of DMSP usage by E. ruthgatesii 8E shows that it can metabolize more DMSP than previously characterized E. acroporae. Further, genomic analysis is being conducted to answer the intriguing question, why does E. ruthgatesii 8E have a disproportionate genome size. Overall, in this study we discovered two novel Endozoicomonas species and is the first study to quantify DMSP consumption and DMS production by a coral-associated dominant bacterium.