荧光标记珊瑚组织来源细菌及其与虫黄藻相互作用的显微观察

Labeling a coral tissue-derived bacterium with stable fluorescence and microscopic observation of the interaction between the labeled strain and Symbiodiniaceae

摘要:

【背景】研究珊瑚-细菌、虫黄藻-细菌的相互作用是解析珊瑚健康机理的关键。对珊瑚共附生细菌进行稳定荧光标记有助于原位观察细菌与虫黄藻或珊瑚的相互作用。当前，对于野生型珊瑚共附生细菌遗传操作体系的研究有限，限制了对细菌与珊瑚、虫黄藻原位互作模式的揭示。【目的】建立一种适合专性海洋细菌的遗传操作体系，利用其对珊瑚组织来源细菌进行绿色荧光蛋白标记，用于研究标记菌株与虫黄藻的相互作用。【方法】通过电穿孔的方式将构建好的广宿主重组质粒转入供体菌(Escherichia coli WM3064)，然后将供体菌与添加海水才可以生长的受体菌SCSIO 12696 (港口球菌科，Porticoccaceae；分离自鹿角杯形珊瑚组织)按供、受体菌细胞数比分别为4:1、2:1、1:1比例混合，在25 °C和30 °C下于改良LB培养基上接合转移。显微观察标记细菌与虫黄藻相互作用。【结果】改良的LB培养基适用于需海水才可生长的专性海洋细菌的接合转移实验。接合转移的效率与供、受体菌的比例及温度有关。确定优化的接合转移条件为：供、受体菌的比例为1:1，温度为30 °C。利用建立的接合转移体系，构建了增强型绿色荧光蛋白标记菌株SCSIO 12696。在激光扫描共聚焦显微镜下能清晰观察到标记菌株SCSIO 12696和虫黄藻在共培养时的相互作用。【结论】建立了适合专性海洋细菌的遗传操作体系，利用其构建荧光蛋白标记菌株可用于虫黄藻-细菌、珊瑚-细菌相互作用的研究，有助于揭示珊瑚共附生细菌的生态作用机制。

Abstract:

[Background] Investigations of coral-bacterial and Symbiodiniaceae-bacterial interactions are crucial for unravelling the mechanisms of coral health maintaining. Labeling coral-associated bacteria with stable fluorescence is useful for visualization of interactions between bacteria and Symbiodiniaceae or coral host in real time. Presently, the lack of the genetic manipulation system of coral-associated bacteria hampers the research on coral-bacterial and Symbiodiniaceae-bacterial interactions. [Objective] We tried to establish a genetic manipulation system that is suitable for obligate marine bacteria, and then label a coral-associated bacterial strain with an enhanced green fluorescent protein gene (eGFP). The interaction between eGFP-labeled strain and Symbiodiniaceae can be visualized. [Methods] The recombinant plasmid was transferred to the donor strain (Escherichia coli WM3064) by electroporation. The donor strain was mixed with the recipient strain SCSIO 12696 (Porticoccaceae, isolated from the tissue of coral Pocillopora damicornis), growth of which requires seawater, at different cell amount ratios (4:1, 2:1, 1:1). The mixed cells were then cocultured on the modified LB medium at 25 °C and 30 °C. The interaction between Symbiodiniaceae and the eGFP-labeled strain was observed by using microscopy. [Results] The modified LB medium met the growth requirement of the obligate marine bacterium SCSIO 12696, and was preferably used in the conjugational transfer experiment. The efficiency of conjugational transfer was influenced by the ratio of the donor and recipient bacterial cells and the incubation temperature. The optimum incubation temperature was 30 °C, and the optimum ratio of donor to recipient bacterial cells was 1:1. Strain SCSIO 12696 was labeled with an eGFP gene by using the conjugational transfer approach established in this study. The interaction between eGFP-labeled SCSIO 12696 and Symbiodiniaceae was clearly observed by using confocal laser scanning microscopy. [Conclusion] A genetic operation system that is suitable for the obligate marine bacteria has been established. The coral-associated bacterial strains can be labeled with fluorescent proteins by using this system. Moreover, the fluorescence-labeled bacteria can be used for investigations of the Symbiodiniaceae-bacterial and coral-bacterial interactions, which is helpful to revealing the ecological function of coral-associated bacteria and its mechanism.