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

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.