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Abstract Chloroplasts are thought to have co-evolved through endosymbiosis, after a cyanobacterial-like prokaryote was engulfed by a eukaryotic cell; however, it is impossible to observe the process toward chloroplasts. In this study, we constructed an experimental symbiosis model to observe the initial stage in the process from independent organisms to a chloroplast-like organelle. Our system of synthetic symbiosis is capable of long-term coculture of two model organisms: a cyanobacterium (Synechocystis sp. PCC6803) as a symbiont and a ciliate (Tetrahymena thermophila) as a host with endocytic ability. The experimental system was clearly defined, because we used a synthetic medium and the cultures were shaken to avoid spatial complexity. We determined the experimental conditions for sustainable coculture, by analyzing population dynamics using a mathematical model. We experimentally demonstrated that the coculture was sustainable for at least 100 generations, through serial transfers. Moreover, we found that cells isolated after the serial transfer improved the probability of coexistence of both species without extinction in re-coculture. The constructed system will be useful for understanding the initial stage of primary endosymbiosis from cyanobacteria to chloroplasts, i.e., the origin of algae and plants.

Chloroplasts originated from cyanobacteria through endosymbiosis. The original cyanobacterial endosymbiont evolved to adapt to the biochemically rich intracellular environment of the host cell while maintaining its photosynthetic function; however, no such process has been experimentally demonstrated. Here, we show the adaptation of a model cyanobacterium, Synechocystis sp. PCC 6803, to a biochemically rich environment by experimental evolution. Synechocystis sp. PCC 6803 does not grow in a biochemically rich, chemically defined medium because several amino acids are toxic to the cells at approximately 1 mM. We cultured the cyanobacteria in media with the toxic amino acids at 0.1 mM, then serially transferred the culture, gradually increasing the concentration of the toxic amino acids. The cells evolved to show approximately the same specific growth rate in media with 0 and 1 mM of the toxic amino acid in approximately 84 generations and evolved to grow faster in the media with 1 mM than in the media with 0 mM in approximately 181 generations. We did not detect a statistically significant decrease in the autotrophic growth of the evolved strain in an inorganic medium, indicating the maintenance of the photosynthetic function. Whole-genome resequencing revealed changes in the genes related to the cell membrane and the carboxysome. Moreover, we quantitatively analyzed the evolutionary changes by using simple mathematical models, which evaluated the evolution as an increase in the half-maximal inhibitory concentration (IC50) and estimated quantitative characteristics of the evolutionary process. Our results clearly demonstrate not only the potential of a model cyanobacterium to adapt to a biochemically rich environment without a significant decrease in photosynthetic function but also the properties of its evolutionary process, which sheds light of the evolution of chloroplasts at the initial stage.