探討馴化於可見光或遠紅光的兩株藍綠菌在混合培養下的基因表現圖譜

Abstract

雖然大多藍綠菌生長於可見光 (λ = 400-700 nm)下（可見光藍綠菌），然而有部分藍綠菌可行遠紅光轉換（遠紅光藍綠菌），藉此吸收遠紅光 (λ = 700-750 nm)行光合作用。我們過去的研究發現當遠紅光藍綠菌與可見光藍綠菌共培養在石英砂管柱時，遠紅光藍綠菌會在管柱較深的區域行使遠紅光轉換，顯示共培養會使遠紅光藍綠菌的生長受到可見光藍綠菌的影響，然而兩種藍綠菌之間是否有交互作用，以及在共培養情況下會有何種反應尚不清楚。 本研究發現共培養對於遠紅光藍綠菌Chlorogloeopsis fritschii PCC 9212與可見光藍綠菌Synechocystis sp. PCC 6803在生長與基因表現圖譜上的影響。生理實驗結果顯示，Synechocystis sp. PCC 6803的生長不論在何種光源下皆不受共培養影響，而Chlorogloeopsis fritschii PCC 9212的生長則會在可見光下受共培養抑制。轉錄體分析顯示Chlorogloeopsis fritschii PCC 9212在可見光下共培養時，離子通道相關基因的表現會顯著上升，而代謝相關基因表現則顯著下降。此外與遠紅光轉換相關的基因表現也在遠紅光下較可見光下顯著上升。另一方面Synechocystis sp. PCC 6803則在遠紅光下共培養時，II型分泌系統以及和壓力反應相關的蛋白質摺疊的基因表現量有顯著上升的趨勢；此外Synechocystis sp. PCC 6803在遠紅光下代謝相關基因表現也顯著低於在可見光下的表現。藍綠菌在共培養下生長受抑制與代謝相關基因的低表現說明Chlorogloeopsis fritschii PCC 9212與Synechocystis sp. PCC 6803之間可能存在著負向交互作用。 透過基因表現圖譜的分析，可以發現在Synechocystis sp. PCC 6803與Chlorogloeopsis fritschii PCC 9212之內可能有數個與共培養相關的基因，如在Synechocystis sp. PCC 6803中與II型分泌系統有關的gspH，以及在Chlorogloeopsis fritschii PCC 9212中與硫酸鹽離子運輸相關的基因。為了進一步了解這些基因在共培養之中參與的功能，需要進一步使用基因編輯技術，而CRISPR-Cpf1正是一種可以運用於藍綠菌的基因編輯方法。然而由於CRISPR-Cpf1尚未於我們實驗室以及遠紅光藍綠菌內實際運用，因此做為測試，我們選擇將Synechococcus sp. PCC 7335的cpeBA基因進行剔除。 研究結果顯示了CRISPR-Cpf1可以在Synechococcus sp. PCC 7335上應用，其中有超過95%的菌落的cpeBA基因被成功剔除。這份技術將可以進一步應用於Synechocystis sp. PCC 6803與Chlorogloeopsis fritschii PCC 9212來研究與共培養相關的基因。 總體來說，本研究讓我們更加了解共培養對於遠紅光藍綠菌與可見光藍綠菌在基因表現圖譜上的影響。此外本研究也成功建立了一個可以利用CRISPR-Cpf1來進行藍綠菌基因編輯的方式，這些發現讓我們對於藍綠菌的共培養有更多理解，也有助於未來與共培養相關的基因的研究。

Although most cyanobacteria grow in visible light (VL, λ = 400-700 nm), some cyanobacteria can perform far-red light photoacclimation (FaRLiP) to use far-red light (FRL, λ = 700-750 nm) for oxygenic photosynthesis. Our previous studies show that when FRL-using cyanobacteria are cocultured with VL-using cyanobacteria in a sand column, FRL-using cyanobacteria distribute at deeper depths by performing FaRLiP. This finding suggests that coculture affects the growth of FRL-using cyanobacteria. However, it remains unknown whether FRL-using cyanobacteria and VL-using cyanobacteria have interactions and how FRL-using cyanobacteria respond in the coculture. In this study, we reveal the coculture effects on the growth and the gene expression profiles of an FRL-using cyanobacterium, Chlorogloeopsis fritschii PCC 9212, and a VL-using model cyanobacterium, Synechocystis sp. PCC 6803. Specifically, the results showed no significant physiological difference in Synechocystis sp. PCC 6803 between the coculture and the monoculture. On the contrary, the growth of Chlorogloeopsis fritschii PCC 9212 was suppressed in VL under coculture conditions. When Chlorogloeopsis fritschii PCC 9212 was cocultured with Synechocystis sp. PCC 6803 in VL, transcriptomic data suggested that in Chlorogloeopsis fritschii PCC 9212, the transcript levels of ion transporters were higher and metabolic processes were lower compared to monoculture. In addition, the transcript levels of the genes for FaRLiP were higher in FRL than in VL. In Synechocystis sp. PCC 6803 under FRL condition, the transcript levels of the type II secretion system and the stress responses, including protein folding, in the coculture were higher than in the monoculture. Moreover, the transcript levels of metabolic processes in FRL were lower compared to VL. The suppression of cyanobacterial growth and the low transcript level of metabolic processes in coculture suggest a potentially negative interaction between Chlorogloeopsis fritschii PCC 9212 and Synechocystis sp. PCC 6803 in VL. Through the analysis of gene expression profiles, several genes associated with coculture were identified in Synechocystis sp. PCC 6803 and Chlorogloeopsis fritschii PCC 9212, including gspH, which is involved in type II secretion in Synechocystis sp. PCC 6803, and genes related to sulfate transporters in Chlorogloeopsis fritschii PCC 9212. Further study on the function of these coculture-related genes requires gene editing, and CRISPR-Cpf1 is an available gene editing method in cyanobacteria. However, since CRISPR-Cpf1 has not been previously applied in our lab or FRL-using cyanobacteria, we selected the cpeBA operon in Synechococcus sp. PCC 7335 as a test case for knockout. Our study successfully demonstrates the application of the CRISPR-Cpf1 gene editing workflow in Synechococcus sp. PCC 7335, resulting in the knockout of the cpeBA operon in over 95% of the cell colonies. This workflow can be further applied to Synechocystis sp. PCC 6803 and Chlorogloeopsis fritschii PCC 9212 for future investigation of coculture-related genes. Overall, our study gives an insight into how coculture affects the gene expression profiles of both FRL-using cyanobacteria and VL-using cyanobacteria under VL and FRL. Additionally, we have successfully established a workflow for gene editing in cyanobacteria using CRISPR-Cpf1. These findings contribute to our understanding of the cyanobacterial coculture and pave the way for further investigations on coculture-related genes.