[A-OS17] Marine microbial ecology

[AOS17-10] Heterogeneous cellular phycoerythrin allocation in the unicellular nitrogen-fixing cyanobacterium, Crocosphaera watsonii

*Takako Masuda^1,2, Grzegorz Konert^2,3, Radek Kaňa², Ondřej Prásil² (1.FRA, 2.CAS, 3.Univ. Turku)

Keywords:Cyanobacteria, Nitrogen fixation, Phycoerythrin, Heterogeneity

Nitrogen, along with iron, is the most important rate-limiting factor in marine primary production. In particular, in subtropical oligotrophic oceans where year-round stratification is well developed and nitrate nitrogen is not easily supplied to the surface from the euphotic zone, cyanobacteria with nitrogen fixing ability that reduces extraneous nitrogen gas to ammonium salts are the starting point of the marine nitrogen cycle and support marine production. The unicellular cyanobacterium Crocosphaerea watasonii is widely distributed in tropical and subtropical oceans. C. watsonii is characterized by a high concentration of phycoerythrin, which is composed of phycoerythrobilin and phycourobilin that absorb short-wavelength light. However, its role is unclear. In this study, C. watsonii was cultured in YBCII medium under 50 µmol photons m^-2 s^-1 12L:12D light conditions and diluted to 1 × 10⁶ cells mL^-1 at least three times a week before observation, and the intracellular distribution of photosynthesis-related proteins was observed three days after dilution. To clarify the effect of the nitrogen source, an ammonium-added group was also prepared, and the same acclimation as above was performed before observation. To observe the intracellular distribution of photosynthesis-related proteins, we first performed three-channel imaging using a confocal microscope (LSM880, Zeiss): (1) phycobilisome fluorescence when phycocyanin was excited (Ex. 633 nm, Em. 641-677 nm), (2) phycoerythrin fluorescence when chlorophyll and phycourobilinin were excited (Ex. 488 nm, Em. 490-758 nm), and (3) chlorophyll fluorescence when chlorophyll and phycourobilinin were excited (Ex. 488 nm, Em. 695-758 nm). The difference in distribution of these signals determined whether phycoerythrin was connected to phycobilisomes or whether phycobilisomes were connected to photosystem II on the thylakoid membrane. If they were not connected, it was determined that phycoerythrin was not providing light energy for photosynthesis. Chlorophyll was excited and lambda scanning was performed in the range of 410 - 495 nm to obtain fluorescence spectra. Observations were performed every 3 - 12 hours for 24 hours, and 58 - 255 cells were analyzed.
Three-channel imaging revealed that the distribution of photosynthesis-related proteins in the cells was localized, with only limited overlap between chlorophyll, phycobilisomes, and phycoerythrin. This suggests that the phycoerythrin involved in photosynthesis is very limited. Furthermore, the intracellular distribution of photosynthesis-related proteins varied between cells and was different between nitrogen-fixing and ammonium-fixing environments. When cultured in a nitrogen-fixing environment, phycoerythrin was often observed to exist alone in the center of the cell, away from the thylakoid membrane. On the other hand, in cells cultured under an ammonium environment, the combination of photosynthesis-related proteins that predominate during the light period differed from cell to cell, and in many cells, the overlap of chlorophyll, phycobilisomes, and phycoerythrin was localized, and the combination of photosynthetic proteins that predominate was chlorophyll, phycobilisomes, or phycobilisomes and phycoerythrin. Interestingly, during the dark period, chlorophyll and phycobilisomes were distributed at the cell periphery, while phycobilisomes and phycoerythrin were distributed inside, revealing that photosynthetic proteins move within the cells during the day and night. This result indicates that only a portion of phycoerythrin is used for photosynthesis, regardless of the chemical form of nitrogen available.
It has been revealed that some cells of C. watsonii cease nitrogen fixation, and it is thought that the presence of cells that cease nitrogen fixation allows the community to save energy and expand its distribution to deeper layers. On the other hand, phycoerythrin is known to absorb the light energy necessary for photosynthesis and also to act as a nitrogen source, but its new role as an antioxidant has also become clear. In this presentation, we will try to discuss the relationship between the ON/OFF of nitrogen fixation and phycoerythrin.