Extracellular vesicle-mediated secretion of chlorophyll biosynthetic intermediates in the cyanobacterium Leptolyngbya boryana

Extracellular vesicles (EVs) are derived from the outer membrane (OM) in Gram-negative bacteria and have diverse physiological functions such as toxin transport, extracellular signal transduction, nutrient acquisition, and horizontal gene transfer. EV-mediated secretion of monovinyl protochlorophyllide (MV-Pchlide), the chlorophyll a (Chl) biosynthetic intermediate, was previously reported in a mutant lacking dark-operative Pchlide reductase in the cyanobacterium Leptolyngbya boryana. This study showed a detailed characterization of EVs from the wild-type (WT) of L. boryana grown under photoautotrophic and dark heterotrophic conditions, focusing on the accumulation of Chl intermediates. WT L. boryana cells produce two types of EVs, low-density EVs (L-EVs) and high-density EVs (H-EVs), both under light and dark conditions. L-EVs and H-EVs showed distinct morphological features and protein compositions. L-EVs from cells grown under both light and dark conditions commonly contained carotenoids, myxol glycoside, and zeaxanthin, as major pigments. Based on the protein compositions of EVs and other cellular membrane fractions, L-EVs and H-EVs are probably derived from low-density OM and high-density OM interacting with cell walls, respectively. Fluorescence detection of pigments was applied to EVs, and the three Chl intermediates, protoporphyrin IX, demetallated Mg-protoporphyrin IX monomethyl ester, pheophorbide, and were commonly detected both L-EVs from light- and dark-grown cells, whereas L-EVs from dark-grown cells contained additional MV-Pchlide and MV-protopheophorbide. These Chl intermediates appear to transfer from the thylakoid membrane to L-EVs via an unknown transport system. Cyanobacterial EVs may play a novel function in alleviating the accumulation of Chl intermediates in cells.