Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Abstract

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models.Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis.Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

Novel protein carrier system based on cyanobacterial nano‐sized extracellular vesicles for application in fish

“…A total volume of 3.6 l of extracellular medium, unless stated otherwise, was filtered through 0.45 μm pore size filters, and further concentrated by ultrafiltration using centrifugal filters with a molecular weight cut‐off of 100 kDa (Millipore, Burlington, MA, USA). A final volume of approximately 15 ml of concentrated extracellular medium was then centrifuged at 100 000 g for 3 h, at 4°C, as previously described (Biller et al ., 2022b). The resulting EVs pellets were then suspended in sterile BG11 medium and stored at −80°C until further analysis.…”

Characterization of membrane vesicles inAlteromonas macleodiiindicates potential roles in their copiotrophic lifestyle

“…The production of MVs has been previously investigated in isolates of several pelagic marine bacteria, such as Prochlorococcus (Biller et al . 2014, 2021), Pelagibacter (SAR11; Biller et al . 2022), and Shewanella (Frias et al .…”

“…The MVs were isolated following the experimental guidelines for definition of EVs and their functions of the International Society for Extracellular Vesicles (Lötvall et al . 2014), and following Biller et al (2022) for filtration size cut-offs for marine bacteria.…”

“…2022). However, to date, MV production has only been shown for few marine bacterial isolates (Biller et al . 2022).…”