Isolation, Quantification, and Visualization of Extracellular Membrane Vesicles in Rhizobia Under Free-Living Conditions

Rhizobia are a group of soil proteobacteria that are able to establish a symbiotic interaction with legumes. These bacteria are capable to fix atmospheric nitrogen into ammonia within specific plant root organs called nodules. The rhizobia-legume interaction is established by a complex molecular dialogue that starts with flavonoids exudated by the plant roots. In response, signaling molecules known as Nod factors (NFs) are secreted by the bacteria. These factors are sensed by specific plant receptors that trigger a downstream signaling cascade leading to rhizobium-specific intracellular colonization of the root hair via the formation of infection threads and the eventual development of nodules on roots. In these organs, rhizobia can fix nitrogen from the atmosphere for the plant in exchange for photosynthates and the appropriate environment for nitrogen fixation. Recently, it has been demonstrated that extracellular membrane vesicles (EMVs) produced by some rhizobia carry NFs. EMVs are proteolipidic structures that are secreted to the milieu from the bacterial membranes and are involved in several important biological processes, including intercellular communication. Thus far, little is known about rhizobia vesicles, and further studies are needed to understand their functions, including their role as transporting vessels of signaling molecules during the process of symbiosis. Here, we present a detailed protocol to isolate high-purity EMVs from free-living cultured rhizobia, test their integrity, and quantify their abundance.