Population Dynamics in the Biogenesis of Single-/Multi-Layered Membrane Vesicles Revealed by Encapsulated GFP-Monitoring Analysis

Various generations of membrane vesicles (MV) have been observed in Escherichia coli in terms of triggering events and populations of single-layered (s)/multi-layered (m) forms. Previously, we proposed a novel mechanism for MV generation triggered by the intracellular accumulation of biopolyester polyhydroxybutyrate (PHB). This was designated as the Polymer Intracellular Accumulation-triggered system for Membrane Vesicle Production (PIA-MVP). Herein, we attempted to determine the conditions for the change in the population between s-MV and m-MV using glucose concentration-dependent PIA-MVP. PIA-MVP was established using the good correlation between the glucose concentration-dependent PHB accumulation and MV generation. Thus, we assumed the presence of a critical glucose concentration could determine the population ratio of s-MV to m-MV, indicating that s-MV generation is a dominant component in the extracellular environment. Cytoplasmic green fluorescent protein (GFP) was used to evaluate the glucose concentration, enabling the selective generation of s-MV. The glucose concentration was determined to be 15 g/L to satisfy this purpose under the culture conditions. In conclusion, we established a biological system allowing us to selectively generate both single- and multi-layered MVs based on PIA-VIP encapsulation of GFP, providing a versatile toolkit to gain insights into the MV generation mechanism and achieve progress in various engineering applications.