Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide

Abstract

Bacteria secrete extracellular membrane vesicles (EMVs). Physiology and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the molecular basis of EMV biogenesis has not yet been fully elucidated. In the previous research, an N-terminus-substituted FAAV peptide labeled with NBD (nFAAV5-NBD) was developed, which can sense a curvature of a lipid bilayer and selectively bind to EMVs in the presence of the cells. Herein, we applied nFAAV5-NBD to a screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. As a result, for 16 and six genes, we found that transposon insertion within or near the gene caused hyper- and hypo-vesiculation, respectively. Targeted gene-disrupted mutants of the identified genes demonstrated that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase {beta}-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, D-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study shows the utility of a novel screening method using a curvature-sensing peptide and fundamental information on the genes related to EMV production. IMPORTANCEConventional methods for isolation and quantification of EMVs are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying a number of genes involved in the EMV production of S. vesiculosa HM13, and the strains obtained in this study would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be useful in the application of this bacterium, such as in the production of useful substances as cargoes of EMVs and in the production of surface-engineered vesicles.