A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, IptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs. (C) 2020 Elsevier Inc. All rights reserved.