Gram-negative bacteria, including Escherichia coil, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell-cell communications and host-microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coil W3110, which is a widely used standard E. coil K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coil W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coil strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coil W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs. (C) 2013 Elsevier Inc. All rights reserved.