Enteropathogenic Yersinia sp. releases plasmid-associated proteins of low molecular mass (26-67 kilodaltons) at 37°C. In this study, the optimum conditions for the release of proteins were assessed and the released proteins (RPs) were analyzed for the manner of release, immunochemical characteristics, and the location of the genes necessary for their synthesis. Protein release was strongly enhanced when growth media were markedly depleted of calcium ions by precipitation with oxalate or chelation with EGTA [ethylene glycol-bis(paminoethyl ether)-N,N,N',N'-tetraacetic acid]. RP yields were greatest when Yersinia spp. were in the exponential growth phase. The RPs appeared to be released from the Yersinia spp. by secretion rather than by pinching off of membrane vesicles, because the RPs did not sediment during high-speed centrifugation nor were they contaminated to any signfficant degree with lipopolysaccharide. Moreover, immunoblot analysis revealed only traces of protein species related to RPs within the outer membranes of plasmid-positive Yersinia spp. grown at 37°C under calcium-restricted conditions. Immunoblot studies also showed that the RPs of Y. enterocolitica serotypes 0:3, 0:8, and 0:9 and the RP of Y. pseudotuberculosis serotype I are highly cross-reactive. Finally, the immunoprecipitates of the products of minicells which harbor Yersinia plasmids were used to demonstrate that at least three proteins immunochemically related to the released fraction were plasmid encoded. These results suggest that at least three of the RPs may be related to or identical with previously described plasmid-encoded Yersinia outer membrane proteins.MATERIALS AND METHODS Bacterial strains and culture conditions. Y. enterocolitica serotypes 0:3 (Y-108 Nalr, plasmid positive), 0:9 (Y-96 Nalr, plasmid positive), and 0:8 (WA-314 Nalr, plasmid positive, and its plasmid-negative derivative WA-cNalr) 561 on August 5, 2020 by guest