Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or lodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides.Little is known about the surface or outer membrane structures of Vibrio cholerae. V. cholerae cells have two types of appendage: a single, polar flagellum enclosed in a sheath which appears contiguous with the outer membrane (5), and pili (1,36). The lipopolysaccharide of V. cholerae is chemically different from that of the family Enterobacteriaceae (13). V. cholerae cytoplasmic membrane and outer membrane are not differentially solubilized by Triton X-100 extraction (18), as are the membranes of Escherichia coli and Pseudomonas aeruginosa. Furthermore, the electrophoretic profile of V. cholerae outer membrane proteins in polyacrylamide gels is distinct from those of other gram-negative organisms. These studies and others (16,17,34) indicate that the outer membrane of V. cholerae is a complex structure containing many proteins.Proteins exposed on the bacterial surface are likely to contribute to colonization of the host, and improved knowledge of the surface proteins should further our understanding of their role in colonization. The surface proteins of several bacterial species have been identified by iodination with membrane-impermeable enzymes (15,20,29,31) or the water-insoluble reagent, 1,3,4,6-tetrachloro-3,6-diphenylglycouril (Iodogen) (33). We used lodogen-coated vessels and a covalently immobilized catalyst (lodo-beads) to radioiodinate whole cells and isolated outer membrane of V. cholerae. The major surface proteins on V. cholerae accessible to radioiodination were identified and shown to correspond, with a single exception, to the proteins in radiolabeled outer membrane obtained by lithium chloride-lithium acetate extraction (14). Outer membrane obtained by sucrose density centrifugation and Triton X-100 extraction (18) of labeled whole cells was shown to closely resemble the lithium-extracted outer membrane.MATERIALS AND METHODS Bacterial strains and media. V. cholerae CA401, originally obtained from C. Lankford (7) and characterized in earlier studies (2,3,9,18), was stored at -70°C in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) contain-* Corresponding author. t Present address: Center for Vaccine Development, University of Maryland Me...