FIG. 1.

Purification of the EPEC NCs. (A) Purification scheme for the EPEC NCs. Whole-cell lysate was prepared from an EPEC ΔespA/A strain grown in DMEM, and the lysate was clarified by centrifugation. The resulting supernatant fraction (sup fr.) was ultracentrifuged, and then a pellet fraction (ppt fr.) (NC fr. I) was recovered. NC fr. I was layered on a sucrose cushion and then ultracentrifuged. The resulting pellet fraction (NC fr. II) was subjected to CsCl density gradient centrifugation, and then 20 fractions (CsCl frs. 1 to 20) were collected from the top of the gradient. For more details, see Materials and Methods. (B) Electron micrographs of negatively stained EPEC NCs. NC frs. I and II and CsCl fractions 3, 7, 11, and 14 were negatively stained and observed by TEM. White arrows indicate the basal bodies of the EPEC NCs, and black arrows indicate ring structures that were presumed to be the BfpB-BfpG complexes (29). Bar, 250 nm.