Analysis of E. coli recombinant strains. Panel A: Whole cell lysates were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with Abs against BpaC. Lane 1, E. coli (pCC1.3); lane 2, E. coli (pCCbpaC). MW markers are shown to the left in kilodaltons. Panel B: Non-permeabilized E. coli strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) and with α-BpaC Abs (red). Bacteria were visualized by microscopy using a Zeiss LSM 510 Meta confocal system. Representative microscopic fields are shown. Panel C: E. coli strains were incubated with epithelial cells for 3-hr. Cells were then washed to remove unbound bacteria, lysed, diluted and spread onto agar plates to enumerate bound bacteria. The results are expressed as the mean percentage (±standard error) of inoculated bacteria attached to epithelial cells. Asterisks indicate that the increased adherence of E. coli (pCCbpaC), compared to that of E. coli carrying the control plasmid pCC1.3, is statistically significant (P value shown in parentheses). Adherence assays were performed in duplicate on at least 4 independent occasions.