Analysis of recombinant E. coli strains. Panel A: Total RNA was isolated from E. coli strains, reverse-transcribed to cDNA, and the relative levels of boaA or boaB transcript were determined by qRT-PCR. Each bar represents 4 different samples collected on 2 separate occasions. The Y-axis corresponds to the levels of boaA or boaB transcript normalized to recA and the error bars correspond to the standard error. Negative controls in which the reverse transcriptase enzyme was not added to reaction mixtures were included in all experiments (data not shown). Panel B: Proteins present in Sarkosyl-insoluble OM protein preparations were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with antibodies against BoaA (lanes 1-3) or BoaB (lanes 4-6). Lanes 1 & 4, E. coli (pCC1.3); lanes 2 & 5, E. coli (pSLboaB); lanes 3 & 6, E. coli (pSLboaA). MW markers are shown to the left in kilodaltons. Panel C: Non-permeabilized E. coli strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) and with α-BoaA or α-BoaB antibodies (red). Bacteria were visualized by microscopy using a Zeiss LSM 510 Meta confocal system. Representative microscopic fields are shown. Panel D: E. coli strains were incubated with A549 and HEp2 cells for 3-hr and with NHBE cultures for 6-hr. Epithelial cells were 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 adhering to epithelial cells. Asterisks indicate that the increased adherence of the indicated strains, compared to E. coli carrying the control plasmid pCC1.3, is statistically significant (P < 0.05). These attachment assays were performed in duplicate on at least 3 separate occasions.