FIG. 7.

W83 retains the ability to assemble 33277-type FimA fimbriae. (A) Construction of fimA-exchanged W83 variant W83fA327. A chimeric DNA fragment comprised of the C-terminal coding region of W83 pgmA, full-length 33277 fimA, the N-terminal coding region of W83 fimB, and intergenic sequences from W83 was generated by a PCR-based overlap extension, as described in Materials and Methods. After the insertion of an ermF cassette, the final construct was introduced into W83 by electroporation. (B) Confirmation of allelic exchange by genomic DNA PCR. Lane 1, amplicons with primers a and f (1,968 bp + 1,095 bp of ermF = 3,063 bp; this amplicon also was sequenced); lane 2, amplicons with specific primers for 33277 fimA (700 bp); lane 3, no 700-bp amplicons with W83 fimA-specific primers. (C) Complementation of the fimA-exchanged W83 with the functional 33277 fimS gene is sufficient for the assembly of FimA fimbriae. The upper panel shows a Western blot of 80°C heat-treated whole-cell proteins from 33277 (lane 1), W83 (lane 2), W83fA327 harboring pTCBex (lane 3), and W83fA327 harboring pTCBex33277fimS (lane 4) probed with anti-33277 FimA antibody. Ladder signals are from polymerized FimA. An arrow indicates the signals from 41-kDa monomers. Lower panels represent the expression profile of each strain by RT-PCR. (D) Morphological observation of fimbrial assembly by transmission electron microscopy. The upper panel shows fimbriate W83fA327 harboring pTCBex33277fimS. The lower panel shows a fimbria-less W83 derivative expressing W83 fimA and 33277 fimS (i.e., strain WFS1 harboring pTCBex33277fimS). Scale bar, 200 nm.