FIG. 1.

Flagellin repression is caused by a protein that is absent from animal mucus and is inhibited by a serine protease inhibitor. (A) Anti-flagellin immunoblot of P. aeruginosa grown for 6 h in M63 (lane 1), mucus (lane 2), heat-inactivated mucus (lane 3), pronase-treated mucus (lane 4), bovine submaxillary mucus (lane 5), and porcine gastric mucus (lane 6). In each experiment approximately equal numbers of bacteria based on the optical density at 600 nm and viable counts were used for the Western blots. Molecular mass markers are shown on the right in kilodaltons. (B) The addition of the serine protease inhibitor PMSF to mucus reestablished flagellin synthesis. The P. aeruginosa PAKfliC::lacZ reporter strain was grown either in M63 or in mucus with (+) or without (−) 1 mM PMSF for 6 h. The addition of mucus yielded a significant (P < 0.0005) reduction in fliC expression. On the other hand, the addition of PMSF to mucus significantly increased fliC transcription. Error bars indicate the means ± the SD. The values given were obtained from three independent experiments. The corresponding flagellin immunoblot is shown on the top. (C) The addition of PMSF reestablished flagellin synthesis in other P. aeruginosa strains. Lane 1, M63; lane 2, mucus; lane 3, mucus with PMSF.