FIG. 1.

Western blot analysis of flagellum expression in P. aeruginosa strains. Whole-cell extracts were prepared from P. aeruginosa strains by culturing cells in 10 ml of LB lacking NaCl at 37°C to an A₅₈₀ of 0.4. The culture was centrifuged (5,000 × g for 10 min), and pellets were suspended in 2% of the original culture volume in fractionation buffer (10 mM Tris HCl [pH 8.0], 100 mM NaCl, 1 mM MgCl₂). A 10-μl sample of this preparation was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting with anti-flagellum B antibodies. Polyclonal antiserum against flagella was elicited in New Zealand White rabbits (Covance) using flagella (0.75 mg) purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Anti-flagellum antibodies were used in Western blots at a dilution of 1:25,000 with chemiluminescent reagents by procedures outlined by the manufacturer (Amersham), and film was exposed for 30 s prior to development. Lane 1, flagella B (250 ng) purified from PAO1. Lanes 2 through 11 contain extracts derived from strains FRD1 (mucA22), FRD875 (mucA22 algD::xylE aacC1), FRD440 (mucA22 algT::Tn501), FRD444 (mucA22 algB::Tn501), FRD810 (mucA22 algR::Ωstr), FRD831 (mucA22 ΔalgR::ΩaacC1), FRD1230 (mucA22 fliC::xylE aacC1), FRD1234 (mucA22 algT::Tn501 fliC::xylE aacC1), FRD1240 (mucA22 algT::Tn501 rpoN::xylE aacC1), and FRD1242 (mucA22 rpoN::xylE aacC1), respectively. The mucoid and motility phenotypes of the strains analyzed in each lane are depicted along the bottom. Motility assays were performed by inoculating a single colony into 0.3% L agar lacking sodium chloride. Following overnight growth at 37°C, motility was assessed qualitatively by examining colonies which spread beyond the point of inoculation (2).