FIG 6.

Secretion of FlgJ into the supernatant is dependent on PL-ring formation. Liquid culture of TH20753 (flgJ8021::3×HA ΔflgHI958 ΔaraBAD941::flgH⁺I⁺ P_flhDC8089::tetR P_tetA fljB^enx vh2) was grown to an OD₆₀₀ of ∼0.3, at which point anhydrotetracycline (ATc) was added to induce flhDC expression and basal body assembly. After 10 min of incubation with ATc, arabinose (Ara) was added to the culture to induce expression of flgH⁺. Samples were collected at 10-min intervals. Translation was arrested by the addition of spectinomycin, and the cells were placed on ice. Pellet and supernatant samples from each time point were subjected to Western blot analysis with probing for FlgJ-HA with anti-HA antibody. Time point zero (t₀) represents samples taken after 10 min of incubation with ATc immediately prior to addition of Ara to the culture. (A) Levels of FlgJ-HA in the whole-cell lysates remained constant for the duration of the experiment. The upper band is full-length FlgJ-HA, and the lower band is presumed to be partially degraded FlgJ-HA. (B) In the supernatant samples, FlgJ-3×HA began to accumulate at ∼30 min after the addition of arabinose and continued to accumulate throughout the remainder of the experiment. In the supernatant, FlgJ-HA is present in the full-length form only. The supernatant of the sample that received ATc and saline (t₇₀ + ATc/−Ara) had no detectable FlgJ-HA.