FIG 3.

The relA mutant is comprised of mostly flagellated cells (A) Representative images of mid-log-phase cultures of wt (BQA057) and ΔrelA (BQA062) strains. The membranes were stained with FM4-64 (red), and flagellin (Hag) was stained with Alexa Fluor 488 C₅ maleimide (green). Both strains harbor a point mutation in the chromosomal copy of hag that creates Hag_T209C. (B) Comparison of flagellin (Hag) protein levels in wt (BQA057), ΔrelA (BQA062), ΔrelA P_relA-relA (BQA080), and Δhag (BQA076) strains. Samples were collected from mid-log-phase cultures grown in CH medium and stained with Alexa Fluor 488 C₅ maleimide. Proteins from cell lysates were separated by SDS-PAGE, stained with Coomassie blue (left), and scanned with a laser scanner to visualize fluorescently labeled protein (right). The arrowhead indicates flagellin. (C) (Top) Western blot analysis showing flagellin levels associated with the culture supernatants (left; S) and cell pellets (right; P) of the indicated strains grown in CH medium to mid-log phase: wt (BJH001), ΔrelA (BQA009), ΔrelA P_relA-relA (BQA068), ΔsigD (BQA022), and Δhag (BQA076). It was necessary to dilute the cell pellet lysates to the indicated dilutions in order to assess the levels of protein associated with each sample without overexposure. (Bottom) SigA protein served as a loading control for the cell pellet samples.