Loss of PefI-SrgD induces transient but sufficient Class II/III and III activation to restore flagellar biosynthesis in Δ hha Δ ydgT. Promoter activity at each transcriptional class in wild type, Δhha ΔydgT, ΔpefI-srgD and Δhha ΔydgT ΔpefI-srgD was measured as fluorescence intensity using plasmid-based GFP reporters. A promoterless GFP reporter construct was used as a negative control (first panel). Fluorescence intensity (485/525 nm) and OD600 was measured at 15 min intervals over 19 h. Data represents fluorescence intensity normalized to OD600 (RLU/OD600). GFP transcriptional reporter assay data is representative of three independent experiments and quantified as means and standard errors (at the 3 h time point for PflhD, P < 0.05 for wt vs. Δ pefI-srgD and wt vs. Δhha ΔydgT ΔpefI-srgD; ANOVA, Newman-Keuls multiple comparison test). After 3-5 hours, PflhD-gfp activity in the quadruple mutant reached the maximum detection limit of the fluorescence reader. Data is shown for 12 hours rather than for 19 hours for the remaining flagellar reporters as there was no change in the fluorescence levels from 12-19 hours.