Fig. 1.

Analysis of the requirement of FlgR/DNA interactions for σ⁵⁴-dependent flagellar gene expression. (A) Domain organization of E. coli NtrC, C. jejuni WT FlgR, and C. jejuni FlgR_ΔCTD. The phosphorylated aspartic acids (D or D51; P denotes phosphoryl group) in receiver domains are indicated. (B and C) EMSAs to analyze P_flgDE2 DNA binding by FlgR proteins. (B) WT FlgR and FlgR_ΔCTD were used (from left to right) at 0, 0.1, 0.25, 0.5, 0.75, and 1 μM. (C) Increasing ratios of unlabeled P_flgDE2 DNA (1:1, 5:1, or 10:1) or P_gyrA DNA (1:1 or 10:1) to labeled P_flgDE2 DNA were incubated with 1 μM of FlgR proteins. (D) Expression of flgDE2::astA with 5′ promoter truncations in WT C. jejuni or flgR_ΔCTD, ΔrpoN (Δσ⁵⁴), or ΔflgR mutants. flgDE2::astA expression levels are reported as arylsulfatase units ± SD. All transcriptional fusions were on plasmids, except for the fusion at the native chromosomal flgDE2 locus (chr). The 5′ ends of fusions on plasmids are indicated relative to the transcriptional start site (arrow). Black boxes indicate the −24 and −12 σ⁵⁴ binding sites essential for σ⁵⁴-dependent gene expression.