Interaction of the Atypical Prokaryotic Transcription Activator FlhD2C2 with Early Promoters of the Flagellar Gene Hierarchy

The transcriptional activator FlhD2C2 is the master regulator of bacterial flagellum biogenesis and swarming migration, activating the “early” class II promoters of the large flagellar gene hierarchy. Using primer extensions, band-shift assays, and enzymatic and chemical footprinting, we describe the binding of the FlhD2C2 heterotetramer to the promoter regions of four class II flagella operons, fliAZ, flhBA and the divergent flgAMN and flgBCD(EFGHIJ). Each of the promoter regions was bound by a single heterotetramer, i.e. the flgAMN and flgBCD operons are characterised by a single FlhD2C2 binding site. Binding affinity differed, and correlated with previously reported promoter strength and order of activation. Methylation protection and interference, and depurination and depyrimidation interference provided a detailed map of critical bases within a common 46–59 bp DNaseI footprint overlapping the promoter −35 sequences. These data and compilation of the 12 known class II promoter sequences of Escherichia coli, Proteus mirabilis and Salmonella typhimurium allowed determination of a FlhD2C2 binding site with pseudo symmetry, comprising two 17–18 bp inverted repeats, each a consensus FlhD2C2 box, separated by a 10–11 bp spacer. DNaseI hypersensitivity indicated that binding may cause a conformational change in the promoter regions. Only the FlhC subunit can bind DNA independently, but the specificity and stability of the interaction is strengthened by FlhD. Here, photo-crosslinking established that both FlhC and the stabilising FlhD contact the DNA within the FlhD2C2 tetramer. Our data suggest that specificity of recognition and stability of the FlhD2C2/DNA complex require protein–protein interaction and interaction of both FlhC and FlhD subunits with DNA. These characteristics of the FlhD and FlhC subunits in the FlhD2C2/DNA complex are strikingly atypical of prokaryotic regulators.