The role of FlbD in regulation of flagellar gene transcription in Caulobacter crescentus

The flagellar (fla) genes in Caulobacter crescentus are organized into a regulatory hierarchy of four levels (I-IV) in which transcription of the class III and class IV genes late in the cell cycle from σ⁵⁴-dependent promoters depends on expression of the class II genes above them. The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. We have been particularly interested in understanding the function and regulation of one such transcription factor, FlbD. FlbD belongs to the NtrC family of bacterial response regulators that catalyse the initiation of transcription by σ⁵⁴ RNA polymerase (Eσ⁵⁴) and its function is required for transcription of the class III and IV fla genes. Here we show that purified FlbD binds to ftr elements that are required for transcription from the σ⁵⁴-dependent class III flbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4). Dimethylsulphate footprinting assays demonstrated that FlbD makes base-specific contacts at highly conserved guanine nucleotides in each half site of the ftr sequences. In a reconstituted in vitro transcription system using E. coli Eσ⁵⁴, we found that FlbD was clearly capable of driving transcriptional initiation from the flbG promoter and that this activity relied on the ftr1 binding site. Several observations suggest that phosphorylation plays a role in the regulation of FlbD activity. First, we found that a mutant form of FlbD (FlbD^S140F) corresponding to the substitution found in a constitutively active NtrC protein (NtrC^S160F), displayed a greater potential for activating Eσ⁵⁴-dependent transcription than the wildtype protein. We also observed that high energy-phosphate-containing molecules stimulate transcription activation by the wild type FlbD. Together, these results suggest that FlbD is responsible for mediating fla gene transcription initiation by Eσ⁵⁴ and that covalent modification is likely to play a role in governing FlbD activity during the cell cycle.