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 o f the class III and class IV genes late in the cell cycle from sigma(5 4)-dependent promoters depends on expression of the class II genes abo ve them. The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factor s. 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 in itiation of transcription by sigma(54) RNA polymerase (E sigma(54)) an d its function is required for transcription of the class III and IV f la genes. Here we show that purified FlbD binds to ftr elements that a re required for transcription from the sigma(54)-dependent class III f lbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4). Dimethylsulphate footprinting assays demonstrate d that FlbD makes base-specific contacts at highly conserved guanine n ucleotides in each half site of the ftr sequences. In a reconstituted in vitro transcription system using E. coli E sigma(54), we found that FlbD was clearly capable of driving transcriptional initiation from t he flbG promoter and that this activity relied on the ftr1 binding sit e. Several observations suggest that phosphorylation plays a role in t he regulation of FlbD activity. First, we found that a mutant form of FlbD (FlbD(S140F)) corresponding to the substitution found in a consti tutively active NtrC protein (NtrC(S160F), displayed a greater potenti al for activating E sigma(54)-dependent transcription than the wildtyp e protein. We also observed that high energy-phosphate-containing mole cules stimulate transcription activation by the wild type FlbD. Togeth er, these results suggest that FlbD is responsible for mediating fla g ene transcription initiation by E sigma(54) and that covalent modifica tion is likely to play a role in governing FlbD activity during the ce ll cycle.