GLOBAL REGULATION OF A SIGMA(54)-DEPENDENT FLAGELLAR GENE FAMILY IN CAULOBACTER-CRESCENTUS BY THE TRANSCRIPTIONAL ACTIVATOR FLBD

Biosynthesis of the Caulobacter crescentus polar flagellum requires th e expression of a large number of flagellar (fla) genes that are organ ized in a regulatory hierarchy of four classes (I to IV). The timing o f fla gene expression in the cell cycle is determined by specialized f orms of RNA polymerase and the appearance and/or activation of regulat ory proteins. Here we report an investigation of the role of the C. cr escentus transcriptional regulatory protein FlbD in the activation of sigma(54)dependent class III and class IV fla genes of the hierarchy b y reconstituting transcription from these promoters in vitro. Our resu lts demonstrate that transcription from promoters of the class III gen es flbG, flgF, and flgI and the class TV gene fljK by Escherichia coli E sigma(54) is activated by FlbD or the mutant protein FlbD(S140F) (w here S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro s tudies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transc ription regulation) sequence elements. We have now: identified multipl e ftr sequences that are conserved in both sequence and spatial archit ecture in all known class III and class IV promoters. These newly iden tified ftr elements are positioned ca. 100 bp from the transcription s tart sites of each sigma(54)-dependent fla gene promoter, and our stud ies indicate that they play an important role in controlling the level s of transcription from different class III and class TV promoters. We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo. Thus, our results suggest that FlbD, which is encoded by the cl ass II flbD gene, is a global regulator that activates the cell cycle- regulated transcription from all identified sigma(54)-dependent promot ers in the C. crescentus fla gene hierarchy.