Global regulation of a sigma 54-dependent flagellar gene family in Caulobacter crescentus by the transcriptional activator FlbD

Abstract

Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV). The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins. Here we report an investigation of the role of the C. crescentus transcriptional regulatory protein FlbD in the activation of 54 -dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro. Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fljK by Escherichia coli E 54 is activated by FlbD or the mutant protein FlbD S140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements. We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters. These newly identified ftr elements are positioned ca. 100 bp from the transcription start sites of each 54 -dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV 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 class II flbD gene, is a global regulator that activates the cell cycleregulated transcription from all identified 54 -dependent promoters in the C. crescentus fla gene hierarchy.The dimorphic bacterium Caulobacter crescentus generates two functionally and structurally distinct progeny cells after each cell division: a motile swarmer cell with a single polar flagellum and a sessile stalked cell. The new swarmer cell is formed during division of the stalked cell and requires the sequential assembly of surface structures during the cell cycle at the stalk-distal pole, including a single flagellum. The C. crescentus flagellum, like other bacterial flagella, can be divided into three basic parts: the basal body, a short curved structure called the hook, and the external flagellar filament (22). Flagellum biosynthesis requires the activity of ca. 50 flagellar (fla) genes that have been assigned to one of four classes (I to IV) arranged in a regulatory hierarchy on the basis of their time of expression in the cell cycle, promoter architecture, and results of epistasis experiments. The flagellum is assembled from the inside of the cell to the outside in the order basal body-hook-flagellar filament, and the sequence of fla gene expression in the C. crescent...

Characterization of flagella genes of Agrobacterium tumefaciens, and the effect of a bald strain on virulence

“…5, underlined t residues). This arrangement is similar to class III and class IV fla gene promoters of Caulobacter crescentus (Wu et al, 1995). In our case, the conserved GC is 11 bp upstream of the transcriptional start and the conserved GG doublet is 10 bp farther upstream; such motifs are considered to be the minimal requirement for 54 promoters (Kustu et al, 1989).…”

Characterization of Enhancer Binding by the Vibrio cholerae Flagellar Regulatory Protein FlrC

“…The other FlrC binding sites within the genome are probably more degenerate from the flaAp binding site sequence, reflecting the lower levels of transcription necessary for the other FlrC-dependent genes. Thus, although FlrC activates multiple promoters, different levels of FlrC-dependent transcription are achieved in part by the strength of FlrC binding to the individual promoters, as has also been suggested for the FlrC homologue from Caulobacter crescentus, the flagellar regulator FlbD (42). It follows from this logic that enhancer strength also depends on strength of FlrC binding; the flaAp sites constitute an enhancer because FlrC binds more strongly to these sites, and the higher occupancy of FlrC at these sites facilitates FlrC-RNAP contacts, even from a distance.…”

Signal transduction mechanisms in Caulobacter crescentus development and cell cycle control

“…However, in vitro activation of class II promoters by the Ec 73 RNA polymerase indicates that these promoters are in fact recognized by the C. crescentus housekeeping sigma factor [44]. Cell cycle control of class II promoters would thus be mediated by transcriptional regulators rather than alternative sigma factors.…”

“…The FlbD protein is a homolog of the E. coli c 54 activator protein NtrC [77,78]. FlbD has a C-terminal DNA-binding motif which allows speci¢c interaction with the enhancer sequences of class III and class IV £agellar promoters [67,72,73,79]. FlbD is strictly required for the activity of these promoters both in vitro and in vivo [66,67,72,73,78,79].…”