Ak. Benson et al., THE ROLE OF FLBD IN REGULATION OF FLAGELLAR GENE-TRANSCRIPTION IN CAULOBACTER-CRESCENTUS, Research in microbiology, 145(5-6), 1994, pp. 420-430
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.