Multiple control of flagellum biosynthesis in Escherichia coli: Role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon
O. Soutourina et al., Multiple control of flagellum biosynthesis in Escherichia coli: Role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon, J BACT, 181(24), 1999, pp. 7500-7508
Little is known about the molecular mechanism by which histone-like nucleoi
d-structuring: (H-NS) protein and cyclic AMP-catabolite activator protein (
CAP) complex control bacterial motility, In the present paper, we show that
cip and hns mutants are nonmotile due to a complete lack of flagellin accu
mulation, This results from a reduced expression in vivo of fliA and fliC,
which encode the specific flagellar sigma factor and flagellin, respectivel
y, Overexpression of the flhDC master operon restored, at least in part, mo
tility in crp and hns mutant strains, suggesting that this operon is the ma
in target for both regulators. Binding of H-NS and CAP to the regulatory re
gion of the master operon was demonstrated by gel retardation experiments,
and their DNA binding sites were identified by DNase I footprinting assays.
In vitro transcription experiments showed that CAP activates flhDC express
ion while H-NS represses it. In agreement with this observation, the activi
ty of a transcriptional fusion carrying the flhDC promoter was decreased in
the crp strain and increased in the hns mutant, In contrast, the activity
of a transcriptional fusion encompassing the entire flhDC regulatory region
extending to the ATG translational start codon was strongly reduced in bot
h hns and cry mutants. These results suggest that the region downstream of
the +1 transcriptional start site plays a crucial role in the positive cont
rol by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complem
entation of the nonmotile phenotype in a crp mutant by activation-deficient
CAP mutated proteins and characterization of cfs, a mutation resulting in
a CAP-independent motility behavior, demonstrate that CAP activates flhDC t
ranscription by binding to its promoter and interacting,vith RNA polymerase
.