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Journal of Bacteriology, December 1999, p. 7500-7508, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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,1
A.
Kolb,2
E.
Krin,1
C.
Laurent-Winter,3
S.
Rimsky,2
A.
Danchin,1 and
P.
Bertin1,*
Unité de Régulation de
l'Expression Génétique,1
Unité de Physico-Chimie des Macromolécules
Biologiques (URA1773 CNRS),2 and
Laboratoire de Chimie Structurale des
Macromolécules,3 Institut Pasteur,
75724 Paris Cedex 15, France
Received 12 July 1999/Accepted 4 October 1999
Little is known about the molecular mechanism by which histone-like
nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator
protein (CAP) complex control bacterial motility. In the present paper,
we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a
reduced expression in vivo of fliA and fliC,
which encode the specific flagellar sigma factor and flagellin,
respectively. Overexpression of the flhDC master operon
restored, at least in part, motility in crp and
hns mutant strains, suggesting that this operon is the main
target for both regulators. Binding of H-NS and CAP to the regulatory
region 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 expression while H-NS represses it. In
agreement with this observation, the activity 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 both hns
and crp mutants. These results suggest that the region
downstream of the +1 transcriptional start site plays a crucial role in
the positive control by H-NS of flagellum biosynthesis in vivo.
Finally, the lack of complementation 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 transcription by binding to its promoter and
interacting with RNA polymerase.
*
Corresponding author. Mailing address: Unité de
Régulation de l'Expression Génétique, Institut
Pasteur, 28 rue du Dr. Roux, 75724 Paris cedex 15, France. Phone: 33 (0) 1 40 61 35 56. Fax: 33 (0) 1 45 68 89 48. E-mail:
phbertin{at}pasteur.fr.
Journal of Bacteriology, December 1999, p. 7500-7508, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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