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

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 .