Functions of the subunits in the FlhD(2)C(2) transcriptional master regulator of bacterial flagellum biogenesis and swarming

In enterobacteria like Salmonella, biogenesis of cell surface flagella need ed for motility is dependent upon the master operon flhDC at the apex of th e flagellar gene hierarchy. The operon products FLhD and FLhC act together in a FLhD(2)C(2) heterotetramer to induce flagellar gene transcription, whi le FLhD also represses cell septation. The flhDC operon is pivotal to diffe rentiation into elongated hyperflagellated swarm cells that undergo multice llular migration, most strikingly in Proteus. We set out to establish the m echanism of action of the FLhD(2)C(2) multimer. In Proteus swarm cell extra cts, all the FLhC was assembled into the FLhD(2)C(2) transcription activato r, but FLhD additionally formed approximately equimolar amounts of a FLhD, homodimer. Both FLhD and FLhC subunits homodimerised in vivo and in vitro, suggesting that self-interactions stabilise the FLhD(2)C(2) complex. The Fl hC and FLhD subunit proteins were separately expressed and purified, and th e FLhD(2)C(2) heterotetramer was reconstituted in vitro. Purified FLhC boun d specifically and cooperatively to the promoter region of the flhDC-regula ted flhB flagellar gene in the absence of FLhD. Purified FLhD was unable to bind this target DNA, but binding by the FLhD(2)C(2) complex was approxima tely tenfold greater than the FLhC subunit alone, suggesting that FLhD pote ntiated the FLhC/DNA interaction. Ln support of this possibility, pre-incub ation of FLhC with FlhD reduced the apparent dissociation constant, KD, for the FLhC/DNA complex from 100 nM to 13 nM. Furthermore, in competition ass ays, FLhD substantially increased the specificity of DNA recognition by Flh C, and also stabilised the resultant labile protein/DNA complex, prolonging its half-life from around two minutes to more than 40 minutes. FLhD(2)C(2) is therefore an atypical prokaryotic transcription activator in which inte raction of the FlhC subunit with DNA target sequences is enhanced by the co expressed helper subunit FLhD. (C) 2000 Academic Press.