Functions of the subunits in the FlhD2C2 transcriptional master regulator of bacterial flagellum biogenesis and swarming

Abstract

In enterobacteria like Salmonella, biogenesis of cell surface flagella needed for motility is dependent upon the master operon flhDC at the apex of the flagellar gene hierarchy. The operon products FlhD and FlhC act together in a FlhD2C2 heterotetramer to induce flagellar gene transcription, while FlhD also represses cell septation. The flhDC operon is pivotal to differentiation into elongated hyperflagellated swarm cells that undergo multicellular migration, most strikingly in Proteus. We set out to establish the mechanism of action of the FlhD2C2 multimer. In Proteus swarm cell extracts, all the FlhC was assembled into the FlhD2C2 transcription activator, but FlhD additionally formed approximately equimolar amounts of a FlhD2 homodimer. Both FlhD and FlhC subunits homodimerised in vivo and in vitro, suggesting that self-interactions stabilise the FlhD2C2 complex. The FlhC and FlhD subunit proteins were separately expressed and purified, and the FlhD2C2 heterotetramer was reconstituted in vitro. Purified FlhC bound specifically and cooperatively to the promoter region of the flhDC-regulated flhB flagellar gene in the absence of FlhD. Purified FlhD was unable to bind this target DNA, but binding by the FlhD2C2 complex was approximately tenfold greater than the FlhC subunit alone, suggesting that FlhD potentiated the FlhC/DNA interaction. In support of this possibility, pre-incubation of FlhC with FlhD reduced the apparent dissociation constant, KD, for the FlhC/DNA complex from 100 nM to 13 nM. Furthermore, in competition assays, FlhD substantially increased the specificity of DNA recognition by FlhC, and also stabilised the resultant labile protein/DNA complex, prolonging its half-life from around two minutes to more than 40 minutes. FlhD2C2 is therefore an atypical prokaryotic transcription activator in which interaction of the FlhC subunit with DNA target sequences is enhanced by the coexpressed helper subunit FlhD.