Direct interaction of FliX and FlbD is required for their regulatory activity in Caulobacter crescentus

FliX and FlbD form stable complexes, which can stand the interference of 2.65 M NaCl. The stability of FliX and FlbD was affected by the co-existence of each other. Five FliX mutants (R71A, L85K, Δ117-118, T130L, and L136K) were created by site-directed mutagenesis in conserved regions of the protein. All mutants were successfully expressed in both wild-type and ΔfliX Caulobacter strains. All but FliXL85K could rescue the motility and cell division defects of a ΔfliX mutant strain. The ability of FliX to regulate the transcription of class II and class III/IV flagellar promoters was fully diminished due to the L85K mutation. Co-immunoprecipitation experiment revealed that FliXL85K was unable to physically interact with FlbD.FliX interacts with FlbD and thereby directly regulates the activity of FlbD in response to flagellar assembly. Mutations in highly conserved regions of FliX could severely affect the recognition between FliX and FlbD and hence interrupt the normal progression of flagellar synthesis and other developmental events in Caulobacter.Caulobacter crescentus undergoes a series of programmed differentiation events within each cell cycle and generates two dissimilar progeny cells, a motile swarmer cell possessing a single polar flagellum and a sessile stalked cell. A hallmark of this asymmetric cell division event is the temporal expression and asymmetric targeting of regulatory proteins as well as proteins comprising cellular structures such as the flagellum [1-5]. Over fifty genes are required for flagellar biogenesis in C. crescentus, and their temporal and spatial expression is regulated by both cell cycle events and the progression of flagellum assembly. Epistasis experiments have revealed that flagellar gene expression is subject to a regulatory hierarchy that reflects the assembly sequence of major flagellum sub-structures [6-15]. The expression of the early flagellar genes (class II) encoding components of basal body switch, MS-ring, and flagellum-