Abstract
BackgroundThe temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. The physical interaction of these two proteins represents an alternative mechanism for regulating the activity of σ54 transcription factors. This study is to characterize the interaction of the two proteins and the consequences of the interaction on their regulatory activity.ResultsFliX 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.ConclusionsFliX 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.
Highlights
The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX
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
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
Summary
The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. 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. The expression of the early flagellar genes (class II) encoding components of basal body switch, MS-ring, and flagellum-specific type-three secretion system (TTSS) is regulated by the timed synthesis and phosphorylation of the transcription factor CtrA [16,17,18]. The polar assembly of the MS-ring/switch/TTSS complex is required, in turn, for the transcription of genes (class III) encoding structures such as the rod, outer membrane rings, and the hook [8,10,13,14]. During C. crescentus flagellar biogenesis two different regulatory checkpoints link structural assembly to flagellar gene expression
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