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

Abstract

Background: 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. 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.

Results: 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.

Conclusions: 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.

Figure 1

Proteins bound to the sepharose beads coated with histidine-tagged FliX. Purified FliX-His was conjugated to sepharose beads prior to incubation with cell lysis of LS107. The bead complexes were boiled with the sample buffer and were subject to SDS-PAGE analysis. The identities of the five major bands were determined by mass spectrometry.

Figure 2

Stability assays of FliX and FlbD. Samples were periodically removed from cell cultures after the addition of chloramphenicol. Cell pellets were analyzed by SDS-PAGE followed by immunoblotting using anti-FlbD (upper panels) and anti-FliX (lower panels) antibodies.

Figure 3

Site-directed mutagenesis of C. crescentus FliX. Homologs of C. crescentus FliX are aligned with CLUSTAL W 1.81 and are shaded with BOXSHADE 3.3.1. Black, identical residues; grey, similar residues; asterisks, sites of mutation. C._ cau: C. crescentus, R. rub: Rhodospirillum rubrum, B. jap: Bradyrhizobium japonicum, M. mag: Magnetospirillum magnetotacticum, and R. _pal: Rhodopseudomonas palustris.

Figure 4

Analysis of the cellular contents of the FliX mutants and FlbD. Total proteins of LS107 and JG1172 cells expressing various fliX alleles were analyzed by SDS-PAGE prior to immunoblotting using anti-FlbD (upper panels) and anti-FliX (lower panels) antibodies.

Figure 5

Motility of the cells harboring various fliX alleles. Cells were inoculated in motility agar and were incubated at 31°C for 3 days. Motile cells swarming away from the points of inoculation are visible as halos. Host strains containing no plasmid reside at the center of each plate.

Figure 6

Effects of fliX alleles on the transcription of flagellar genes. Wild-type fliX and mutant alleles were introduced to LS107 or JG1172 cells containing reporter genes fliF-lacZ (A) or fliK-lacZ (B). Results of five independent experiments.

Figure 7

Allele fliX_L85K was unable to rescue the cell division defect of JG1172. Cells harvested from overnight cultures were mounted on poly-L-lysine coated slides and examined by differential interference contrast (DIC) microscopy.

Figure 8

Co-immunoprecipitation of FlbD and the FliX mutants. Extracts of JG1172 cells expressing various fliX alleles were incubated with agarose beads coated with anti-FlbD antibodies. Proteins bound to the bead complexes were detected using anti-FliX antibodies following SDS-PAGE electrophoresis. The immunoblot was developed to an extended period of time to visualize the band of FliX_Δ117-118 (indicated by the arrow).