Direct interaction of FliX and FlbD is required for their regulatory activity in Caulobacter crescentus
© Xu et al; licensee BioMed Central Ltd. 2011
Received: 10 February 2011
Accepted: 2 May 2011
Published: 2 May 2011
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.
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-specific type-three secretion system (TTSS) is regulated by the timed synthesis and phosphorylation of the transcription factor CtrA [16–18]. 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]. Finally, the complete construction of these class III-encoded structures are required to derepress the translation of flagellin mRNA (class IV), leading to the assembly of flagellar filament structure [19–22]. Thus, during C. crescentus flagellar biogenesis two different regulatory checkpoints link structural assembly to flagellar gene expression.
The transcription of class III and IV flagellar genes requires σ54-containing RNA polymerase and the DNA binding protein, integration host factor (IHF) [23–28]. Transcription of these flagellar genes is under cell cycle control and, late in the cell cycle, is restricted to the swarmer cell compartment of the predivisional cell. This temporal and spatial transcription is regulated by FlbD, a σ54 transcription factor [29–34]. The conserved receiver domains of this class of proteins are usually phosphorylated by a cognate sensor histidine kinase, which in turn stimulates oligomerization and DNA-binding of these proteins at enhancer sequences. Rather than phosphrylation, FlbD activity is regulated by FliX, a conserved trans-acting factor that is present in polarly flagellated α-proteobacteria and has no demonstrated histidine kinase activity [35–38]. Caulobacter strains bearing mutations in fliX are non-motile and do not transcribe class III and IV flagellar genes. Gain-of-function mutations in FlbD can by-pass the transcriptional requirement for FliX, suggesting that FliX is a trans-acting factor rather than a structural component of the flagellum . Additionally, FliX enhances FlbD-activated transcription in vitro by stimulating purified FlbD to form higher-order oligomers . Interestingly, overexpression of FliX suppresses FlbD-activated transcription in vivo, and a mutant allele of fliX, fliX 1, has been isolated that can by-pass the early flagellar assembly requirement for class III and IV transcription . These observations suggest that upon the complete assembly of an early class II flagellar basal body structure, FliX switches from a negative to a positive regulator of FlbD.
The physical interaction of FliX and FlbD represents a novel mechanism for regulating the activity of a σ54 transcription factor . Here, we describe a genetic and biochemical analysis dissecting the role of FliX in regulating FlbD activities. We present evidence that FliX and FlbD are in stable complexes under physiological conditions. Furthermore, we show that highly-conserved regions of FliX are critical for its productive interaction with FlbD and for proper regulation of flagellar gene expression in response to the progression of flagellar assembly.
Bacterial strains and plasmids
Bacterial strains and plasmids used in this study
Strains or plasmids
Genotypes or descriptions
syn-1000, bla-6, amps derivative of NA1000
Stephens et al. 
syn-1000 bla-6 ΔfliX
Muir et al. 
Ohta et al. 
Rp4-2, Tc::Mu, Km::Tn7
Simon et al. 
F-ompT gal [dcm] [lon] hsdS B (rB- mB-; an E. coli B strain) with DE3, a λ prophage carrying the T7 RNA polymerase gene
derivative of pET-21b carrying histidine-tagged FliX under the control of T7 promoter, Apr
Muir & Gober 
broad host range cloning vector, multicopy, Cmr
Kovach et al. 
derivative of pBBR1MCS carrying PCR generated 894 bp BamHI-HindIII fragment containing fliX gene and its promoter
derivative of pZXfliX, codon 71 cgcc→gcc
derivative of pZXfliX, codon 85 ctg→aag
derivative of pZXfliX, deletion of codon 117 and 118
derivative of pZXfliX, codon 130 acc→ctg
derivative of pZXfliX, codon 136 ctg→aag
derivative of pBBR1-MCS expressing fliX 1, an allele of fliX carrying a point mutation at the sixteenth codon (R16G), a frame shift at codon 141, and an extended carboxyl terminus of 67 amino acids
Muir et al. 
fliF-lacZ transcriptional reporter vector, Tcr
Wingrove & Gober 
fliK-lacZ transcriptional reporter vector, Tcr
Gober & Shapiro 
Identification of FliX-bound proteins with mass spectrometry
About 1.64 g of CNBr-activated sepharose 4B beads (GE Healthcare, Piscataway, NJ, USA) were swelled and washed as recommended by the manufacture and incubated overnight with 36.6 mg of histidine-tagged FliX (FliX-His) that was prepared as previously described . After incubation at 4°C with end-over-end rotation, the bead complexes were alternately washed with acidic buffer (0.1 M acetate, 0.5 M NaCl, pH 4.0) and alkaline buffer (90 mM Tris·Cl, 0.5 M NaCl, pH 8.5) for 3 cycles. Such prepared sepharose-FliX complexes were then conditioned by PBS buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) and stored at 4°C for later use. Meanwhile, 5 liters of C. crescent LS107 culture was harvested by centrifugation, resuspended in 100 ml of PBS buffer, lysed by French Press, and centrifuged at 26,690 g for 1 h. The supernatant was mixed with the above sepharose-FliX complexes and incubated at 4°C for overnight with gentle rocking. Cell extract was then removed by centrifugation. The pellet containing the sepharose bead complexes was washed with 20 ml of PBS buffer for three times and resuspended in 5 ml of the same buffer. An aliquot of 100 μl was removed and boiled with loading buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The gel was visualized with Coomassie staining. The apparent bands were excised, partially digested with trypsin, and were analyzed by electrospray ionization (ESI)-ion trap mass spectrometry at Stanford University http://mass-spec.stanford.edu/.
Stability assays of FliX and FlbD
Protein synthesis in cultures grown to mid-log phase was inhibited by addition of chloramphenicol to a final concentration of 3 mg/ml. One milliliter of cell culture was taken at 0, 15, 30, and 45 min after the addition of the antibiotic. Cell pellets were electrophoresed in 12% (w/v) polyacrylamide gels and were analyzed using anti-FlbD or anti-FliX antibodies.
Site-directed mutagenesis of fliX
A fragment of 894 bp covering the coding sequence of fliX and its promoter region was amplified by PCR from C. crescentus chromosome and was inserted into pBBR1MCS to give raise to pZXfliX, which was then used as the template to create fliX mutants. For every mutant to be generated, two pairs of primers were designed to amplify the two fragments covering each side of the desired mutation site. The intended mutation sequence was overhung at the 5' end of the downstream fragment. For the convenience of manipulation, BamHI recognition sequence was engineered at the 5' end of the upstream fragment, and HindIII at the 3' end of the downstream fragment. The two fragments were then phosphorylated, treated with BamHI or HindIII, and inserted into pBBR1MCS to generate pZX series plasmids (Table 1). All mutants were confirmed by DNA sequencing.
Protein expression analysis of FlbD and the FliX alleles
Overnight cultures of C. crescentus were transferred to fresh PYE media at a ratio of 1 to 10 (v/v) and were allowed to grow at 31°C until mid-log phase. Culture biomass was measured as optical density at 600 nm (OD600), normalized, and was subject to 14% (w/v) SDS-PAGE. After electrophoresis, protein profiles were transferred to nitrocellulose membranes and were detected using anti-FliX or anti-FlbD antibodies purified with affinity columns (AminoLink® Plus Immobilization Kit, Thermo Fisher Scientific Inc., Rockford, IL, USA).
Measurement of the transcription of flagellar genes
The pZX serial plasmids bearing various fliX mutants were introduced into the wild-type strain LS107 or the ΔfliX stain JG1172 via conjugation, along with the reporter genes fliF-lacZ or fliK-lacZ. β-Galactosidase activity was measured as described previously .
Cells in middle log stage were harvested, normalized, and treated with 5 mg/ml lysozyme. The clear cell extract was incubated with Agarose-Protein A beads (Roche Applied Science, Indianapolis, IN, USA) to eliminate non-specific associated proteins. The pre-cleared cell lysate was then incubated overnight with Agarose-Protein A-anti-FlbD complexes prepared as instructed by the manufacturer. After extensive washing, the bead complexes were spun down, resuspended in SDS-PAGE sample buffer and were subjected to electrophoresis followed by immunoblotting with anti-FliX antibodies.
FlbD forms stable in vivocomplex with FliX
Interaction between FlbD and FliX is required for stabilizing each other in vivo
Site-directed mutagenesis of FliX
Role of conserved FliX residues in protein expression
Role of conserved FliX residues in flagellar synthesis
Effect of conserved FliX residues on cell morphology
Role of conserved FliX residues in interaction with FlbD
The interaction between FliX and FlbD, two class II flagellar regulatory proteins in Caulobacter, represents a novel mechanism for regulating the activity of a σ54 transcription factor. The activities of many such factors are regulated by the phosphorylation of a conserved aspartate residue in their receiver domains [42, 43]. However, the receiver domain of FlbD diverges substantially from others . For example, it lacks some key residues necessary for the phosphorylation process . No corresponding cognate histidine kinase for FlbD has been identified so far, and FlbD is active in the absence of phosphorylation [30, 34]. In addition, purified FliX can regulate FlbD-activated transcription in vitro, probably by affecting the oligomerization state of FlbD . In this study, we further demonstrated that through a remarkably high affinity, the two proteins bind to each other to perform their regulatory activity and to escape the fate of premature degradation. Mutations in conserved regions of FliX could interrupt the recognition between the two and hence their activity.
The observed low concentrations of FliXL85K, FliXΔ117-118, and FliX 1 in JG1172 cells may be caused by their intrinsically low expression levels or their short half-life, or a combination of both. DNA or mRNA sequences of the alleles may carry intrinsic defects that inhibit the transcription or translation efficiency of the mutated genes. It is also possible that the mutations unfortunately expose target sites to intracellular proteases, making the gene products prone to degradation. Lack of protection from FlbD may also play a role in the case of FliXL85K. No matter what might be the main cause, the final result is that the cellular levels of the three are about the same (Figure 4). Nevertheless, their differential binding affinities to FlbD lead to dramatically different physiological outcomes. FliXL85K completely losts the ability to interact with FlbD and exerts no influence to FlbD-mediated cellular processes. The fair amount of cellular FliXL85K (Figure 4) does not benefit the ΔfliX host in any observable way (Figure 5, 6 and 7). The mutation must have altered the gross structure of FliX and thus prevented an effective binding to FlbD. FliXΔ117-118 can still interact with FlbD to a certain degree; therefore, it is largely functional in regulating FlbD activity (Figure 5 and 6). With a strong affinity to FlbD, FliX 1 becomes constitutively active; it turns on the transcription of class III/IV genes in the absence of the class II basal body [37, 38]. The other three mutations, R71A, T130L, and L136K cause no significant effect to the expression of FliX, the interaction with FlbD, and hence the regulatory activity of the two partners.
Since the three dimensional structure of FliX (or a homolog) remains to be solved, it is still unclear which residues or regions of FliX and FlbD are in direct contact. An alanine scanning analysis should be helpful to probe the structural basis of the interaction. Relevant insights could also be gathered by selection for extragenic suppressors in FlbD that are specific to loss-of-function FliX alleles like FliXL85K.
Direct association of FliX and FlbD is required for their regulation on flagellar synthesis and other developmental events in Caulobacter. FliX and FlbD form high affinity complexes under physiological conditions, which is essential for the in vivo stability of each protein. Highly conserved regions of FliX are critical for binding to FlbD. Mutations in these regions could severely impact the recognition between the two and compromise their regulatory activity.
We are grateful to Dr. Jill Zeilstra-Ryalls at BGSU for helpful discussions. This work was supported by Public Health Service Grant GM48417 from the National Institutes of Health to JWG.
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