An intrinsically disordered linker controlling the formation and the stability of the bacterial flagellar hook
© Samatey et al. 2017
Received: 26 July 2017
Accepted: 10 October 2017
Published: 27 October 2017
In a macro-molecular complex, any minor change may prove detrimental. For a supra-molecular nano-machine like the bacterial flagellum, which consists of several distinct parts with specific characteristics, stability is important. During the rotation of the bacterial flagellar motor, which is located in the membrane, the flagella rotate at speeds between 200 and 2000 rpm, depending on the bacterial species. The hook substructure of the bacterial flagellum acts as a universal joint connecting the motor to the flagellar filament. We investigated the formation of the bacterial flagellar hook and its overall stability between the FlgE subunits that make up the hook and attempted to understand how this stability differs between bacteria.
An intrinsically disordered segment plays an important role for overall hook stability and for its structural cohesion during motor rotation. The length of this linker segment depends on the species of bacteria; for Salmonella enterica and Campylobacter jejuni it is approximately 37 and 54 residues, respectively. Few residues of the linker are conserved and mutating the conserved residues of the linker yields non-flagellated cells. In the case of Campylobacter, which rotates its flagella at a speed much higher than that of Salmonella, shortening the linker leads to a rupture of the hook at its base, decreasing cell motility. Our experiments show that this segment is required for polymerization and stability of the hook, demonstrating a surprising role for a disordered region in one of the most finely tuned and closely studied macromolecular machines.
This study reveals a detailed functional characteristic of an intrinsically disordered segment in the hook protein. This segment evolved to fulfill a specific role in the formation of the hook, and it is at the core of the stability and flexibility of the hook. Its length is important in the case of bacteria with high-speed rotating flagella. Finding a way of disrupting this linker in Campylobacter might help in preventing infections.
Additional file 3: Video S1. Results of the molecular modeling calculations showing the flexibility of the ID-Rod-Stretch of FlgE from C. jejuni. (MOV 895 kb)
Lys32 of Salmonella FlgE is required for hook assembly
The strain expressing FlgE with all four conserved and two semi-conserved residues substituted with alanine residues was non-motile due to the absence of flagella (Fig. 4a, f). However, this FlgE mutant protein was synthesized and secreted at more than twice the amount of wild-type FlgE (Fig. 4b, c). This suggests that the FlgE protein with the substitutions T28A, G30A, F31A, K32A, F38A, and M41A was also being exported un-polymerized, which produced a similar but more severe phenotype to that shown with the K32A substitution alone.
The ID-Rod-Stretch is required for hook assembly and mobility
To study the role of the ID-Rod-Stretch in the assembly and function of the hook, we divided it into short segments of five amino acid residues and mutated them by exchanging them with alanine residues. We assumed that it would reveal the regions that are required for hook structural integrity and those that are required for hook mobility. The first five residues mutated were Ala27-Thr28-Tyr29-Gly30-Phe31, the second set of five residues mutated were Lys32-Ser33-Gly34-Thr35-Ala36, and so on. In some cases, the residue was already alanine. The results are summarized in Additional file 5.
The strains that synthesized the FlgE AAAAA(42–46) mutant protein was fully motile and made flagella normally (Fig. 6a, b). These amino acid residues are located in the middle of the ID-Rod-Stretch in the connecting loop region (Additional file 2). Strains that synthesized FlgE AAAAA(37–41), FlgE AAAAA(47–51), and FlgE AAAAA(52–6) mutant proteins were significantly less motile than wild-type and produced decreased amounts of flagella (Fig. 6, Additional file 6). These mutants include the residues that comprise the two ID-Rod-Stretch N- and C-terminal region anti-parallel β-strands. Examination by dark-field microscopy revealed that some cells within the populations were motile, but were less so than wild-type. The amount of the FlgE AAAAA(37–41) mutant protein in the supernatant broth was approximately 40% that of wild-type (Fig. 6d) and yet the amount of this protein in the cell pellet was slightly more than in wild-type (Fig. 6c). If cells do not undergo a switch from rod/hook-type export substrates to filament-type export substrates , FlgE will continue to be produced, which may account for more FlgE than wild-type.
Campylobacter FlgE has a long ID-Rod-Stretch vital for hook stability
The ID-Rod-Stretch of FlgE from C. jejuni strain 81116 has an insertion of approximately 20 residues in its middle compared to S. enterica LT2 FlgE. This region was shown to be involved in inter-subunit interactions in the hook . To study the importance of this insertion, a strain of C. jejuni was made where the flgE gene was changed by deleting the codons for 21 amino acid residues from Gln46 to Ile66. The deletion was designed based on the structure of C. jejuni FlgE .
Based on the micrographs that we have of the wild-type unbroken hook attached to cells and the broken hooks of the ID-Rod-Stretch deletion mutant strain, the wild-type hook of C. jejuni has a mean length of 105 ± 8 nm (N = 7), while the broken hooks have an average length of 85 ± 15 nm (N = 23). Almost intact hooks were attached to the filament. We did not see any breakage of the hook without filament, which makes us suspect that, in the absence of the filament, the hook will probably not break. The point of breakage is close to the rod-hook junction. During the growth of the flagellum, the hook assembles normally, followed by the completion of the flagellum. In the case of a shortened ID-Rod-Stretch in the hook, interactions between FlgE and FlgG proteins at the rod-hook interface and between FlgE proteins in the hook are reduced. As the filament is being assembled, the load on the hook gradually weakens the rod-hook connection, inducing breakage of the hook.
Intrinsically disordered peptides often interact with many different proteins  and are known for their involvement in important processes in cells . Mutations in the disordered regions induce a reduction in these interactions as well as dysfunction in cells, which often lead to diseases. In the bacterial flagellum, the ID-Rod-Stretch will not only connect two different domains within the protein, but will also interact with several neighboring molecules in both the hook and rod.
Both mutations F31A and F38A decrease the motility, but less severely than the effect of the mutation K32A. However, the exchange of all four conserved amino acid residues, Thr28, Gly30, Lys32, and Phe38, and the two semi-conserved residues, Phe31 and Met41, of the ID-Rod-Stretch with alanine residues has a cumulative effect that results in non-flagellated cells of S. enterica. Among these residues, Thr28, Gly30 and Phe31 are close to Lys32. Substitutions to alanine residues at these positions accentuate the effect of the K32A substitution, leading to non-flagellated cells. These mutations should have the same effect on all ID-Rod-Stretch with lengths similar to that of S. enterica.
The ID-Rod-Stretch can be divided in two classes. The first class, made of short ID-Rod-Stretch, is found only within some FlgE proteins, such as in S. enterica, and is made of approximately 30 amino acid residues (Additional file 2). The second class, with long ID-Rod-Stretch, is found in all FlgG proteins and in a few FlgE proteins, such as in C. jejuni, and is made of approximately 60 amino acid residues (Additional file 2). Mutating the conserved residues from the short ID-Rod-Stretch will produce non-flagellated, non-motile cells. However, these mutations will not have the same drastic effect on the long ID-Rod-Stretch due to the extensive set of interactions with other molecules (Fig. 8c). These interactions do not exist in the case of a short ID-Rod-Stretch.
C. jejuni strains encoding FlgE AAAAA(27–31) and FlgE AAAAA(32–36) mutant proteins are non-motile for similar reasons. These amino acid substitutions affect some of the conserved residues and might directly affect the stability of the β-strands. These regions of the ID-Rod-Stretch are very important for the assembly of the hook. The strain encoding FlgE AAAAA(42–46) is fully motile because, in S. enterica, amino acid residues Phe42 to Lys46 of the ID-Rod-Stretch do not interact with other FlgE molecules. We predict that this will be the same for all bacteria with a ID-Rod-Stretch of similar length to that of S. enterica. The strains that produce FlgE with the mutations FlgE AAAAA(37–41), FlgE AAAAA(47–51), and FlgE AAAAA(52–56) are significantly less motile than wild-type because these segments of the ID-Rod-Stretch play important roles in the interaction with other subunits of FlgE in the hook (Fig. 8b).
In the hook of S. enterica, the ID-Rod-Stretch of FlgE interacts with three molecules of FlgE, while in the hook of C. jejuni, the ID-Rod-Stretch of FlgE interacts with five molecules of FlgE (Fig. 8b, c, Additional file 7). In FlgE from C. jejuni, the ID-Rod-Stretch is 20 residues longer than that of S. enterica. Deletion of these extra residues prevents interactions with two molecules of FlgE from different protofilaments (Fig. 8d). One of these molecules is located in the neighboring protofilament at the “-16” position, while the second molecule is located two protofilaments away at the “-10” position (Fig. 8c, d). These interactions cannot be found in the hook of bacteria with a shorter ID-Rod-Stretch, such as S. enterica (Fig. 8b), since their ID-Rod-Stretch does not extend to interact with more FlgE molecules due to a shorter amino acid sequence. Disrupting these sets of interactions reduces the cohesion of the hook of C. jejuni. In C. jejuni cells expressing FlgE with a shorter ID-Rod-Stretch, flagella, while being assembled normally, detach themselves from the cell by breaking at the site of the hook (Fig. 7). Bacterial flagellar motors rotate at between 100 and 2000 Hz , and the hook undergoes conformational changes while rotating about its axis [27, 28]. The interactions between FlgE molecules must ensure the stability of the hook while enabling these conformational changes. The intrinsic disorder of the long C. jejuni ID-Rod-Stretch may be particularly important in this context for enabling adjacent hook monomers to maintain favorable interactions throughout the variety of positions and of conformations encountered during rotation, by providing multiple, nearly isoenergetic conformations that can be accessed as the hook rotates. The flagellar motor of C. jejuni is known to produce a very high torque compared to the flagellar motor of other bacteria . This high torque could be the reason behind FlgE having a long ID-Rod-Stretch. In a recent study, Fujii et al.  increased the length of the ID-Rod-Stretch of the hook of S. enterica through the insertion of 18 residues taken from the distal rod protein, FlgG, of S. enterica. The authors found that the polyhook assessed became as straight as the rod, concluding that the hook became as rigid as the rod. Our explanation is that the hook became less flexible due to this insertion, but not as rigid as the rod. We have shown that the ID-Rod-Stretch is at the center of stability and flexibility of the hook. However, there is a difference between the reduced flexibility and the rigidity found in the rod. The latter may be, partly, due to the presence of a long ID-Rod-Stretch. In our study, the native hook of C. jejuni, with its ID-Rod-Stretch similar to that found in the rod, is fully motile and flexible. If the hook of Campylobacter was as rigid as the rod, it would not have been able to function as a universal joint and the flagellum would not have been able to rotate about its axis. The length of the ID-Rod-Stretch does not change the flexibility of the hook such as to induce a rigid structure similar to that of the rod.
More generally, deleting approximately 20 residues that are inserted in the long ID-Rod-Stretch in both FlgE or FlgG will reduce the interactions between molecules that stabilize the hook or the distal rod, as shown here for the case of C. jejuni (Figs. 7 and 8c, d). This reduction of interactions will destabilize the hook, or the rod, and the flagellum may be ripped off from its base (Fig. 7 h, i).
The hook disordered segment is important for both the formation and stability of the hook, and thus for cell motility. In the case of pathogenic bacteria, such as C. jejuni, which also uses its flagella to secrete toxins, we believe that targeting the ID-Rod-Stretch could be a strategy to prevent toxin secretion into host cells.
Protein sequence alignment
Sequence alignment was performed with Clustal Omega .
Disorder prediction of the ID-Rod-Stretch
Prediction of disordered regions was performed using online software Predictor of Natural Disordered Regions (PONDR) . The prediction shows that a part of the segment of C. jejuni FlgE, residues Thr55 to Arg77, has the highest probability of being intrinsically disordered.
To assess the level of disorder present in the D0–D1 linker of various flagellar components, we generated and analyzed an ensemble of optimized loop structures using the loopmodel module of Rosetta 2016.46.59086 . Fragment libraries were generated for the full-length protein of each target using the Robetta web server , and thousands of loop models (2000 for S. enterica FlgE and 3500 for C . jejuni FlgE) were subsequently obtained using the quick_ccd modeling method and refine_ccd refinement method. We generated loop models for S. enterica FlgE (residues 25–64), C. jejuni FlgE (residues 30–82), and S. enterica FliC (residues 30–44), each in the context of a single full-length protein monomer. Built models were aligned to the corresponding initial structure by the D0 helices (which had been kept rigid during the modeling process), and Cα RMSDs calculated between the aligned, modeled loop and the initial structure using the MDAnalysis package . Structural figures and movies were generated using VMD 1.9.3 .
Strains and culture conditions
Bacterial strains and plasmids used in this study are listed in Additional file 8. The following strains and plasmids have been previously described: S. enterica strains SJW1103 , JR501 , and SJW1368 ; C. jejuni strains 81116 , CB991 , and CB-A9 ; and plasmids pKD13 , pKD46 , pCP20 ; pTrc99A-FF4 , and pCB956 . S. enterica and E. coli strains were cultured using “Luria–Bertani” broth (LB) or agar at 37 °C . C. jejuni strains were cultured on Mueller–Hinton agar (Difco, Detroit, MI, USA) and incubated at 42 °C under microaerophilic conditions (85% N2, 10% CO2, and 5% O2) in a Tri-Gas incubator. For E. coli, ampicillin (50 μg mL–1) was added to media where appropriate. For Salmonella, kanamycin (50 μg mL–1) and ampicillin (100 μg mL–1) were added to media as required. For Campylobacter, trimethoprim (5 μg mL–1), vancomycin (10 μg mL–1), kanamycin (50 μg mL–1), and apramycin (60 μg mL–1) antibiotics were used where appropriate.
Oligonucleotides used in the plasmid constructions are listed in Additional file 9. The Salmonella flgE expression plasmid pCB954 was made as follows: the flgE gene was amplified in a PCR reaction with primers Fd-NdeI-flgESe and Rv-BamHI-flgESe and strain SJW1103 genomic DNA template. The PCR product and plasmid pTrc99A-FF4 were digested with Nde I and BamH I restriction enzymes. The digested PCR product and plasmid DNA were ligated using T4 DNA ligase. Other plasmids were derived using site-directed mutagenesis with QuikChange Lightning site-directed mutagenesis kits (Agilent, USA). Standard molecular biology procedures were followed .
Construction of Salmonella mutant strains
To make a ΔflgE::FRT null mutant strain the Lambda Red homologous recombination method of Datsenko and Wanner  was used. Briefly, an FLP-flanked kananamycin-resistance cassette with ends homologous to flgE gene was amplified in a PCR reaction with primers Fd-flgESe-FKF and Rv-flgESe-FKF and plasmid pKD13 as template.
Construction of Campylobacter mutant strains
To make the C. jejuni FlgE ID-Rod-Stretch deletion-mutant strain CB-A137, the parent strain CB991, bearing a ΔflgE::KmR allele, was transformed naturally with pCB-A128 suicide vector DNA, as previously described [19, 44]. PCR was used to confirm that double-crossover homologous recombination had occurred within the rRNA cluster. PCR products were sequenced by chain-termination dideoxynucleotide sequencing using a BigDye Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, USA). Strain CB-A137 encodes a flgE ID-Rod-Stretch deletion mutant gene within the rRNA gene cluster. The rRNA gene cluster has been previously determined to be an appropriate location for insertion of genes for stable gene expression in C. jejuni [45, 46].
Motility of Salmonella strains were examined using soft tryptone agar plates consisting of 0.35% w/v agar containing the appropriate antibiotics . Plates were stab-inoculated with colonies of a fresh transformation and incubated at 30 °C for the desired time. Experiments were repeated at least four times for each strain. For the isolation of suppressor mutant strains with increased motility, colonies were streaked through the soft tryptone agar plate and incubated at 30 °C for up to 5 days. The suppressor mutant strains were purified by inoculation onto fresh media.
Motility of Campylobacter was examined in Mueller–Hinton motility media, which contained 0.4% w/v agar . The strains were grown in Mueller–Hinton broth at 42 °C for 24 h, the optical density of each culture was normalized to OD600 nm 0.5 and 1 μL was stab-inoculated into Mueller–Hinton motility media. Motility phenotypes were examined after incubation of strains at 42 °C for the desired time.
Flagellar protein immunoblotting
Immunoblotting of exported flagellar proteins and proteins remaining in the cell pellet was performed similarly as described previously [49, 50]. For Salmonella, cultures were grown in LB at 37 °C until early stationary phase (OD600 nm 1.5). Culture broth (1.5 mL) was sampled and centrifuged at 10,000× g for 10 min. Cell pellets were frozen, and 1.4 mL of supernatant broth was put into a clean tube and centrifuged at 20,400× g for 30 min. Clarified supernatant broth (1.3 mL) was taken and 150 μL of trichloroacetic acid solution was added to precipitate proteins. Supernatant solution samples were centrifuged at 16,100× g for 30 min in order to obtain protein pellets. The protein pellets from the supernatant solutions and thawed cell pellets were suspended in Tris.Cl-SDS buffer or 2× SDS loading buffer (7 M Urea, 0.1 M Tris.Cl (pH 6.8), 140 mM sodium dodecyl sulfate, 5% v/v β-mercaptoethanol, 0.2% w/v bromophenol blue) to an equivalent of 27 μL mL–1, at OD600 nm 1.5 (i.e. the cell pellets were suspended in 40 μL 2× SDS loading buffer).
For Campylobacter, strains were grown in Mueller–Hinton broth containing antibiotics as required and grown at 42 °C under microaerophilic conditions for 24 h. The optical density varied from culture to culture between OD600 nm 0.4 and 1.2. Cells and proteins from the supernatant broth were harvested by centrifugation of 1.5 mL culture broth, as described above for Salmonella cultures. The proteins from the supernatant solutions and cell pellets were suspended in Tris.Cl-SDS buffer or 2× SDS loading buffer to an equivalent of 80 μL mL–1, at OD600 nm 1.0.
Samples were loaded onto the wells of a 4–20% w/v Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad, USA) and subjected to electrophoresis at 120 V for approximately 1 h. Western Breeze chromogenic detection kits were used to detect proteins following manufacturer’s instructions (Thermo Fisher Scientific, USA). Band densities were quantified using a ChemiDoc XRS+ gel documentation system with Image Lab Software (Bio-Rad, USA). The following amounts of sample were loaded per well to detect the proteins (the dilutions of sera containing polyclonal antibodies are indicated in parenthesis): Salmonella FlgE, 10 μL supernatant protein or 0.5 μL cell pellet samples (S. enterica FlgE antibodies, 1:10,000 dilution); Salmonella FliC, 5 μL supernatant protein or 0.5 μL cell pellet samples (S. enterica FliC antibodies, 1:20,000 dilution); Campylobacter FlgE, 10 μL supernatant protein or 10 μL cell pellet samples (C. jejuni FlgE antibodies, 1:10,000 dilution); and Campylobacter flagellin, 2 μL supernatant protein or 2 μL cell pellet samples (C. jejuni FlaA antibodies, 1:5000 dilution). Sera from rabbits containing polyclonal antibodies reactive towards S. enterica FlgE or FliC were generously provided by T. Minamino (Osaka University). Sera from rabbits containing polyclonal antibodies reactive towards C. jejuni FlgE and FlaA proteins were made in this study.
Salmonella strains were examined by electron microscopy similarly as described previously . Cultures were grown in LB with antibiotics as required to late exponential phase (OD600 nm 1.0) at 37 °C. Cell suspensions were spotted on Mextaform HF-34 200-mesh carbon-coated copper grids and the cells were stained with 1% phosphotungstic acid at pH 7. Grids were examined using a JEM-1230R transmission electron microscope (JEOL, Ltd., Japan) at 100 kV. At least 30 negatively stained cells were examined for each strain. Flagella numbers per cell for each strain were compared using a Mann–Whitney U test. Box plots were prepared using BoxPlotR (http://shiny.chemgrid.org/boxplotr/).
Campylobacter strains were examined by electron microscopy similarly as described by Hendrixson and DiRita . Briefly, the strains were grown on Mueller–Hinton agar, containing appropriate antibiotics, at 42 °C under microaerophilic conditions for 36 h. Cells were gently scraped from the surface of the plate using a sterile plastic inoculating loop and stained with phosphotungstic acid as described above for Salmonella.
We thank all the members of the Trans-membrane Trafficking Unit (OIST) for the discussions about the project. We thank Tohru Minamino (Osaka University, Japan) for providing us with polyclonal antibodies to Salmonella FlgE and FliC proteins. We thank Toshio Sasaki and Toshiaki Mochizuki (OIST) for help with electron microscopy. We are grateful to Eric Martz (University of Massachusetts, Amherst, USA) for critical reading of the manuscript and for preparing the interactive pages on http://proteopedia.org/w/Samatey/5. We thank Olga A. Elisseeva (Okinawa Institute of Science and Technology Graduate University, Japan) for the critical discussions and for reading the manuscript. We are grateful to Keiichi Namba (Osaka University, Japan) for his support. We are grateful to Olesya Gusachenko (University of St Andrews, UK) for producing the schematic illustration of the bacterial flagellum.
This work was supported by a direct funding from OIST to FAS.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional files.
Interactive 3D views of ID-Rod-Stretch and its interactions are reported here: http://proteopedia.org/w/Samatey/5
FAS and CSB designed the mutations and experiments. CSB and IVM performed the experiments. ASK studied the disordered segment using online prediction software. PLF made the molecular modeling calculations. CSB and FAS wrote the manuscript with input from all the authors. FAS was responsible for the overall project strategy and management. All authors read and approved the final manuscript.
The authors declare no competing financial interests.
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