Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori

  • Jack M. Botting ,

    Contributed equally to this work with: Jack M. Botting, Shoichi Tachiyama

    Roles Formal analysis, Investigation, Writing – original draft, Writing – review & editing

    Affiliation Department of Microbiology, University of Georgia, Athens, Georgia, United States of America

    ⨯
  • Shoichi Tachiyama ,

    Contributed equally to this work with: Jack M. Botting, Shoichi Tachiyama

    Roles Formal analysis, Investigation

    Affiliations Microbial Sciences Institute, Yale University, West Haven, Connecticut, United States of America, Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, United States of America

    ⨯
  • Katherine H. Gibson,

    Roles Formal analysis, Investigation, Writing – review & editing

    Current address: Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

    Affiliation Department of Microbiology, University of Georgia, Athens, Georgia, United States of America

    ⨯
  • Jun Liu,

    Roles Formal analysis, Funding acquisition, Supervision, Writing – review & editing

    Affiliations Microbial Sciences Institute, Yale University, West Haven, Connecticut, United States of America, Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, United States of America

    ⨯
  • Vincent J. Starai,

    Roles Supervision

    Affiliation Department of Microbiology, University of Georgia, Athens, Georgia, United States of America

    ⨯
  • Timothy R. Hoover

    Roles Conceptualization, Funding acquisition, Supervision, Writing – review & editing

    trhoover@uga.edu

    Affiliation Department of Microbiology, University of Georgia, Athens, Georgia, United States of America

    ⨯

Abstract

Flagella-driven motility is essential for Helicobacter pylori to colonize the human stomach, where it causes a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. H. pylori has evolved a high-torque-generating flagellar motor that possesses several accessories not found in the archetypical Escherichia coli motor. FlgV was one of the first flagellar accessory proteins identified in Campylobacter jejuni, but its structure and function remain poorly understood. Here, we confirm that deletion of flgV in H. pylori B128 and a highly motile variant of H. pylori G27 (G27M) results in reduced motility in soft agar medium. Comparative analyses of in-situ flagellar motor structures of wild-type, ΔflgV, and a strain expressing FlgV-YFP showed that FlgV forms a ring-like structure closely associated with the junction of two highly conserved flagellar components: the MS and C rings. The results of our studies suggest that the FlgV ring has adapted specifically in Campylobacterota to support the assembly and efficient function of the high-torque-generating motors.

Citation: Botting JM, Tachiyama S, Gibson KH, Liu J, Starai VJ, Hoover TR (2023) FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori. PLoS ONE 18(11): e0287514. https://doi.org/10.1371/journal.pone.0287514

Editor: Eric Cascales, Centre National de la Recherche Scientifique, Aix-Marseille Université, FRANCE

Received: March 28, 2023; Accepted: June 7, 2023; Published: November 17, 2023

Copyright: © 2023 Botting et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data has been deposited in the Electron Microscopy Data Bank (https//www.ebi.ac.uk/emdb/). Entry code for the deposited data is EMD-40219.

Funding: This work was supported by National Institutes of Health grants AI140444 (TRH), AI1469077 (TRH), AI087946 (JL), and AI132828 (JI). The URL for NIH is https://www.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The human gastric pathogen Helicobacter pylori, a member of the phylum Campylobacterota, uses a cluster of polar sheathed flagella for motility, which is required for colonization in animal models for infection [1, 2]. The bacterial flagellum is organized into three basic parts, referred to as the basal body, hook, and filament [3–5]. The basal body contains a rotary motor powered by a transmembrane ion gradient and generates the torque needed for motility [6].

The torque-generating stator of the flagellar motor consists of a MotA pentamer and MotB dimer [7, 8]. MotB contains an N-terminal transmembrane domain, a plug/linker region, and a C-terminal periplasmic region that fixes the stator to the peptidoglycan layer through interactions with the peptidoglycan layer and P ring protein FlgI [9, 10]. MotA has four transmembrane helices and together with MotB forms a proton channel in the inner membrane [7, 8]. When not engaged with the motor, the MotB plug blocks proton flow through the stator [11]. Following incorporation of the stator into the motor, MotB undergoes a conformational change that unplugs the stator and promotes dimerization of the periplasmic domain to allow binding of the protein to peptidoglycan [12]. Proton flow through the stator is thought to power rotation of the MotA pentamer around the stationary MotB dimer [7, 8]. The C ring, which consists of the proteins FliG, FliM, and FliN (plus FliY in Campylobacterota) [13], forms the rotor of the flagellar motor [14, 15]. Torque from the stator units is transferred to the rotor through electrostatic interactions between conserved acidic and basic residues in MotA and the FliG HelixTorque, an α-helix in the C-terminal domain of the protein [8]. The MS ring interfaces with the C ring through interactions between the C-terminal, cytoplasmic domain of FliF (FliFC) and the N-terminal domain of FliG (FliGN) [16, 17]. Engagement of MotA with the FliG HelixTorque results in rotation of the C ring/MS ring rotor complex, and the resulting torque is transmitted through the rod and hook to the filament, which acts as a propeller to push the cell forward [18, 19].

The MS ring is formed by the FliF protein and is one of the earliest flagellar structures assembled [20]. FliF is embedded in the inner membrane by two transmembrane helices that flank a large periplasmic region and contains the ring-building motif (RBM) domains RBM1, RMB2, RBM3a, and RBM3b, plus the β-collar domain [21, 22]. In addition to interacting with the C ring, the MS ring houses the flagellar type III secretion system (T3SS) that transports subunits of the axial components (e.g., rod, hook, and filament proteins) of the flagellum across the cell membrane [23]. Interestingly, FliF forms rings of mixed symmetry: an inner ring having 23-fold symmetry that interacts with the export gate of the T3SS and an outer ring with 34-fold symmetry that matches the symmetry of the C ring [24, 25].

Campylobacter jejuni is a member of the phylum Campylobacterota and is closely related to H. pylori. Gao and co-workers identified FlgV as a previously uncharacterized protein required for motility in C. jejuni and showed that the protein interacted with FliF in a co-immunoprecipitation assay [26]. However, the exact structure and function of FlgV remain to be defined. We report here that H. pylori and other members of the phylum Campylobacterota have FlgV homologs. Deleting flgV in H. pylori resulted in a reduction in the number of flagella per cell (i.e., hypoflagellation) and significantly impaired motility of the strain in soft agar medium. The hypoflagellation phenotype of the ΔflgV mutant did not fully account for the impaired motility of the strain, suggesting that FlgV plays roles in both flagellar assembly and function. Comparative analysis of in-situ structures of flagellar motors of H. pylori wild-type and ΔflgV mutants revealed that FlgV forms a ring closely associated with the MS ring and FliGN. We hypothesize that the FlgV ring is an adaptation that allows the large-diameter, high-torque-generating motors of members of the phylum Campylobacterota to assemble and operate efficiently.

Materials and methods

Bacterial strains and growth conditions

Escherichia coli NEB TURBO cells were used for cloning and plasmid construction and grown in LB liquid or agar medium supplemented with ampicillin (100 μg/ml) or kanamycin (30 μg/ml) when appropriate. H. pylori strains and plasmids used in this study are listed in Table 1. H. pylori strains were grown microaerobically in an atmosphere consisting of 10% CO2, 4–6% O2, and 84–86% N2 at 37°C on tryptic soy agar supplemented with 5% horse serum (TSA-HS) or Columbia agar supplemented with 5% sheep blood. Liquid cultures of H. pylori were grown in brain heart infusion (BHI) medium supplemented with 5% heat-inactivated horse serum with shaking in serum bottles in an atmosphere consisting of 5% CO2, 10% H2, 10% O2, and 75% N2. H. pylori growth medium was supplemented with kanamycin (30 μg/ml) or sucrose (5%) when appropriate.

thumbnail
Download:
Table 1. Plasmids and H. pylori strains used in this study.

https://doi.org/10.1371/journal.pone.0287514.t001

Construction of H. pylori ΔflgV mutants

All primers used for PCR in the construction of H. pylori mutants are listed in S1 Table. Plasmids and H. pylori stains used in the study are listed in Table 1. Genomic DNA from H. pylori G27 was purified using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA) and used as the PCR template to construct the suicide vectors used to generated deletion mutants. PCR was carried out using Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA), and the resulting amplicons were incubated with Taq polymerase (Promega) at 72°C for 10 min to add 3’-A overhangs for T/A cloning with pGEM-T Easy plasmid (Promega). A 610 bp DNA fragment upstream of flgV was amplified using PCR primers 85 and 86, and a 600 bp downstream DNA fragment was amplified using PCR primers 87 and 88. PCR SOEing was used to join the upstream and downstream regions, and the resulting amplicon was cloned into pGEM-T Easy to generate plasmid pKHG25. Plasmid pKHG25 was cut at unique XhoI and NheI restriction sites and ligated with a kanR-sacB cassette [27] to generate plasmid pKHG26. The suicide vector pKHG26 was introduced into H. pylori G27M or H. pylori B128 by natural transformation. Transformants in which the kanR-sacB cassette on the plasmid had replaced the chromosomal copy of flgV by allelic exchange were selected on TSA-HS supplemented with kanamycin. Replacement of the kanR-sacB cassette with flgV in kanamycin-resistant transformants was confirmed by PCR, and the resulting strains were designated KHG40 (B128 mutant) and KHG45 (G27M mutant). The suicide vector pKHG25 was introduced into strains KHG40 and KHG45 by natural transformation, and sucrose-resistant transformants in which the unmarked flgV deletion carried in plasmid pKHG25 had replaced the kanR-sacB cassette in the chromosomal copy of flgV by allelic exchange were isolated by plating onto TSA-HS supplemented with 5% sucrose. Sucrose-resistant colonies were screened for sensitivity to kanamycin, and the deletion of flgV in a kanamycin-sensitive isolate was confirmed by PCR and DNA sequencing (Eton Bioscience, Research Triangle Park, NC, USA) of the resulting amplicon. The resulting G27M and B128 ΔflgV mutants were designated KHG48 and KHG62, respectively.

Complementation of flgV mutation.

To facilitate complementation assays in H. pylori, we modified the shuttle vector pHel3 [28] for Golden Gate assembly using tandem sites for the Type IIS restriction enzyme BspQ1. The modified plasmid, which we designated pHel3-GG, contains the fliF promoter and the ureA ribosome-binding site upstream of the tandem BspQ1 sites where the cloned gene is introduced. Primers 151 and 152 were used to amplify flgV from H. pylori G27 genomic DNA. The primers introduced BspQ1 sites immediately upstream and downstream of the start and stop codons of flgV, respectively. The resulting amplicon and pHel3-GG were digested together with BspQ1 (New England Biolabs) for 1 h at 50°C, after which the amplicon and vector were ligated using Fast-Link DNA ligase (Biosearch Technologies, Hoddesdon, UK) for 30 min at room temperature. The reaction mix was incubated with additional BspQ1 for 1 h at 50°C, then used for transformation of E. coli. A plasmid containing the expected insert was identified following restriction enzyme digestion, and the identity of the insertion was confirmed by DNA sequencing (Eton Biosciences). The resulting plasmid, pJMB36, was introduced into KHG48 by natural transformation to generate strain JMB79.

Site-directed mutagenesis of flgV.

Specific mutations were introduced into flgV carried on plasmid pJMB36 using the primer pairs 153 and 154, 155 and 156, or 157 and 158 together with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) per the manufacturer’s instructions. The resulting plasmids, pJMB39 and pJMB38, were individually introduced into strain KHG48 by natural transformation to generate strains JMB81 and JMB82, respectively.

Construction of H. pylori strain expressing FlgV-YFP fusion.

PCR primers 85 and 74 amplified flgV and an upstream flanking region totaling 877 bp, omitting the stop codon of flgV. Primers 75 and 76 were used to amplify yfp and added a sequence encoding a flexible linker (Gly-Ser-Ala-Gly-Ser-Gly) that joined flgV and yfp. Primers 77 and 78 amplified a 512 bp downstream sequence of flgV. PCR SOEing was used to join all three fragments, and the resulting amplicon was cloned into pGEM-T Easy to generate suicide vector pKHG35. Plasmid pKHG35 was introduced to strain KHG45 by natural transformation, and sucrose-resistant transformants in which the kanR-sacB cassette in the chromosomal copy of flgV had been replaced with the flgV-yfp fusion by allelic exchange were isolated on TSA-HS supplements with kanamycin. The presence of the flgV-yfp fusion in the flgV locus was confirmed by PCR, and the strain was designated KHG58.

Motility assay

Motility of H. pylori strains in a soft agar medium consisting of Mueller-Hinton broth, 10% heat-inactivated horse serum, 20 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.0), and 0.4% noble agar was tested as described [27]. Diameters of the swim halos emanating from the point of inoculation in the soft agar medium were measured 7 d post-inoculation. A minimum of three replicates were done for each strain. Mean values for swim halo diameters were calculated, and a two-sample t test was used to determine statistical significance.

Transmission electron microscopy

H. pylori cultures were grown to late-log phase (A600 = ∼1.0) in BHI supplemented with 10% heat-inactivated horse serum. Cells from the cultures were collected by centrifugation, fixed with formaldehyde and glutaraldehyde, and negatively stained with uranyl acetate as described [27]. Cells were visualized using a JEOL JEM 1011 transmission electron microscope operated at 80 kV.

Genome sequencing and analysis

A genomic library of H. pylori G27M was prepared with the Illumina iTruSeq adaptor kit from 500 ng of gDNA and sequenced at the University of Georgia Genomics Facility by Illumina sequencing. Sequence analysis was performed using the breseq computational pipeline [29]. A reference genome was constructed by mapping reads on an NCBI genome for H. pylori G27 (Accession no.: NC_011333.1). Reads were mapped to the reference genome, and a minimum variant frequency of 0.8 was used to call variants and identify SNPs. Variants and SNPs in the strains were compared manually to identify those present only in the suppressor strains.

Protein modeling and structure analysis

Tertiary structures of FlgV homologs and protein-protein interactions were predicted using the AlphaFold2 (ColabFold v1.5.1) tool in UCSF ChimeraX 1.5 [30]. Superimpositions of protein structures were performed using the Matchmaker tool in UCSF ChimeraX 1.5. All other visualizations and analyses of protein structures were conducted in UCSF ChimeraX 1.5.

Cryo-electron tomography (cryo-ET)

H. pylori strains were grown on Columbia agar plates supplemented with 5% horse red blood cells at 37°C in 10% CO2 conditions. For the complemented strains, the medium was supplemented with 30 μg/mL of kanamycin to maintain the shuttle vector. Bacteria from the agar medium were resuspended in phosphate buffer saline (PBS) and mixed with 10 nm of BSA gold tracers (Aurion, Wageningen, NL). The mixtures were deposited on freshly grow-discharged cryo-EM grids (Quantifoil R2/1, Cu 200, Ted Pella, Inc., Redding, CA, USA) and then frozen into liquid ethane using a manual plunger.

Cryo-ET data collection and processing.

Frozen-hydrated specimens were visualized in a 300kV Titan Krios electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a K3 summit direct detection camera and a BioQuantum energy filter (Gatan, Pleasanton, CA, USA). Tilt series were acquired at 42,000x magnification (corresponding to a pixel size of 2.148 Å at the specimen level) using SerialEM [31] and FastTomo script [32] based on a dose-symmetric scheme at defocus ∼4.5 μm. The stage was tilted from -48° to +48° at 3° increments. The total accumulative dose for each tilt series was ∼60 e-/Å2. MotionCor2 was used for drift correction [33]. Gold tracer beads were tracked to align all image stacks using IMOD [34]. Gctf was used for all aligned stacks to estimate defocus [35], and then the ctfphaseflip function in IMOD was used for contrast transfer function (CTF) correction.

Subtomogram averaging.

Tomo3D [36] was used for 3D reconstruction, and a total of 268, 420, 224, 332, and 214 tomograms were reconstructed from the aligned tilt series of the wild-type, ΔflgV with ΔfliL (G27M), ΔflgV (B128), FlgV-YFP, and complemented strains, respectively. Binned 6 × 6 × 6 tomograms using the simultaneous iterative reconstructive technique (SIRT) were used to select 824, 2523, 654, 1255, and 659 flagellar motors at the bacterial pole from the wild-type, ΔflgV with ΔfliL (G27M), ΔflgV (B128), FlgV-YFP, and complemented strains, respectively. After particle picking, tomograms with weighted back projection (WBP) were used for the initial subtomogram average structures using i3 suite [37, 38]. Binned 4 × 4 × 4 subtomograms were used to refine the intact motor structures. Classification was used to remove non-flagellated motors.

Results

FlgV homologs are widespread among members of the phylum Campylobacterota

H. pylori 26695 was reported to lack a FlgV homolog [26], but this report was based on an incorrect annotation of the flgV locus that identified an open reading frame in the opposite orientation [39]. A subsequent analysis of the region revealed an open reading frame between flhG and fliA in the same orientation as these genes, and the gene was given the locus tag HP1033b (Fig 1A) [40]. The revised annotation of the flhG-fliA region was consistent with the genomes of other H. pylori strains, including H. pylori G27, where HPG27_395 is the HP1033b homolog. A blastp analysis revealed homology between HPG27_395 and C. jejuni FlgV, although the sequence similarity between the two proteins is low (30% identity/53% similarity over 89.4% of total length; Table 2). HPG27_395 and C. jejuni FlgV are of similar lengths (104 and 118 amino acid residues, respectively), and both are encoded in genes located between flhG and fliA (Fig 1B). FlhG is a MinD-like protein that controls flagellum number in many polar-flagellated bacteria [41, 42], while FliA (σ28) is an alternative sigma factor needed for transcription of flagellar genes that are required late in the flagellum assembly pathway [43–45]. HPG27_395 and C. jejuni FlgV are predicted integral membrane proteins with similar membrane topologies (S1 Fig), and the tertiary structures of HPG27_395 and C. jejuni FlgV predicted by the AlphaFold2 tool in ChimeraX [30] are very similar (Fig 1C). Taken together, these data strongly suggest that HPG27_395 and C. jejuni FlgV are structurally and functionally equivalent, and hence we refer to HPG27_395 as FlgV.

thumbnail
Download:
Fig 1. Organization of operons containing flgV in H. pylori and representative members of the phylum Campylobacterota.

(A) Organization of genes within operon containing flgV (red) in H. pylori 26695. The smaller arrows upstream of aroQ and within flhG indicate identified transcriptional start sites in H. pylori 26695 [46]. Flagellar genes in the operon are flhF (dark blue), flhG (purple), fliA (green), fliM (dark gold), and fliY (black). Other genes are aroQ (gold), folK (light blue), and three genes of unknown function (HP1036, yellow; HP1029, dark olive; and HP1028, navy blue). (B) Synteny of flhFGflgV in representative genera of Campylobacterota. Homologous genes are color coded as indicated in panel A. Genes absent from the H. pylori 26695 flhFGflgV operon include pepQ (light green), potential flgJ homolog (dark red), potential flgN homolog (dark orange), hypothetical protein (white), fliE (light blue), flgB (pink), and fliK (peach). In A. butzleri, flhFGflgV appears to be part of a larger operon of ∼30 flagellar and chemotaxis genes. (C) Tertiary structures of FlgV proteins from H. pylori, C. jejuni, and A. butzleri predicted by the AlphaFold2 tool in ChimeraX [30]. The N-terminus (N) and C-terminus (C) of the protein are indicated. Regions in blue indicate a high confidence for predicted structure, while regions in yellow indicate a lower confidence for predicted structure.

https://doi.org/10.1371/journal.pone.0287514.g001

thumbnail
Download:
Table 2. C. jejuni FlgV homologs in various genera of Campylobacterota identified by blastp analysis.

https://doi.org/10.1371/journal.pone.0287514.t002

A blastp search of the National Center for Biotechnology Information (NCBI) database revealed that homologs of C. jejuni FlgV are widespread among members of the phylum Campylobacterota (Table 2). In each case, the flgV homolog is located between flhG and fliA (Fig 1B). Although a blastp analysis failed to identify a FlgV homolog in Arcobacter butzleri 7h1h, the genome of the strain has a gene immediately downstream of flhG that encodes a protein similar in size, predicted membrane topology, and predicted tertiary structure to that of C. jejuni FlgV (Fig 1C). The failure to identify a FlgV homolog in A. butzleri from the blastp analysis was not unexpected as the Arcobacter flagellar motor proteins were reported to branch from the root of the Campylobacterota motor clade [47].

FlgV is required for robust motility of H. pylori in soft agar medium

To determine if FlgV was required for flagellum function in H. pylori, we initially deleted flgV in H. pylori G27 and examined the motility of the resulting mutant in soft agar medium. Although the △flgV mutant produced a smaller swim halo than the G27 parental strain, the parental strain produced a relatively small swim halo, which limited our confidence in ascribing a motility defect to the △flgV mutant. To address this issue, we isolated a highly motile variant of H. pylori G27 from a motility enrichment and designated the motile variant as G27M. Whole genome sequencing of H. pylori G27M indicated that the strain has eighty single nucleotide polymorphisms (SNPs) and indels compared to an NCBI genome for H. pylori G27. The vast majority of the SNPs and indels presumably resulted from repeated passage of the G27 parental strain in the laboratory rather than during enrichment for enhanced motility. The SNPs and indels are in the coding regions of eight genes and twenty-nine pseudogenes (S2 Table), and twenty are in intergenic regions (S3 Table). G27M has intragenic mutations in two flagellar genes, flgH (encodes L ring protein) and fliL (encodes a motor protein). The mutation in flgH changed Gly-178 to Cys, while the mutation in fliL was a nonsense mutation that introduced a stop codon at codon 78 (Gln-78 to stop) (S2 Table). FliL is an integral membrane protein involved flagellar motor function in various bacteria [48–55]. In-situ structures of the H. pylori and Borrelia burgdorferi flagellar motors showed that the C-terminal periplasmic domain of FliL (FliLC) forms a ring that surrounds the plug/linker regions of the MotB dimer in its extended, active form [53, 56].

Deleting flgV in H. pylori G27M resulted in hypoflagellation and a significant motility defect in soft agar medium (Fig 2A and 2B). Introducing flgV on the shuttle vector pHel3 into the G27M △flgV mutant complemented the motility and hypoflagellation defects, indicating that loss of FlgV was responsible for these phenotypes in the ΔflgV mutant.

thumbnail
Download:
Fig 2. Motility and flagellation phenotypes of the H. pylori G27M ΔflgV mutant.

(A) Motility of H. pylori G27M, ΔflgV mutant (G27M ΔflgV), and ΔflgV mutant complemented with plasmid-borne copies of wild-type flgV (G27M ΔflgV pflgV), flgV allele encoding FlgVF15A,F16A variant (G27M ΔflgV pflgVF15A/F16A), or flgV allele encoding FlgVE71A,E72A variant (G27M ΔflgV pflgVE71A,E72A). Strains were stab inoculated into soft agar medium, and diameters of the resulting swim halos were measured following 7 d incubation. Mean values for swim halo diameters of each strain were normalized to the mean swim halo value for H. pylori G27M. Number of technical replicates for the strains ranged from 3 to 12. Error bars indicate the standard deviation of the mean. Asterisks indicate swim halo diameters that differed significantly from those of G27M (p-value <0.00001). Statistical analysis of the data was done using a two-sample t test. (B) Flagella were counted for at least 100 cells per strain. Asterisks indicate numbers of flagella per cell that were significantly different (p-value <0.00001) as determined using a Mann-Whitney U test. (C) Prediction aligned error (PAE) score for the model for interactions between FlgV and FliF. Both the x- and y-axis indicate the position of individual amino acids. The numbers on the x-axis indicate the approximate amino acid positions for FliF. The uncertainty in the predicted distance of two amino acids is color coded from blue (0 Ǻ) to white (30 Ǻ), as indicated by the bar at the top of the figure. The upper left quadrant corresponds to errors in the distances of residues within FlgV, and the lower right quadrant to errors within FliF. Transmembrane helices in FlgV and FliF are indicated by the red bars at the top of the figure. Note the lower error for the distances of the transmembrane helices of the two proteins as seen in the lower left and upper right quadrants. (D) AlphaFold2 predictions of protein-protein interactions between transmembrane helices of FliF (blue) and FlgV (pink) modeled in ChimeraX [30]. Regions highlighted in red are predicted points of contact between the proteins. Predicted distances between residues Phe-15 and Phe-16 in FlgV and residues Phe-466 and Tyr-467 in FliF are indicated. FliF C-terminal transmembrane helix (FliF TMC), FliF N-terminal transmembrane helix (FliF TMN), first transmembrane helix in FlgV (FlgV TM1), second transmembrane helix in FlgV (FlgV TM2).

https://doi.org/10.1371/journal.pone.0287514.g002

Since G27M has mutations in flgH and fliL, we wished to determine if the phenotype of the G27M ΔflgV mutant is dependent on the mutations in these genes. To address this issue, we deleted flgV in H. pylori B128, which has wild-type flgH and fliL alleles, and characterized the resulting mutant. As observed with the G27M △flgV mutant, the B128 ΔflgV mutant was hypoflagellated and displayed a motility defect in soft agar medium (S2 Fig), indicating that the motility and flagellation defects in the G27M ΔflgV mutant were not reliant on the mutations in flgH or fliL.

Conserved phenylalanine residues in the H. pylori FlgV transmembrane region are required for flagellum assembly

Alignment of the H. pylori G27 FlgV sequence with the sequences of FlgV homologs from nine representative species of Campylobacterota genera revealed few conserved amino acid residues (S3 Fig). A notable exception, however, was a GFFxG motif in the first transmembrane helix of H. pylori FlgV that was present in all the FlgV homologs we examined. GxxxG motifs in transmembrane regions are often involved in protein-protein interactions [57]. The Phe residues in the GFFxG motif of H. pylori FlgV (Phe-15 and Phe16) were changed to Ala residues to examine the potential role of these conserved residues in FlgV function. In addition to introducing alanine substitutions in the conserved Phe-15 and Phe-16 residues, the conserved residue Glu-71 together with adjacent amino acid residue Glu-72 were also changed to Ala residues. Glu-71 and Glu-72 are located in the C-terminal domain of FlgV (FlgVC).

Both the FlgVF15A,F16A and FlgVE71A,E72A variants supported robust motility of the G27M ΔflgV mutant in soft agar medium (Figs 2A and S4). The strain expressing the FlgVE71A,E72A variant was well flagellated and had significantly more flagella per cell compared to the G27M ΔflgV parental strain (Fig 2B). By contrast, the strain expressing the FlgVF15A,F16A variant was hypoflagellated (Fig 2B), and about half the cells examined lacked flagella. The FlgVF15A,F16A variant failed to suppress the flagellation defect of the ΔflgV mutant but did rescue motility in the ΔflgV mutant, indicating that the hypoflagellation of the ΔflgV mutant does not account fully for its motility defect. Thus, FlgV plays apparent roles in both flagellar assembly and function.

Since FliF is a FlgV interaction partner [26], we modeled potential interactions between FlgV and FliF using AlphaFold2. The FlgV-FliF interaction predicted with the highest confidence involved the transmembrane helices of the proteins (Fig 2C). Interactions between the transmembrane helices involved contact at several points, including an interface between the conserved Phe-15 and Phe-16 in FlgV and Phe-466 and Tyr-467 in FliF, which are located in the transmembrane helix near the C-terminus (Fig 2D). At this position, the chains were predicted to be within 4.4 Å of each other, and each of the four residues was a predicted contact point, with at least 15 Å2 of buried solvent-accessible surface area. Modeling interactions of the FlgVF15A,F16A variant with FliF revealed that Phe-466 and Tyr-467 in FliF and Ala-16 in FlgV no longer served as contact points, and the distances between the chains increased from 4.4 Å to between 5.8 and 8.9 Å. Alignment of the predicted C-terminal transmembrane helices of FliF homologs from representatives of various genera of Campylobacterota indicated that H. pylori FliF residues Phe-466 and Tyr-467 are well conserved across diverse Campylobacterota genera (S5 Fig).

FlgV forms a ring-like structure near the junction of the MS and C rings

The motility defects of the H. pylori G27M and B128 △flgV mutants suggested that the mutant motor lacks a component critical for optimum motility. To address this hypothesis, we compared in-situ structures of the motors of wild-type H. pylori G27, H. pylori G27M ΔflgV mutant, H. pylori B128 ΔflgV mutant, and H. pylori G27M ΔflgV mutant complemented with flgV. In-situ structures of the motors were generated by cryo-ET and subtomogram averaging. The motor of wild-type H. pylori G27 (Fig 3A) has a globular density on the cytoplasmic side of the inner membrane near the junction of the C ring and MS ring that is absent from the motors of the G27M ΔflgV and B128 ΔflgV mutants (Fig 3B and 3C). A corresponding density at the junction of the C ring and MS ring was noted previously for the C. jejuni flagellar motor [58]. The globular density corresponds to a ring-like structure (Fig 3E) absent from the motors of the B128 ΔflgV and G27M ΔflgV mutants (Fig 3F and 3G). These data suggest that the previously uncharacterized H. pylori motor ring is formed by FlgV.

thumbnail
Download:
Fig 3. In-situ structures of the flagellar motor in H. pylori G27 wild-type and flgV mutant cells.

(A-D) Medial slices through in-situ structures of G27 WT and several flgV mutant motors determined by cryo-ET and subtomogram averaging, respectively. A ring-like structure (highlighted by red arrows) in the G27 WT motor (A) is absent as shown by white arrows in the B128 ΔflgV mutant (ΔflgVB128) (B) and G27M ΔflgV mutant (ΔflgVG27M) (C). (D) Extra densities highlighted by green arrows are adjacent to the ring-like structure (red arrows) in G27M expressing FlgV-YFP. (E-H) Perpendicular cross sections at the top of the C ring through the G27 WT and flgV mutant motor structures corresponding to the panels (A-D), respectively. Note the absence of electron densities corresponding to the FlgV ring from two ΔflgV mutants (F, G). (I-L) Different perpendicular cross sections at the FliL ring level through the WT and flgV mutant motor structures corresponding to the panels (A-D), respectively. Note the absence of the FliL ring from the motors of the two mutants derived from strain G27M (K, L). OM, outer membrane; IM, inner membrane.

https://doi.org/10.1371/journal.pone.0287514.g003

To further assess whether FlgV does indeed form the novel motor ring, we expressed a FlgV-yellow fluorescent protein (YFP) fusion from the native flgV locus in H. pylori G27M. The motility of the strain expressing the FlgV-YFP fusion protein in soft agar medium did not differ from that of G27M (S6 Fig). The motor structure of the G27M strain expressing the FlgV-YFP fusion protein showed that the globular density near the C ring is present (Fig 3D), as well as the ring structure internal to the C ring (Fig 3H). An additional globular density was associated with the motor of the G27M strain expressing the FlgV-YFP fusion protein (Fig 3D), which corresponded to YFP. These data provide compelling evidence that FlgV forms the motor ring structure internal to the C ring, which we hereafter refer to as the FlgV ring. Consistent with the nonsense mutation in fliL identified from the whole genome sequencing of G27M (S2 Table), motors of strains derived from G27M lacked the FliLC ring (Fig 3K and 3L).

Closer examination of the FlgV ring showed that the cytoplasmic portion of the protein forms a ring with 21-fold symmetry that is approximately 33 nm in diameter (Fig 4C). The cytoplasmic portion of FliF, FliGN, and FlgVC are also closely associated and located within the same plane (Fig 4C and 4E, and 4F). Symmetries of the FlgV ring and FliGN (and C ring) differ though, as the latter has 39-fold symmetry (Fig 4C and 4F).

thumbnail
Download:
Fig 4. In-situ structure of the H. pylori FlgV ring.

(A) Cartoon of the H. pylori flagellar motor. The FlgV ring (cyan) is located near the junction of the C ring (yellow) and MS ring (light green). Core motor components (flagellar protein export apparatus, rod, hook, P ring, and L ring) are shown in gray. Stator proteins MotA and MotB are shown in orange. FliL is shown in pink. H. pylori motor accessories are shown in dark blue and include the cage-like structure, outer disk, and basal disk. Outer membrane (OM); inner membrane (IM). (B) Medial slice through subtomogram averaged structure of the H. pylori G27 flagellar motor. (C) Perpendicular cross section at the top of the C ring through the FlgV ring. Note the 39-fold symmetry of the C ring and 21-fold symmetry of the FlgV ring. (D-F) Isosurface renderings of H. pylori motor from different vantage points. (D) Exterior side view of the motor showing the basal disk, cage, and C ring. (E) View of motor tilted to show structures exposed on the cytoplasmic side of the inner membrane and location of FlgV ring (cyan) relative to MS ring (dark blue), C ring and FliGN (both in yellow), and MotA pentamers of the stator units (orange). (F) Perpendicular cross-sectional view of motor from the cytoplasmic side of the inner membrane.

https://doi.org/10.1371/journal.pone.0287514.g004

Discussion

The H. pylori flagellar motor, as well as the motors of many other bacteria, have accessories not found in E. coli and S. enterica motors [47, 58–61]. Identifying and characterizing the proteins that form these motor accessories is essential for revealing the unique adaptations that have evolved to support specific flagellar functions in different bacterial species. We report here a previously uncharacterized H. pylori motor accessory formed by FlgV. Consistent with the previous report that C. jejuni FlgV interacts with FliF [26], this FlgV ring is intimately associated with the MS ring in the H. pylori motor (Figs 3 and 4). More importantly, we found that the FlgV ring is located at the junction between the MS and C rings. Given that the MS ring and C ring are highly conserved and essential for flagellar assembly and function, the FlgV ring likely plays an important role through interactions with key components of the motor. FlgV homologs are found in many members of the phylum Campylobacterota (Table 2), and we expect the motors of these bacteria to contain the FlgV ring.

The synteny of flhFG and flgV within Campylobacterota genomes is notable as it is maintained in diverse genomic contexts (Fig 1B). In many Campylobacterota species, including H. pylori, flhFGflgV is immediately upstream of the flagellar genes fliAMY. As in H. pylori, the flhFGflgV locus in the A. butzleri genome is part of a larger flagellar operon, but the surrounding flagellar genes differ from those in the genomes of H. pylori and other Campylobacterota (Fig 1B). FlhF is a GTPase belonging to the signal recognition particle family that includes Ffh and FtsY, which direct protein substrates to the Sec system for secretion or insertion in the cell membrane [62]. FlhF cycles between a GTP-bound form that facilitates assembly of the MS ring at the cell pole in many polar-flagellated bacteria, and an inactive GDP-bound or apo-form [42]. In polar-flagellated bacteria, mutations in flhF typically result in hypoflagellation and the mislocalization of flagella to non-polar sites [63–66]. FlhG controls flagella number by stimulating the GTPase activity of FlhF, and mutations in flhG often result in hyperflagellation [67–71].

As in other bacteria, deletion of flhF in H. pylori results in hypoflagellation and the mislocalization of flagella to non-polar sites. Given that the ΔflgV mutants in both the G27M and B128 backgrounds were hypoflagellated (Figs 2B and S2B), we hypothesize that FlgV works in conjunction with FlhF to facilitate assembly of the MS ring in H. pylori. We did not observe the mislocalization of flagella to non-polar sites in the △flgV mutants though, which suggests that FlgV is not required for FlhF activity.

An intriguing question is why might FlgV be needed for MS ring assembly in H. pylori but not in S. enterica? The answer may lie in the complicated assembly of the MS ring, which involves the complex arrangement of subunits and mixed symmetries built from a single polypeptide chain [24]. While the symmetries of the H. pylori FliF rings have not been defined, it is safe to assume that the outer ring has a 39-fold symmetry that matches the symmetry of the C ring (Fig 4C and 4F) given that the S. enterica outer ring matches the 34-fold symmetry of its C ring. We expect that the H. pylori FliF inner ring has the same 23-fold symmetry found in the S. enterica FliF inner ring since it houses the export gate of the flagellar T3SS, which is highly conserved across different bacterial species. The greater number of FliF subunits in the H. pylori MS ring compared to the S. enterica MS ring may impose barriers in assembling an inner ring with a 23-fold symmetry. FlgV may overcome these barriers and facilitate building of the FliF rings. For example, interactions between the transmembrane helices of specific FlgV and FliF monomers may confine the movement of these FliF subunits and promote assembly of the inner ring from their RBM2s. In the absence of FlgV, FliF rings with incorrect symmetries may be formed that interfere with assembly of the T3SS export gate or adversely affect interactions between FliFC and FliGN. Such outcomes could result in flagellum assembly being aborted or might mark the flagellum for disassembly.

In addition to its apparent role in MS ring assembly, FlgV plays a role in motor function, as supported by the observation that the FlgVF15A,F16A variant failed to suppress the flagellation defect of the G27M ΔflgV mutant but did support robust motility of the mutant (Fig 2A and 2B). These findings indicate that the hypoflagellation of the G27M ΔflgV mutant does not account for its decreased motility as the strain expressing the FlgVF15A,F16A variant displayed a similar degree of hypoflagellation but was highly motile. The MS ring not only houses the T3SS export gate but also interfaces with the C ring to form the rotor. The connection between the MS ring and C ring involves FliGN folding around two helices of FliFC near its C-terminus [17]. The MS ring-C ring junction is critical for flagellar function, and FlgV may enhance the motility of H. pylori in soft agar medium by stabilizing this junction. Stabilizing the MS ring-C ring junction may hold particular importance for bacteria that have high-torque-generating motors, such as H. pylori and other Campylobacterota. Another possible mechanism by which the FlgV ring optimizes motor function is by facilitating productive interactions between the FliG HelixTorque and MotA. There is precedence for auxiliary proteins modulating interactions between the stator and rotor, as the Bacillus subtilis motor clutch proteins EpsE and MotI act to disengage the stator units from the rotor [72, 73].

Alternatively, the FlgV ring may assist the switch complex in controlling the rotation direction of the rotor. In the bacterial chemotaxis system, CheY-phosphate induces a directional switch that causes the FliG HelixTorque to engage the opposite side of the MotA pentamer [8, 74, 75], resulting in the MotA pentamer powering rotation of the rotor in the clockwise direction. It is possible that the FlgV ring aids in motor reversals important for agar-based motility. Examining the swimming behavior of the H. pylori △flgV mutants and △flgV motile variants should allow us to distinguish between these models for FlgV function.

Supporting information

S1 Fig. Predicted membrane topology of C. jejuni and H. pylori FlgV homologs.

The amino acid sequences of C. jejuni 81–176 and H. pylori G27 FlgV homologs were analyzed using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) to predict transmembrane topology of the proteins. Predicted transmembrane helices are indicated in orange, regions of the protein predicted to be exposed on the cytoplasmic side of the membrane are indicated by the pink line, and regions of the protein predicted to be exposed on the periplasmic side of the membrane are indicated by the blue line.

https://doi.org/10.1371/journal.pone.0287514.s001

(TIF)

S2 Fig. Motility and flagellation phenotypes of H. pylori B128 ΔflgV mutant.

(A) Motilities of H. pylori B128 wild type and H. pylori B128 ΔflgV mutant in soft agar medium. Strains were stab inoculated into soft agar medium, and diameters of the resulting swim halos were measured following 7 d incubation. Bars indicate mean values for swim halo diameters. Three replicates were done for each strain. Error bars indicate standard deviation of the mean. The swim halo diameter of the ΔflgV mutant differed significantly from that of wild type (p-value <0.00001). Statistical analysis of the data was done using a two-sample t test. (B) Flagella were counted for at least 95 cells for H. pylori B128 wild type and H. pylori B128 ΔflgV. Distribution of the number of flagella per cell for ΔflgV mutant differed significantly from that of wild type (p-values <0.00001). Statistical significance for differences in the distribution of the number of flagella per cell were determined using a Mann-Whitney U test.

https://doi.org/10.1371/journal.pone.0287514.s002

(TIF)

S3 Fig. Alignment of amino acid sequences of FlgV homologs.

Amino acid sequences of FlgV homologs from H. pylori G27 (Hpy), Wolinella succinogenes DSM 1740 (Wsu), Campylobacter jejuni 81–176 (Cje), Lebetimonas natshushimae (Lna), Caminibacter mediatlanticus (Cme), Nautilia profundicola AM-H (Npr), Sulfurimonas autotrophica DSM 16294 (Sau), and Hydrogenimonas thermophila (Hth) were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Transmembrane helices 1 and 2 (TM-1 and TM-2) for H. pylori FlgV are indicated and were predicted using Phobius (https://www.ebi.ac.uk/Tools/pfa/phobius/). The conserved GFFxG motif is indicated. Arrows indicate H. pylori Glu-71 and Glu-72. Numbers indicate the lengths of the FlgV homologs.

https://doi.org/10.1371/journal.pone.0287514.s003

(TIF)

S4 Fig. Electron micrographs and motility of H. pylori G27M strains used in the study.

(A) Transmission electron micrographs of representative flagellated cells of H. pylori G27M; H. pylori G27M ΔflgV mutant; and H. pylori G27M ΔflgV mutant bearing the pHel3 vector that expresses wild-type FlgV, FlgVF15A/F16A, or FlgVE71A/E72A. (B) Motility of the H. pylori G27M derived strains in soft agar medium. Photograph of motility plate was taken 7 d post-inoculation.

https://doi.org/10.1371/journal.pone.0287514.s004

(TIF)

S5 Fig. Alignment of amino acid sequences of predicted C-terminal transmembrane helices of FliF homologs.

FliF homologs are from Arcobacter butzleri 7h1h (Abu), Hydrogenimonas thermophila (Hth), Sulfurimonas autotrophica DSM 16294 (Sau), Sulfuricurvum kujiense DSM 16994 (Sku), Campylobacter jejuni 81–176 (Cje), Sulfurospirillum multivorans DSM 12446 (Smu), Helicobacter pylori G27 (Hpy), Thiovulum sp. ES (Tsp), Wolinella succinogenes DSM 1740 (Wsu), and Helicobacter hepaticus ATCC 51449 (Hhe). Alignment was done using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Transmembrane helices were predicted using Phobius (https://www.ebi.ac.uk/Tools/pfa/phobius/). Arrows indicate H. pylori FliF Phe-466 and Tyr-467. Numbers indicate the length of the predicted transmembrane helices.

https://doi.org/10.1371/journal.pone.0287514.s005

(TIF)

S6 Fig. Motility of H. pylori G27M expressing FlgV-YFP.

Motilities of H. pylori G27M expressing wild-type FlgV (G27M) or FlgV-YFP fusion protein (G27M::flgV-yfp) in soft agar medium. Strains were stab inoculated into soft agar medium, and diameters of the resulting swim halos were measured following 7-d incubation. Bars indicate mean values for swim halo diameters. Six to eight replicates were done for each strain. Error bars indicate standard deviation of the mean. The mean swim halo diameters of the two strains did not differ significantly from each other (p-value = 0.37). Statistical analysis of the data was done using a two-sample t test.

https://doi.org/10.1371/journal.pone.0287514.s006

(TIF)

S1 Table. Primers used in this study.

https://doi.org/10.1371/journal.pone.0287514.s007

(DOCX)

S2 Table. Intragenic mutations identified in H. pylori G27M.

https://doi.org/10.1371/journal.pone.0287514.s008

(DOCX)

S3 Table. Intergenic mutations identified in H. pylori G27M.

https://doi.org/10.1371/journal.pone.0287514.s009

(DOCX)

Acknowledgments

We thank Jennifer Aronson for critical reading of the manuscript.

References

  1. 1. Eaton KA, Morgan DR, Krakowka S. Motility as a factor in the colonisation of gnotobiotic piglets by Helicobacter pylori. J Med Microbiol. 1992;37:123–7. pmid:1629897
  2. 2. Ottemann KM, Lowenthal AC. Helicobacter pylori uses motility for initial colonization and to attain robust infection. Infect Immun. 2002;70(4):1984–90. pmid:11895962
  3. 3. Berg HC. The rotary motor of bacterial flagella. Annu Rev Biochem. 2003;72:19–54. pmid:12500982
  4. 4. Chevance FF, Hughes KT. Coordinating assembly of a bacterial macromolecular machine. Nat Rev Microbiol. 2008;6(6):455–65. pmid:18483484
  5. 5. Macnab RM. How bacteria assemble flagella. Annu Rev Microbiol. 2003;57:77–100. pmid:12730325
  6. 6. Manson MD, Tedesco P, Berg HC, Harold FM, Van der Drift C. A protonmotive force drives bacterial flagella. Proc Natl Acad Sci U S A. 1977;74(7):3060–4. pmid:19741
  7. 7. Deme JC, Johnson S, Vickery O, Aron A, Monkhouse H, Griffiths T, et al. Structures of the stator complex that drives rotation of the bacterial flagellum. Nat Microbiol. 2020;5(12):1553–64. pmid:32929189
  8. 8. Santiveri M, Roa-Eguiara A, Kuhne C, Wadhwa N, Hu H, Berg HC, et al. Structure and function of stator units of the bacterial flagellar motor. Cell. 2020;183(1):244–57 e16. pmid:32931735
  9. 9. Hizukuri Y, Kojima S, Homma M. Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor. J Biochem. 2010;148(3):309–18. pmid:20576620
  10. 10. Hizukuri Y, Morton JF, Yakushi T, Kojima S, Homma M. The peptidoglycan-binding (PGB) domain of the Escherichia coli pal protein can also function as the PGB domain in E. coli flagellar motor protein MotB. J Biochem. 2009;146(2):219–29. pmid:19364805
  11. 11. Hosking ER, Vogt C, Bakker EP, Manson MD. The Escherichia coli MotAB proton channel unplugged. J Mol Biol. 2006;364(5):921–37. pmid:17052729
  12. 12. Hu H, Santiveri M, Wadhwa N, Berg HC, Erhardt M, Taylor NMI. Structural basis of torque generation in the bi-directional bacterial flagellar motor. Trends Biochem Sci. 2022;47(2):160–72. pmid:34294545
  13. 13. Henderson LD, Matthews-Palmer TRS, Gulbronson CJ, Ribardo DA, Beeby M, Hendrixson DR. Diversification of Campylobacter jejuni flagellar C-ring composition impacts its structure and function in motility, flagellar assembly, and cellular processes. mBio. 2020;11(1). pmid:31911488
  14. 14. Lowenthal AC, Hill M, Sycuro LK, Mehmood K, Salama NR, Ottemann KM. Functional analysis of the Helicobacter pylori flagellar switch proteins. J Bacteriol. 2009;191(23):7147–56. pmid:19767432
  15. 15. Tang H, Braun TF, Blair DF. Motility protein complexes in the bacterial flagellar motor. J Mol Biol. 1996;261(2):209–21. pmid:8757288
  16. 16. Lynch MJ, Levenson R, Kim EA, Sircar R, Blair DF, Dahlquist FW, et al. Co-folding of a FliF-FliG split domain forms the basis of the MS:C fing interface within the bacterial flagellar motor. Structure. 2017;25(2):317–28. pmid:28089452
  17. 17. Xue C, Lam KH, Zhang H, Sun K, Lee SH, Chen X, et al. Crystal structure of the FliF-FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS-C ring in the bacterial flagellum. J Biol Chem. 2018;293(6):2066–78. pmid:29229777
  18. 18. Blair DF, Berg HC. The MotA protein of E. coli is a proton-conducting component of the flagellar motor. Cell. 1990;60(3):439–49. pmid:2154333
  19. 19. Stolz B, Berg HC. Evidence for interactions between MotA and MotB, torque-generating elements of the flagellar motor of Escherichia coli. J Bacteriol. 1991;173(21):7033–7. pmid:1938906
  20. 20. Jones CJ, Macnab RM. Flagellar assembly in Salmonella typhimurium ‐ Analysis with temperature-sensitive mutants. J Bacteriol. 1990;172(3):1327–39. pmid:2407720
  21. 21. Bergeron JR. Structural modeling of the flagellum MS ring protein FliF reveals similarities to the type III secretion system and sporulation complex. PeerJ. 2016;4:e1718. pmid:26925337
  22. 22. Ueno T, Oosawa K, Aizawa S. Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium. J Mol Biol. 1994;236(2):546–55. pmid:8107139
  23. 23. Minamino T, Imada K, Namba K. Mechanisms of type III protein export for bacterial flagellar assembly. Mol Biosyst. 2008;4(11):1105–15. pmid:18931786
  24. 24. Johnson S, Fong YH, Deme JC, Furlong EJ, Kuhlen L, Lea SM. Symmetry mismatch in the MS-ring of the bacterial flagellar rotor explains the structural coordination of secretion and rotation. Nature Microbiol. 2020;5(7):966–75. pmid:32284565
  25. 25. Tan J, Zhang X, Wang X, Xu C, Chang S, Wu H, et al. Structural basis of assembly and torque transmission of the bacterial flagellar motor. Cell. 2021;184(10):2665–79 e19. pmid:33882274
  26. 26. Gao B, Lara-Tejero M, Lefebre M, Goodman AL, Galan JE. Novel components of the flagellar system in epsilonproteobacteria. mBio. 2014;5(3):e01349–14. pmid:24961693
  27. 27. Chu JK, Zhu S, Herrera CM, Henderson JC, Liu J, Trent MS, et al. Loss of a cardiolipin synthase in Helicobacter pylori G27 blocks flagellum assembly. J Bacteriol. 2019;201(21):e00372–19. pmid:31427391
  28. 28. Heuermann D, Haas R. A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation. Mol Gen Genet. 1998;257(5):519–28. pmid:9563837
  29. 29. Deatherage DE, Barrick JE. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Meth Mol Biol. 2014;1151:165–88. pmid:24838886
  30. 30. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–9. pmid:34265844
  31. 31. Mastronarde DN. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol. 2005;152(1):36–51. pmid:16182563
  32. 32. Xu A, Xu C. FastTomo: A SerialEM script for collecting electron tomography data. bioRxiv. 2021.
  33. 33. Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods. 2017;14(4):331–2. pmid:28250466
  34. 34. Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol. 1996;116(1):71–6. pmid:8742726
  35. 35. Zhang K. Gctf: Real-time CTF determination and correction. J Struct Biol. 2016;193(1):1–12. pmid:26592709
  36. 36. Agulleiro JI, Fernandez JJ. Tomo3D 2.0—exploitation of advanced vector extensions (AVX) for 3D reconstruction. J Struct Biol. 2015;189(2):147–52. pmid:25528570
  37. 37. Winkler H. 3D reconstruction and processing of volumetric data in cryo-electron tomography. J Struct Biol. 2007;157(1):126–37. pmid:16973379
  38. 38. Winkler H, Zhu P, Liu J, Ye F, Roux KH, Taylor KA. Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes. J Struct Biol. 2009;165(2):64–77. pmid:19032983
  39. 39. Tomb J-F, White O, Keflavage AR, Clayton RA, Sutton GG, Fleischmann RD, et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997;388:539–47. pmid:9252185
  40. 40. Niehus E, Gressmann H, Ye F, Schlapbach R, Dehio M, Dehio C, et al. Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori. Mol Microbiol. 2004;52(4):947–61. pmid:15130117
  41. 41. Kojima S, Terashima H, Homma M. Regulation of the single polar flagellar biogenesis. Biomolecules. 2020;10(4):533. pmid:32244780
  42. 42. Schuhmacher JS, Thormann KM, Bange G. How bacteria maintain location and number of flagella? FEMS Microbiol Rev. 2015;39(6):812–22. pmid:26195616
  43. 43. Aldridge P, Hughes KT. Regulation of flagellar assembly. Curr Opin Microbiol. 2002;5(2):160–5. pmid:11934612
  44. 44. McCarter LL. Regulation of flagella. Curr Opin Microbiol. 2006;9(2):180–6. pmid:16487743
  45. 45. Smith TG, Hoover TR. Deciphering bacterial flagellar gene regulatory networks in the genomic era. Adv Appl Microbiol. 2009;67:257–95. pmid:19245942
  46. 46. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, et al. The primary transcriptome of the major human pathogen Helicobacter pylori. Nature. 2010;464(7286):250–5. pmid:20164839
  47. 47. Chaban B, Coleman I, Beeby M. Evolution of higher torque in Campylobacter-type bacterial flagellar motors. SciRep. 2018;8(1):97. pmid:29311627
  48. 48. Attmannspacher U, Scharf BE, Harshey RM. FliL is essential for swarming: motor rotation in absence of FliL fractures the flagellar rod in swarmer cells of Salmonella enterica. Mol Microbiol. 2008;68(2):328–41. pmid:18284590
  49. 49. Jenal U, White J, Shapiro L. Caulobacter flagellar function, but not assembly, requires FliL, a non-polarly localized membrane protein present in all cell types. J Mol Biol. 1994;243(2):227–44. pmid:7932752
  50. 50. Mengucci F, Dardis C, Mongiardini EJ, Althabegoiti MJ, Partridge JD, Kojima S, et al. Characterization of FliL proteins in Bradyrhizobium diazoefficiens: lateral FliL supports swimming motility, and subpolar FliL modulates the lateral flagellar system. J Bacteriol. 2020;202(5). pmid:31843800
  51. 51. Motaleb MA, Pitzer JE, Sultan SZ, Liu J. A novel gene inactivation system reveals altered periplasmic flagellar orientation in a Borrelia burgdorferi fliL mutant. J Bacteriol. 2011;193(13):3324–31. pmid:21441522
  52. 52. Suaste-Olmos F, Domenzain C, Mireles-Rodriguez JC, Poggio S, Osorio A, Dreyfus G, et al. The flagellar protein FliL is essential for swimming in Rhodobacter sphaeroides. J Bacteriol. 2010;192(23):6230–9. pmid:20889747
  53. 53. Tachiyama S, Chan KL, Liu X, Hathroubi S, Peterson B, Khan MF, et al. The flagellar motor protein FliL forms a scaffold of circumferentially positioned rings required for stator activation. Proc Natl Acad Sci U S A. 2022;119(4). pmid:35046042
  54. 54. Takekawa N, Isumi M, Terashima H, Zhu S, Nishino Y, Sakuma M, et al. Structure of Vibrio FliL, a new stomatin-like protein that assists the bacterial flagellar motor function. mBio. 2019;10(2):e00292–19. pmid:30890608
  55. 55. Zhu S, Kumar A, Kojima S, Homma M. FliL associates with the stator to support torque generation of the sodium-driven polar flagellar motor of Vibrio. Mol Microbiol. 2015;98(1):101–10. pmid:26103585
  56. 56. Guo S, Xu H, Chang Y, Motaleb MA, Liu J. FliL ring enhances the function of periplasmic flagella. Proc Natl Acad Sci U S A. 2022;119(11):e2117245119. pmid:35254893
  57. 57. Unterreitmeier S, Fuchs A, Schaffler T, Heym RG, Frishman D, Langosch D. Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs. J Mol Biol. 2007;374(3):705–18. pmid:17949750
  58. 58. Beeby M, Ribardo DA, Brennan CA, Ruby EG, Jensen GJ, Hendrixson DR. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold. Proc Natl Acad Sci U S A. 2016;113(13):E1917–26. pmid:26976588
  59. 59. Chang Y, Moon KH, Zhao X, Norris SJ, Motaleb MA, Liu J. Structural insights into flagellar stator-rotor interactions. Elife. 2019;8:e48979. pmid:31313986
  60. 60. Qin Z, Lin WT, Zhu S, Franco AT, Liu J. Imaging the motility and chemotaxis machineries in Helicobacter pylori by cryo-electron tomography. J Bacteriol. 2017;199(3):e00695–16. pmid:27849173
  61. 61. Zhu S, Nishikino T, Takekawa N, Terashima H, Kojima S, Imada K, et al. In situ structure of the Vibrio polar flagellum reveals distinct outer membrane complex and its specific interaction with the stator. J Bacteriol. 2019;202(4):e00592–19. pmid:31767780
  62. 62. Halic M, Beckmann R. The signal recognition particle and its interactions during protein targeting. Curr Opin Struct Biol. 2005;15(1):116–25. pmid:15718142
  63. 63. Balaban M, Joslin SN, Hendrixson DR. FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation and biosynthesis in Campylobacter jejuni. J Bacteriol. 2009;191(21):6602–11. pmid:19717591
  64. 64. Green JC, Kahramanoglou C, Rahman A, Pender AM, Charbonnel N, Fraser GM. Recruitment of the earliest component of the bacterial flagellum to the old cell division pole by a membrane-associated signal recognition particle family GTP-binding protein. J Mol Biol. 2009;391(4):679–90. pmid:19497327
  65. 65. Murray TS, Kazmierczak BI. FlhF is required for swimming and swarming in Pseudomonas aeruginosa. J Bacteriol. 2006;188(19):6995–7004. pmid:16980502
  66. 66. Pandza S, Baetens M, Park CH, Au T, Keyhan M, Matin A. The G-protein FlhF has a role in polar flagellar placement and general stress response induction in Pseudomonas putida. Mol Microbiol. 2000;36(2):414–23. pmid:10792727
  67. 67. Balaban M, Hendrixson DR. Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni. PLoS Pathog. 2011;7(12):e1002420. pmid:22144902
  68. 68. Campos-Garcia J, Najera R, Camarena L, Soberon-Chavez G. The Pseudomonas aeruginosa motR gene involved in regulation of bacterial motility. FEMS Microbiol Lett. 2000;184(1):57–62. pmid:10689166
  69. 69. Correa NE, Peng F, Klose KE. Roles of the regulatory proteins FlhF and FlhG in the Vibrio cholerae flagellar transcription hierarchy. J Bacteriol. 2005;187(18):6324–32. pmid:16159765
  70. 70. Dasgupta N, Arora SK, Ramphal R. fleN, a gene that regulates flagellar number in Pseudomonas aeruginosa. J Bacteriol. 2000;182(2):357–64. pmid:10629180
  71. 71. Kusumoto A, Kamisaka K, Yakushi T, Terashima H, Shinohara A, Homma M. Regulation of polar flagellar number by the flhF and flhG genes in Vibrio alginolyticus. J Biochem. 2006;139(1):113–21. pmid:16428326
  72. 72. Blair KM, Turner L, Winkelman JT, Berg HC, Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science. 2008;320(5883):1636–8. pmid:18566286
  73. 73. Subramanian S, Gao X, Dann CE, 3rd, Kearns DB. MotI (DgrA) acts as a molecular clutch on the flagellar stator protein MotA in Bacillus subtilis. Proc Natl Acad Sci U S A. 2017;114(51):13537–42. pmid:29196522
  74. 74. Carroll BL, Nishikino T, Guo W, Zhu S, Kojima S, Homma M, et al. The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching. Elife. 2020;9:e61446. pmid:32893817
  75. 75. Chang Y, Zhang K, Carroll BL, Zhao X, Charon NW, Norris SJ, et al. Molecular mechanism for rotational switching of the bacterial flagellar motor. Nat Struct Mol Biol. 2020;27(11):1041–7. pmid:32895555