Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors
2022; Springer Nature; Volume: 41; Issue: 10 Linguagem: Inglês
10.15252/embj.2021109523
ISSN1460-2075
AutoresMohammed Kaplan, Catherine M. Oikonomou, Cecily R. Wood, Georges Chreifi, Poorna Subramanian, Davi R. Ortega, Yi‐Wei Chang, Morgan Beeby, Carrie L. Shaffer, Grant J. Jensen,
Tópico(s)Genomics and Phylogenetic Studies
ResumoArticle18 March 2022free access Transparent process Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors Mohammed Kaplan Mohammed Kaplan Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Catherine M Oikonomou Catherine M Oikonomou orcid.org/0000-0003-2312-4746 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Cecily R Wood Cecily R Wood orcid.org/0000-0003-1259-3862 Department of Veterinary Science, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Methodology, Writing - review & editing Search for more papers by this author Georges Chreifi Georges Chreifi Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Poorna Subramanian Poorna Subramanian Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Davi R Ortega Davi R Ortega Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Yi-Wei Chang Yi-Wei Chang orcid.org/0000-0003-2391-473X Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Morgan Beeby Morgan Beeby Department of Life Sciences, Imperial College London, London, UK Contribution: Data curation, Writing - review & editing Search for more papers by this author Carrie L Shaffer Carrie L Shaffer orcid.org/0000-0002-7457-7422 Department of Veterinary Science, University of Kentucky, Lexington, KY, USA Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Grant J Jensen Corresponding Author Grant J Jensen [email protected] orcid.org/0000-0003-1556-4864 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA Contribution: Conceptualization, Resources, Formal analysis, Funding acquisition, Writing - review & editing Search for more papers by this author Mohammed Kaplan Mohammed Kaplan Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Catherine M Oikonomou Catherine M Oikonomou orcid.org/0000-0003-2312-4746 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Cecily R Wood Cecily R Wood orcid.org/0000-0003-1259-3862 Department of Veterinary Science, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Methodology, Writing - review & editing Search for more papers by this author Georges Chreifi Georges Chreifi Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Poorna Subramanian Poorna Subramanian Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Davi R Ortega Davi R Ortega Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Yi-Wei Chang Yi-Wei Chang orcid.org/0000-0003-2391-473X Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Contribution: Data curation, Writing - review & editing Search for more papers by this author Morgan Beeby Morgan Beeby Department of Life Sciences, Imperial College London, London, UK Contribution: Data curation, Writing - review & editing Search for more papers by this author Carrie L Shaffer Carrie L Shaffer orcid.org/0000-0002-7457-7422 Department of Veterinary Science, University of Kentucky, Lexington, KY, USA Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, USA Contribution: Data curation, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Grant J Jensen Corresponding Author Grant J Jensen [email protected] orcid.org/0000-0003-1556-4864 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA Contribution: Conceptualization, Resources, Formal analysis, Funding acquisition, Writing - review & editing Search for more papers by this author Author Information Mohammed Kaplan1, Catherine M Oikonomou1, Cecily R Wood2, Georges Chreifi1, Poorna Subramanian1, Davi R Ortega1, Yi-Wei Chang3, Morgan Beeby4, Carrie L Shaffer2,5,6 and Grant J Jensen *,1,7 1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA 2Department of Veterinary Science, University of Kentucky, Lexington, KY, USA 3Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 4Department of Life Sciences, Imperial College London, London, UK 5Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA 6Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, USA 7Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA *Corresponding author. Tel: +626 395 8827; E-mail: [email protected] The EMBO Journal (2022)41:e109523https://doi.org/10.15252/embj.2021109523 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine. Synopsis The assembly of bacterial flagellar motors is an “inside-out” process, starting from the inner membrane. Cryo-ET reveals novel structures that surround the cytoplasmic C-ring of high-torque motors that intriguingly, are only associated with certain assembly stages and are not present in the fully assembled motor. Transient cytoplasmic rings surround the motors of various Beta-, Gamma-, and Epsilon-proteobacteria during assembly. These rings appear to surround the FliG-ring of the assembling motors. In Helicobacter pylori, these rings are dependent on components of the flagellar type III secretion system. Introduction Having undergone billions of years of optimization through natural selection, the bacterial flagellum represents a rich system to study how biological nanomachines are assembled at the macromolecular level. A prime example of a multi-component complex that is built through a tightly regulated self-assembly process (Macnab, 2003), the flagellum consists of a long extracellular filament driven by a cell envelope-embedded motor at its base, connected through a flexible joint known as the hook (Fig EV1). Click here to expand this figure. Figure EV1. Schematic of the Escherichia coli flagellar motor A schematic representation of the E. coli flagellum highlighting its major parts and their constituent proteins. The same color code is used in all main and supplemental figures, except Appendix Fig S3. Download figure Download PowerPoint Our current understanding of how this structure assembles is based primarily on the popular model organisms Escherichia coli and Salmonella enterica (both Gammaproteobacteria). Flagellar biogenesis is believed to proceed in an inside-out fashion (Fig 1), beginning with the assembly of a part of the flagellar type III secretion system (fT3SS) known as the core complex (fT3SScc), an integral inner membrane (IM) complex consisting of five proteins (FliP, FliQ, FliR, FlhB, and FlhA) (Abrusci et al, 2013). A sixth protein, FliO, is believed to be required for the assembly of the fT3SScc but does not form part of the complex (Fabiani et al, 2017; Fukumura et al, 2017). The assembly of the fT3SScc initiates with FliP, which forms a pentameric platform on which FliQ, FliR, and FlhB assemble to create a FliP5FliQ4FliR1FlhB1 subcomplex upon which a nonameric FlhA ring is built (Fabiani et al, 2017; Kuhlen et al, 2018; Minamino et al, 2019; Milne-Davies et al, 2021). The C-terminal domains of both FlhA and FlhB remain in the cytoplasm (Fig 1, top). Figure 1. Current understanding of flagellar assembly based on the canonical systems of Escherichia coli and Salmonella enterica A schematic summary of our current understanding of how E. coli and S. enterica assemble the fT3SScc (top) and flagellum and the various flagellar complexes formed during this process. See also (Jones & Macnab, 1990; Kubori et al, 1992; Macnab, 2003; Li & Sourjik, 2011; Fabiani et al, 2017). The color code for the different parts of the flagellum is used in all subsequent schematics (except Appendix Fig S3), and labels are shown only in some schematics to avoid redundancy. HFJPs = hook-filament junction proteins. Download figure Download PowerPoint Upon formation of the fT3SScc, the MS-ring (FliF) and C-ring (FliG, FliM, and FliN) assemble, followed by the cytoplasmic fT3SS ATPase (FliH, FliI, and FliJ) inside the C-ring. Biogenesis of the fT3SS promotes secretion of additional periplasmic and extracellular components across the IM including the flagellar rod (FliE, FlgB, FlgC, FlgF, and FlgG), hook (FlgE), hook-filament junction proteins (FlgK and FlgL), filament capping protein (FliD), and filament subunits (FliC) (Macnab, 2003) (Fig 1, bottom). The periplasmic P-ring (FlgI) and L-ring (FlgH) which assemble around the rod are secreted through the IM via the Sec pathway (Macnab, 2003). Additional chaperones and capping proteins assist in the assembly process. While all known motors share this conserved core, various embellishments are found in different species, highlighting the continuous evolution of this nanomachine (Chen et al, 2011; Zhao et al, 2014; Qin et al, 2017; Chaban et al, 2018; Kaplan et al, 2019a). The C-ring, MS-ring, rod, hook, and filament make up the rotor of the motor. Torque is generated by a ring of IM-embedded ion channels known as stators which translate the flux of ions into rotation of the C-ring (also called the torque ring) through interaction with FliG (Chang et al, 2020; Deme et al, 2020; Santiveri et al, 2020). While the majority of motors use H+- or Na+-dependent stators, others can use different cations (Terahara et al, 2012; Ito & Takahashi, 2017). In addition, stators have also been discovered that can use either Na+ or H+ in a pH-dependent manner, as well as motors with dual stator systems (Terahara et al, 2008; Thormann & Paulick, 2010). The motors of E. coli and S. enterica have dynamic stators that continually associate and disassociate from the rotor in response to the external environment (Lele et al, 2013). The motors of some other species generate higher torque with wider, more highly-occupied stator rings stabilized by periplasmic scaffolds and chaperones that directly interact with the stators to maintain their integrity (Beeby et al, 2016; Kaplan et al, 2019a; Ribardo et al, 2019). The assembly of the flagellum is believed to proceed sequentially, with the addition of each component building upon the previous one (Li & Sourjik, 2011). Additional mechanisms evolved to control the lengths of the different sections of the flagellum. For example, the periplasmic rod (driveshaft) is believed to grow until it hits the outer membrane (OM) (Cohen et al, 2017). FliK is thought to limit the length of the extracellular hook to ~ 50–55 nm (Erhardt et al, 2011; Kodera et al, 2015; Guse et al, 2020). Different models have been proposed for the assembly of the extracellular filament. First, it was proposed that the secreted subunits interact head-to-tail inside the channel of the flagellum and that the folding of subunits at the tip of the growing filament provides a pulling force on the head-to-tail chain (Evans et al, 2013). A more recent model suggests that filament assembly is instead governed by an injection-diffusion mechanism in which the growth kinetics decrease quadratically, thus providing an explanation for why growth does not continue indefinitely (Chen et al, 2017; Hughes, 2017; Renault et al, 2017). For a recent review see (Renault et al, 2019). Our current understanding of flagellar assembly comes mainly from biochemistry and negative-staining electron microscopy of purified motors, and lower-resolution light microscopy of cells (Jones & Macnab, 1990; Kubori et al, 1992; Li & Sourjik, 2011). Higher-resolution electron cryo-tomography (cryo-ET) of native motors inside intact cells captured late assembly stages in Borrelia burgdorferi (Zhao et al, 2013) and non-canonical Gammaproteobacteria (Kaplan et al, 2019b), but images of the flagellar assembly process from its earliest stages (the fT3SScc and the MS-ring) inside the cell are still lacking. Here, we investigated how flagellar motors are built in situ using cryo-ET. We identified early flagellar intermediates that precede the formation of the C-ring and rod and discovered that in the absence of the torque ring protein, FliG, the MS-ring does not assemble. Moreover, we identified novel cytoplasmic rings that surround the FliG torque ring during assembly. These ring(s) are built before the C-ring is fully assembled and persist until the stator ring assembles, but are not found in the fully assembled flagellum. By imaging mutants of candidate genes, we discovered that the fT3SScc proteins FliO and FliQ are required for the formation of the novel cytoplasmic rings. Results We identified flagellar motor assembly intermediates in electron cryotomograms of several Proteobacteria species: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Shewanella oneidensis, Pseudomonas aeruginosa, and Pseudomonas fluorescens (Table EV1). These species have various flagellar arrangements: monotrichous, amphitrichous, and lophotrichous (see Table EV1). For convenience, we refer to flagellar intermediates by the names indicated in Fig 1. In some cases, established terms are available, such as the fT3SScc, basal body (BB), and hook-basal-body (HBB) complex. For the others, we named them according to either the last component to join the complex or its dominant structural feature. Thus, we have the MS-complex, the C-complex, the R-complex, and the P-complex. Table 1 summarizes the number of examples we identified for each of these complexes in each species. Note that schematics and interpretation of complexes where not enough examples were found to produce a subtomogram average are tentative. In addition to collecting new cryotomograms for this study, we also mined the Jensen lab tomography database (Ding et al, 2015; Ortega et al, 2019), which contains more than 40,000 cryotomograms of ~ 90 bacterial species collected in the course of various projects. Table 1. Summary of the number of examples of each flagellar intermediate identified in each species investigated in this study. Species No. of tomograms MS-complex C-complex R-complex P-complex Basal body HBB Motor without collar Flagellum Helicobacter pylori 46 11 3 – – 9 1 – 34 Helicobacter pylori fliP* 76 64 15 – – – – – – H. pylori ∆flgS fliP* 84 21 2 – – – – – – Helicobacter pylori ∆fliM fliP* 265 121 – – – – – – – Helicobacter pylori ∆fliG fliP* 47 – – – – – – – – Helicobacter pylori ∆fliO fliP* 267 47 15 – – – – – – Helicobacter pylori ∆fliQ fliP* 220 94 3 – – – – – – Helicobacter pylori ∆fliF fliP* 455 – – – – – – – – Shewanella oneidensis ∆flgH 59 – – – 13 – – – – Campylobacter jejuni ∆flhBC 19 4 15 – – – – – – Campylobacter jejuni ∆flhAC 54 8 30 – – – – – – Pseudomonas fluorescens 32 – – 2 – 2 – – 4 Hylemonella gracilis 76 5 1 1 35 1 7 8 86 Novel transient cytoplasmic rings surround the FliG ring during the assembly of high-torque motors in Beta- and Epsilonproteobacteria In Hylemonella gracilis, a Betaproteobacterium whose thin cells yield high-quality cryo-ET images, we identified eight classes of flagellar motor intermediates corresponding to the MS-complex, the C-complex, the R-complex, the P-complex, BB, HBB, otherwise full flagella lacking the periplasmic collar characteristic of this species (see (Chen et al, 2011)), and full flagella with the periplasmic collar (Fig 2 and Movies EV1 and EV2). For many of these classes, we were able to collect enough examples for subtomogram averaging to reveal greater detail (Fig 2A and D, and F–H and Appendix Fig S1). We observed a novel ~55-nm-wide ring located in the cytoplasm 12 nm below the IM (green arrows in Fig 2A–F). This ring was present at the earliest stage of assembly we observed, the MS-complex, and persisted through assembly of the hook. The ring was absent only in the two final complexes where the filament was assembled, either without the periplasmic collar surrounding the P-ring (Fig 2G) or with the collar present and the stators assembled (the fully assembled flagellum) (Fig 2H). Figure 2. The flagellar assembly pathway of the Betaproteobacterium Hylemonella gracilis A–H. Slices through electron cryotomograms of H. gracilis cells (left panels), central slices through subtomogram averages (STA, middle panels), and schematic representations (right panels) representing the various assembly stages observed. White arrow in (A) points to the MS-complex; green arrows point to the novel 55-nm-wide cytoplasmic ring. Scale bars in (H) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels. Empty panels represent stages where not enough particles were found to produce a STA. OM, outer membrane; IM, inner membrane. Download figure Download PowerPoint The position of the P-ring in P-complexes was variable (Fig EV2), which explains why the P-ring was not as well-resolved in the subtomogram average of this complex (Fig 2D). We noted that the OM of H. gracilis frequently undulates, with the distance between the OM and IM varying significantly around the cell (Fig EV3). Both S. enterica and E. coli tether their OM to the PG with a protein known as Braun’s lipoprotein (Lpp) (Miller & Salama, 2018). The H. gracilis genome lacks an Lpp homolog, raising the question of how this bacterium controls its periplasmic space and whether this is related to the variable location of the P-ring. It has recently been shown that some β-barrel proteins can perform the same function as Lpp in some Gram-negative bacteria (Godessart et al, 2020; Sandoz et al, 2020). Click here to expand this figure. Figure EV2. The variable position of the P-ring in Hylemonella gracilis P-complexes A–C. Slices through electron cryotomograms of H. gracilis showing P-complexes. The variable distance between the MS- and P-rings is indicated (15, 20 and 25 nm, respectively). Scale bars are 50 nm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. The undulating outer membrane of Hylemonella gracilis A–C. Slices through electron cryotomograms of H. gracilis highlighting undulating OM (indicated by red arrows). Scale bars are 100 nm. Download figure Download PowerPoint We next examined other high-torque motors, in the Epsilonproteobacteria Helicobacter pylori and Campylobacter jejuni (Fig EV4). In cryotomograms of H. pylori, in addition to fully assembled flagella, we identified the MS-complex, the C-complex, the BB, and the HBB (Fig 3A–D). Interestingly, the MS-complex contained two cytoplasmic rings (one electron-dense and one weaker) surrounding the FlhAC densities close to the IM, as well as two additional larger cytoplasmic rings with diameters of 58 nm and 75 nm, located 17 nm below the IM (Fig 3A, dark and light green rings). These lower rings were not present at the corresponding position in fully assembled motors (Qin et al, 2017) (Fig EV4), or in the BB complex. They were also not readily apparent in cryotomograms of the C-complex, although we did not find enough examples of the complex for subtomogram averaging to definitively confirm this. In fully assembled H. pylori flagella, a periplasmic structure called the cage surrounds the rod and the MS-ring and stabilizes the stator ring (Qin et al, 2017). In individual particles, we observed a density that may correspond to the lower part of this cage in the C-complex, suggesting that it is assembled before the rest of the basal body (Fig 3B and Movie EV3). Click here to expand this figure. Figure EV4. The flagellar motors of the species investigated in this study A–E. Central slices through the STAs of fully assembled high-torque motors in species in which we identified novel cytoplasmic rings interacting with the FliG ring during assembly. The H. pylori motor is from (Qin et al, 2017), C. jejuni motor from (Beeby et al, 2016), S. oneidensis and P. aeruginosa motors from (Kaplan et al, 2019a). F, G. Slices through electron cryotomograms of P. fluorescens highlighting the presence of a basal body (F) and a disassembly PL-subcomplex (G, yellow dotted circle), showing the similarity of the motor of this species to that of P. aeruginosa. Data information: Scale bars (A–F) 10 nm, (G) 20 nm. Download figure Download PowerPoint Figure 3. Flagellar assembly intermediates in Helicobacter pylori A–D. Slices through electron cryotomograms of H. pylori (left panels) indicating the presence of various flagellar intermediates. Middle panels show central slices through STAs. Right panels show schematic representations. Question marks in the schematic in (C) indicate that it is unclear whether the densities surrounding the upper part of the C-ring are the same as the novel cytoplasmic rings seen in the MS-complex. Empty panels indicate that there were not enough examples to produce a STA. E, F. Cryotomogram slices, STAs, and schematics of H. pylori fliP* cells showing the MS-complex (E) and the C-complex (F). The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel. White arrows in (A-F) point to the particles. G, H. Central slices through the STAs of the MS-complexes of the motile H. pylori (G) and H. pylori fliP* mutant (H) with the diameters of their various constituent rings indicated. Data information: Scale bars are 50 nm for cryotomogram slices and 20 nm for STAs. Download figure Download PowerPoint In a non-motile strain of H. pylori which does not produce FliP due to a naturally occurring point mutation (Chang et al, 2018) (henceforth referred to as fliP*), we identified many MS- and C-complexes (Fig 3E and F and Appendix Fig S2). Subtomogram averaging revealed that these complexes are similar to those in motile cells (Fig 3A–D) but lack the FlhAC densities (Fig 3E and F). A ring surrounding the MS-ring, previously putatively attributed to FliL in the fully assembled motor (Qin et al, 2017), was seen associated with the MS-complex (Fig 3E). In addition, the entire MS-complex in the fliP* strain was narrower than in motile cells (Fig 3G and H). For example, the two lower cytoplasmic rings in the fliP* strain had diameters of 53 and 67 nm, respectively, compared to diameters of 58 and 75 nm in motile cells. A subtomogram average of the C-complex in the fliP* strain confirmed that the lower cage and the stator ring are assembled at this stage and that the novel cytoplasmic rings are no longer present, or at least not in the same location they occupy in the MS-complex (Fig 3F). The improved quality of the subtomogram average of the MS-complex in the fliP* strain allowed us to tentatively assign densities to FliF and FliG (Fig EV5) and compare our structure to available high-resolution structures of the purified MS-ring (Johnson et al, 2020; Kawamoto et al, 2021). Manually fitting the high-resolution structure of the purified MS-ring from S. enterica (PDB 6SCN) (Johnson et al, 2020) into the average allowed us to provisionally assign densities to different periplasmic domains including the drive-shaft-housing ring (the Beta collar), the C-ring template (the ring building motif 3 (RBM3) ring), and the RBM2+RBM1 rings. Additionally, fitting the crystal structure of FliG (which is known to interact with FliF) from Aquifex aeolicus (PDB 3HJL) (Lee et al, 2010) suggested that the dense ring directly surrounding the (here absent) FlhAC densities probably contains the C terminus of FliF (FliFC) and the N- and middle domains of FliG (FliGMN), while the peripheral ring contains the C terminus of FliG (FliGC) (Fig EV5). Click here to expand this figure. Figure EV5. Tentative component assignment to various MS-complex rings in Helicobacter pylori Surface rendering of the MS-complex of H. pylori fliP* with the crystal structures of Aquifex aeolicus FliG (PDB 3HJL) and S. enterica MS-ring (PDB 6SCN) docked inside it. RBM, ring-building motif. Download figure Download PowerPoint We also examined cryotomograms of another Epsilonproteobacterium, Campylobacter jejuni (see (Abrusci et al, 2013; Beeby et al, 2016)). In wild-type C. jejuni cells, we found only fully assembled flagella. In mutants lacking the C-terminal domain of either FlhA (ΔflhAC) or FlhB (ΔflhBC), however, which do not assemble full flagella, we identified MS- and C-complexes (Fig 4). In the ΔflhBc strain, the MS-complex comprised the MS-ring, putative FliG densities, the fT3SScc (as indicated by the presence of the cytoplasmic FlhAC densities), and, notably, a ~ 50-nm-wide cytoplasmic ring located ~ 17 nm below the IM (as in H. pylori) (Fig 4A). The same 50-nm ring was also seen in ΔflhAC MS-complexes, which of course lack the cytoplasmic FlhAC densities (Fig 4C). Neither mutant retained the 50 nm cytoplasmic ring in the C-complex (Fig 4B and D). Fully assembled flagella of C. jejuni have two concentric periplasmic discs which assist the assembly of the stator ring (Beeby et al, 2016). Similar to the lower portion of the cage in H. pylori, we observed these discs in the C-complex of both ΔflhBc and ΔflhAC cells (Fig 4B and D). Figure 4. Flagellar intermediates in Campylobacter jejuni mutants A–D. Slices through electron cryotomograms (left panels), central slices through STAs (middle panels), and schematic representations (right panels), of C. jejuni ΔflhBC and ΔflhAC illustrating different flagellar intermediates (highlighted by white arrows). Green arrows in (A) and (C) point to the ~ 50-nm cytoplasmic ring associated with the MS-complex. Scale bars are 50 nm for cryotomogram slices, 20 nm for STAs. Download figure Download PowerPoint To summarize, we found unexpected cytoplasmic rings encircling the FliG ring in high-torque motors of Betaproteobacteria (H. gracilis) and Epsilonproteobacteria (H. pylori and C. jejuni). These rings assemble very early, associating with the MS-complex before the C-ring is built and then disassemble (or at leas
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