In situ structures of periplasmic flagella reveal a distinct cytoplasmic ATPase complex in Borrelia burgdorferi

Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific Type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we combine cryo-electron tomography and mutagenesis approaches to characterize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We define the fT3SS machine by systematically characterizing mutants lacking key component genes. We discover that a distinct cytosolic ATPase complex is attached to the flagellar C-ring through multiple spoke-like linkers. The ATPase complex not only strengthens structural rigidity of the C-ring, but also undergoes conformational changes in concert with flagellar rotation. Our studies provide structural framework to uncover the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the bases for the development of novel therapeutic strategies against several pathogenic spirochetes.


INTRODUCTION
formation of the hook and the filament in the periplasmic space (Zhao et al., 2013). The flagellarspecific type III secretion system (fT3SS) is responsible for the assembly of the periplasmic flagella.
assembles into a homo-hexamer (Imada et al., 2007;Macnab, 2003). FliH probably acts as a negative 93 regulator of the FliI ATPase, and FliJ has a chaperone-like activity that prevents substrate aggregation 94 (Fraser et al., 2003). FliH, FliI, and FliJ coordinately deliver a chaperone-substrate complex to the with FliN on the C-ring (Minamino et al., 2009). FlhA is required for stable anchoring of the FliI 6 ring 98 to the gate (Bai et al., 2014). burgdorferi flhAD158E cells that exhibit non-motile phenotype, or the reduced-motility mutant displayed by the flhAD158N cells (not shown).

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In the ΔflhB mutant, both the ATPase complex and the export apparatus are evident (Fig. 3D), 174 suggesting that FlhB does not contribute to the structure or positioning of the ATPase complex or the 175 export apparatus. In contrast, in the ΔfliP (Fig. 3E, Fig. S4), ΔfliQ and ΔfliR mutants (Fig. S5, S6), the 176 membrane underneath the MS-ring has a flat surface, and the FlhA complex appears to be absent or 177 disordered. Therefore, the structures of these mutants are strikingly different from the WT structure  Fig. 3H). It has been recently of the FliP/Q/R channel. FlhB is not well-defined in our structures, but it is essential for fT3SS 185 function.

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The FlhA complex stabilizes the ATPase complex

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The FliI/FliH ATPase complex appears to be associated with the bottom portion of the C-ring, 189 even in the absence of FlhA. However, the FliI/FliH-associated density in the ΔflhA mutant is 190 indistinct, indicating that the FlhA complex is involved in stabilization of the ATPase complex under 191 the C-ring (Fig. 3C). In addition, the ATPase complex appears to shift away from the 192 implying that there is an interaction between the FlhA and ATPase complexes. Therefore, we propose 193 that the FlhA complex is not essential for the assembly of the ATPase complex, but it provides a 194 docking site to stabilize the ATPase complex. Our result is consistent with previous study that FlhA is 195 required for stable anchoring the FliI 6 ring to the export gate (Bai et al., 2014).

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FliO has a limited role in the flagellar assembly in B. burgdorferi

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FliO is the less conserved among the membrane proteins of the export apparatus. In fact, B. 199 burgdorferi FliO and its Salmonella homolog have very weak sequence identify (13% ; Table S1). We

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The export apparatus has a profound impact on flagellar motor formation in B. burgdorferi

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To understand the overall contribution of the membrane-bound export apparatus proteins to the 208 structure and assembly of the flagellar motor, we generated a quintuple ΔfliP-flhA mutant by deleting fliP, fliQ, fliR, flhB and flhA genes using the Cre-LoxP method (Bestor et al., 2010). As expected, the similar to that of the ΔfliQ motor (Fig. 4C), although the resolution of the image is relatively poor 213 because fewer motors were available for sub-tomogram averaging. Importantly, the ATPase complex 214 (indicated by orange arrow in Fig. 4D) remains in a similar location as in the ΔfliQ or ΔflhA motors, 215 supporting the notion that the export apparatus is dispensable for the formation of the ATPase 216 complex. However, absence of the export apparatus proteins has a significant impact on motor 217 formation, as the number of motors per cell tip is highly variable in the mutants of the export    (Fig. 5). The entire ring fits well into the torus-like density (Fig. 5B, C), suggesting that the FlhA 238 complexes also form a nonameric ring in B. burgdorferi. Three subdomains (SD1, SD3 and SD4) of 239 FlhA C are located inside the nonameric FlhA C ring, whereas the SD2 domain is located outside of the 240 ring (Fig. S7). The distance between the FlhA C ring and the cytoplasmic membrane is about 6 nm. The

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FlhA C is linked to the FlhA trans-membrane domain embedded in the cytoplasmic membrane under 242 the MS-ring. The central channel of the export apparatus appears to be aligned with the central axis of 243 the MS-ring and the ATPase complex (Fig. 5D).

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The ATPase complex can be divided into two major components: a large central hub, and 23 245 spoke-like linkers extending to the C-ring (Fig. 5). The ATPase complex was originally proposed to 246 form a hexamer (Claret et al., 2003;Fan and Macnab, 1996;Imada et al., 2007) and is part of the 247 density beneath the FlhA C ring, as suggested by analysis of a ΔfliI mutant in Campylobacter jejuni 248 (Chen et al., 2011) andin B. burgdorferi (Lin et al., 2015). FliI, FliH, and FliJ are known to form a large 249 complex that delivers the chaperone-substrate complex to the export gate (Fraser et al., 2003; 250 Minamino and Macnab, 2000). B. burgdorferi contains the homologs of these proteins (Fig. S8).

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Therefore, we postulate that the hexametric density is composed mainly of the FliI/FliJ complex.

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Based on its similarity with a portion of the F 0 F 1 -ATPase, the FliI/FliJ complex was modeled by 253 aligning the monomer structures of Salmonella FliI and FliJ to the α 3 β 3 and γ parts of F 0 F 1 -ATPase, 254 respectively (Ibuki et al., 2011;Imada et al., 2007). The pseudo-atomic structure of FliI/FliJ fits well 255 into the spherical density (Fig. 5C). The density of FliJ is not well resolved in our maps, probably 256 because of its small size or dynamic nature (Ibuki et al., 2011). The N-and C-termini of FliJ insert into        318 burgdorferi flagellar motor, the linker between the ATPase and C-ring is likely formed by multiple (305 residues) than its homolog OrgB (170 residues). Thus, the ATPase complex not only provides a large docking platform for substrates recruitment and secretion, but also supports the integrity of the the MS-ring. The rotation of the C-ring is driven by sixteen stators that surround the C-ring and a 326 spirochete-specific periplasmic collar (Moon et al., 2016). In contrast, the pods found in Salmonella 327 injectisomes do not appear to rotate, although OrgB and SpaO likely undergo high turnover with a 328 cytoplasmic pool. These key differences between the fT3SS and vT3SS underline the distinct 329 mechanisms involved in the assembly and function of flagella and injectisomes.

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The assembly of the flagellum can be divided into two distinct processes. The first stage includes 331 the formation of the MS-ring, C-ring, export apparatus, and the stator. The second stage, which 332 includes the assembly of the rod, hook, and filament, is mediated by the fT3SS. It is generally thought 333 that the MS-ring is the first unit assembled and is central to flagellar assembly and function (Kubori et 334 al., 1992). Recently, fluorescence microscopy was used to investigate dynamic protein exchange in the 335 assembled E. coli motor structure. This study suggested that flagellar assembly is initiated by 336 oligomerization of the export protein FlhA, which is followed by the recruitment of the MS-ring

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FliQ, FliR, FlhA and FlhB, motor assembly still occurs with very low efficiency. Taken together, our 344 results imply that most export proteins are involved in the coordinated assembly of the MS-ring and 345 export apparatus, whereas FlhA is not critical in this process, at least, in B. burgdorferi. Interestingly, coordination in the assembly of the basal body and export apparatus might be shared by flagella and 348 injectisomes.
In conclusion, our study reveals unprecedented details about the fT3SS machine in the Lyme components, and document their roles in flagellar structure and function. We present the first 352 structural evidence that the distinct ATPase complex of the fT3SS machine is attached to the flagellar 353 C-ring through multiple spoke-like linkers comprised of FliH. The novel architecture not only 354 strengthens the C-ring, but also enables an optimal translocation of substrates through the ATPase 355 complex and the export apparatus. Remarkably, the ATPase complex together with the C-ring can 356 adopt variable orientations, implying that the fT3SS machine undergoes rotation in concert with the 357 flagellar C-ring. Therefore, our studies not only provide a structural framework for a better 358 understanding of the fT3SSs, but also underscore the striking differences between flagella and their 359 evolutionally related bacterial injectisomes.

EXPERIMENTAL PROCEDURES
Bacterial strains and growth conditions. High-passage Borrelia burgdorferi strain B31A (WT) and its isogenic mutants (Table S2) were grown in BSK-II liquid medium supplemented with 6% rabbit described (52, 53).  (Table S3) contain several overlapping base pairs, as 381 indicated by different colors in Figure S10. In step two, a PCR product was obtained by using primers 382 P1 and P4 and the purified DNA products for upstream fliP and aadA as templates. In step three, the 383 final PCR product was obtained by using primers P1 and P6, and the purified DNA products of   Table S3.   were observed under a dark-field microscope (Zeiss Axio Imager. M1) connected to an AxioCam 437 digital camera. Exponentially growing cells were examined for their shape and motility. Almost all 438 mutants were non-motile and rod-shaped (see Fig. S3 as an example).

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Frozen-hydrated EM sample preparation. The frozen-hydrated specimens were prepared as 440 previously described (Liu et al., 2009). Briefly, B. burgdorferi culture was centrifuged at 5,000 × g for 5 441 minutes. The pellet was suspended with 1.0 ml PBS. The cells were centrifuged again and suspended 442 in 50~80 µl PBS. The cultures were mixed with 10 nm colloidal gold and were then deposited onto 443 freshly glow-discharged, holey carbon grids for 1 min. Grids were blotted with filter paper and then 444 rapidly frozen in liquid ethane, using a homemade gravity-driven plunger apparatus.

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Cryo-electron tomography. Frozen-hydrated specimens were imaged at -170 °C using a Polara 446 G2 electron microscope (FEI) equipped with a field emission gun and a 16 megapixel CCD camera 447 (TVIPS). The microscope was operated at 300 kV with a magnification of 31,000 ×, resulting in an 448 effective pixel size of 5.7 Å after 2×2 binning. Using the FEI "batch tomography" program, low-dose,

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The PCR products are shown as expected.      and MS-ring) structure was from global average applied with 16-fold symmetry. The bottom part (C-918 ring, spoke links and ATPase) structure was from a combine of class averages. Same in Fig. 5 and Fig.   919 6. In order to refine the bottom structure, the top part of those class averages were not aligned and do 920 not show the 16-fold symmetry. The hexagonal "hub" were segmented in chimera, and the FliI-FliH 921 atomic structure (pdb:5B0O) were initially fitted into the segmented density. However, as there is 922 extra density, and 3 to 4 links extend from the hub, we postulate there are more than one FliH 2 . Three 923 more FliH 2 were placed next to the FliI-FliH atomic structure, and they were fitted into the density