GB_GCA_002328585.1 GB_GCA_002407865.1 GB_GCA_001829455.1 GB_GCA_001829495.1 A B GB_GCA_002839375.1 GB_GCA_003246895.1 GB_GCA_002839285.1 GB_GCA_002418835.1 GB_GCA_002425885.1 Turneriella parva DSM 21527 Leptonema illini DSM 21528 GB_GCA_002730875.1 GB_GCA_002729885.1 GB_GCA_002717505.1 GB_GCA_002333425.1 GB_GCA_002786225.1 Leptospira sp. YH101 Leptospira sp. E30 Leptospira biflexa serovar Patoc strain 'Patoc 1 (Paris)' Leptospira yanagawae serovar Saopaulo str. Sao Paulo = ATCC 700523 Leptospira sp. CN6-C-A1 Leptospira sp. E18 Leptospira sp. JW2-C-A2 Leptospira meyeri serovar Hardjo str. Went 5 Leptospira sp. FH2-B-A1 Leptospira terpstrae serovar Hualin str. LT 11-33 = ATCC 700639 Leptospira vanthielii serovar Holland str. Waz Holland = ATCC 700522 Leptospira wolbachii serovar Codice str. CDC Leptospira sp. FH1-B-B1 Leptospira fainei serovar Hurstbridge str. BUT 6 Leptospira inadai serovar Lyme str. 10 Leptospira broomii serovar Hurstbridge str. 5399 Leptospira wolffii serovar Khorat str. Khorat-H2 Leptospira sp. B5-022 Leptospira licerasiae serovar Varillal str. VAR 010 Leptospira sp. MCA2-B-A3 Leptospira sp. ES4-C-A1 Leptospira sp. E8 Leptospira sp. FH4-C-A2 Leptospira venezuelensis Leptospira sp. ATI7-C-A4 Leptospira sp. FH2-B-C1 Leptospira alstonii Leptospira sp. ATI7-C-A5 Leptospira kmetyi serovar Malaysia str. Bejo-Iso9 Leptospira sp. FH4-C-A1 Leptospira alstonii serovar Sichuan str. 79601 Leptospira weilii serovar Ranarum str. ICFT Leptospira noguchii serovar Panama str. CZ214 Leptospira kirschneri serovar Cynopteri str. 3522 CT Leptospira interrogans Leptospira santarosai serovar Shermani str. LT 821 Leptospira mayottensis 200901116 Leptospira borgpetersenii Leptospira alexanderi serovar Manhao 3 str. L 60 Leptospira weilii serovar Topaz str. LT2116 GB_GCA_003497555.1 GB_GCA_000431595.1 Brachyspira pilosicoli P43/6/78 Brachyspira alvinipulli ATCC 51933 Brachyspira sp. G79 Brachyspira murdochii DSM 12563 Brachyspira innocens ATCC 29796 Brachyspira hampsonii Brachyspira hampsonii 30446 Brachyspira hampsonii Brachyspira intermedia PWS/A Brachyspira hyodysenteriae ATCC 27164 Brachyspira suanatina GB_GCA_001829155.1 GB_GCA_002450905.1 Brevinema andersonii GB_GCA_001829395.1 GB_GCA_001829415.1 GB_GCA_001829125.1 GB_GCA_001829315.1 Candidatus Borrelia tachyglossi tachyglossi Borrelia miyamotoi LB-2001 Borrelia hermsii DAH Borrelia anserina Es Borrelia turicatae 91E135 Borrelia coriaceae Co53 Borrelia persica No12 Borrelia hispanica CRI Borrelia crocidurae str. Achema Borrelia duttonii Ly GB_GCA_000808095.1 Borrelia mayonii Borreliella bissettii DN127 Borreliella burgdorferi B31 Borreliella finlandensis Borreliella valaisiana VS116 Borreliella garinii Borreliella japonica Borreliella afzelii HLJ01 Borreliella spielmanii A14S GB_GCA_003454065.1 GB_GCA_001604265.1 GB_GCA_002406055.1 GB_GCA_002452215.1 GB_GCA_002308475.1 GB_GCA_001940825.1 GB_GCA_900321635.1 GB_GCA_003456855.1 GB_GCA_001604275.1 Sphaerochaeta coccoides DSM 17374 GB_GCA_002428625.1 GB_GCA_002839335.1 GB_GCA_002839355.1 GB_GCA_900315435.1 Sphaerochaeta pleomorpha str. Grapes GB_GCA_003446545.1 GB_GCA_003247345.1 GB_GCA_001603105.1 GB_GCA_001604325.1 GB_GCA_001603115.1 GB_GCA_002432715.1 Sphaerochaeta globosa str. Buddy GB_GCA_002688185.1 GB_GCA_003233545.1 GB_GCA_002238925.1 GB_GCA_003170115.1 GB_GCA_003141795.1 GB_GCA_003154555.1 Sediminispirochaeta smaragdinae DSM 11293 Sediminispirochaeta bajacaliforniensis DSM 16054 GB_GCA_003245835.1 GB_GCA_003454605.1 Candidatus Marispirochaeta associata associata Marispirochaeta aestuarii Spirochaeta thermophila DSM 6192 Spirochaeta thermophila DSM 6578 Salinispira pacifica Spirochaeta lutea Spirochaeta africana DSM 8902 GB_GCA_002313505.1 Alkalispirochaeta alkalica DSM 8900 Alkalispirochaeta americana Spirochaeta cellobiosiphila DSM 17781 GB_GCA_003247135.1 GB_GCA_002084805.1 GB_GCA_002408085.1 GB_GCA_003249275.1 GB_GCA_003246505.1 GB_GCA_002084135.1 GB_GCA_002352375.1 GB_GCA_002748225.1 GB_GCA_001829185.1 GB_GCA_002412825.1 GB_GCA_002839245.1 GB_GCA_002839315.1 GB_GCA_003247225.1 GB_GCA_002307245.1 GB_GCA_002839205.1 GB_GCA_002428725.1 GB_GCA_001603165.1 GB_GCA_002383375.1 GB_GCA_003452735.1 GB_GCA_002427685.1 GB_GCA_002435985.1 GB_GCA_002441395.1 GB_GCA_002425765.1 GB_GCA_002307015.1 Treponema caldarium DSM 7334 GB_GCA_003527485.1 GB_GCA_003245635.1 GB_GCA_002293455.1 Treponema azotonutricium ZAS-9 Treponema primitia ZAS-2 Treponema primitia ZAS-1 GB_GCA_003246335.1 GB_GCA_001604385.1 GB_GCA_001604405.1 GB_GCA_003246765.1 GB_GCA_003245785.1 GB_GCA_003252395.1 Treponema pallidum subsp. pallidum str. Sea 81-4 Treponema phagedenis 4A Treponema vincentii F0403 Treponema medium ATCC 700293 Treponema sp. OMZ 838 GB_GCA_002749205.1 Treponema pedis str. T A4 Treponema putidum Treponema denticola ATCC 35405 Treponema denticola SP32 Treponema brennaborense DSM 12168 GB_GCA_001885315.1 Treponema maltophilum ATCC 51939 Treponema lecithinolyticum ATCC 700332 GB_GCA_001603145.1 GB_GCA_002308875.1 GB_GCA_002369025.1 Treponema saccharophilum DSM 2985 GB_GCA_002404895.1 GB_GCA_002368605.1 GB_GCA_002393835.1 GB_GCA_002394465.1 GB_GCA_003447785.1 GB_GCA_002392405.1 GB_GCA_002477955.1 Treponema berlinense GB_GCA_002394685.1 GB_GCA_003455655.1 GB_GCA_002449305.1 Treponema succinifaciens DSM 2489 GB_GCA_002393865.1 GB_GCA_002373205.1 GB_GCA_002448445.1 GB_GCA_002392275.1 GB_GCA_002373325.1 GB_GCA_002396325.1 GB_GCA_002309305.1 GB_GCA_002319875.1 GB_GCA_002309195.1 GB_GCA_900316905.1 GB_GCA_002391235.1 GB_GCA_002399405.1 GB_GCA_002314445.1 Treponema socranskii Treponema socranskii subsp. paredis ATCC 35535 Treponema porcinum GB_GCA_002437725.1 GB_GCA_900317625.1 GB_GCA_002478955.1 GB_GCA_002296965.1 GB_GCA_002315375.1 GB_GCA_900319625.1 Treponema sp. JC4 0.2 Treponema sp. C6A8 GB_GCA_002350445.1 GB_GCA_002395155.1 Treponema bryantii NK4A124 Treponema bryantii Treponema bryantii

Fig. S1 Complete chemosensory class profile of Spirochaetota. The three major taxonomy classes are colored: Leptospirae (red), Brachyspirae (purple) and Spirochaetia (green). Species found in MiST3 are represented by their species and strain name. The profile shows the presence of the chemotaxis classes F1(blue), F8(orange), F2 (green), F7(red), F5 (purple), ACF (brown) and TFP (pink). The systems with CheW-CheRlike are marked with a black outline. Scale bar represents the average number of substitutions per site. An additional high-resolution figure is separately available.

Fig. S2 (A) Sequence logo of representative sequences of the two groups of CheR-containing proteins in the F2 system. (B) Sequence logo of representative sequences of the three groups of CheW-containing proteins in the F2 system. Blue boxes denote the location of variable regions identified in the Td CheW and P5 homologs. In these locations, all regions possess unique conserved residues with the exception of Region 2, which is not at the CheA:CheW ring interfaces. An additional high-resolution figure is separately available.

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Fig. S3 Phylogenetic tree of a non-redundant set of CheW protein sequences in genomes with at least one CheA-F2. The initial classification of CheW classes are mapped to the tree nodes: F2 (red), F5 (purple), F7 (light green), F8 (green) and ACF (blue). The clustering of the CheW classified as class F2 suggests a last common ancestor of CheW-F2 sequences (larger red internal node). This clustering allows us to extrapolate the conservative selection of F2 sequences to other CheW proteins in the cluster to get a more inclusive set. Scale bar represents the average number of substitutions per site.

Fig. S4 (A) Top: The number of arrays and particles selected to generate sub-tomogram averages for each Td strain, and the average resolution of the sub-tomogram averages. Resolution values were calculated at FSC = 0.3 in a masked region that contains the single central receptor hexagon and CheA:CheW ring. Bottom: Example of the masked region used for resolution calculation (WT map). Scale bar is 12 nm. (B) Sub-tomogram averages reveal the orientation of the chemotaxis arrays with respect to the cell axis. Intriguingly, the arrays have a preferred orientation in the cells and this orientation is conserved in WT and the Δ2498 and Δ2498Δ2496 deletion mutants. The small size of arrays in the ΔCheRlike strain inhibits direct observation of the cell axis.

Fig. S5 (A) SEC-MALS of Td CheA and CheW-Rlike. Top: CheA forms a dimer in solution. Bottom: The CheW-Rlike protein is primarily present as a monomer, but associates into a small amount of dimer. There is also a small amount of aggregates present (black arrow). The dRI trace is a black dashed line. The LS trace is grey. (B) The CheW-CheRlike linker is predicted to form a single alpha helix flanked by unordered regions (Jpred). (C) Native mass spectrometry (ESI-MS) experiments indicate that CheW-Rlike (left) and the Td classical CheR protein (right) bind SAM (398.44 Da) in a 1:1 stoichiometric ratio. Example shifts in the spectra are denoted by black dashed lines and match expectations for 1:1 binding. The sequence derived mass of CheW-CheRlike and CheR is 51.784 kDa and 33.802 kDa, respectively. Source data are provided as a Source Data file.

Fig. S6 Capillary chemotaxis assay of T. denticola wild-type and ΔCheRlike strain using two chemoattractants: hemin (left) and glucose (right). The ΔCheRlike strain is missing only the CheRlike domain of CheW-CheRlike. For the non-gradient control, both the capillary tubes and bacterial suspensions contained chemoattractants. Differences in chemoattraction of WT and ΔCheRlike strains are statistically insignificant using a two-tailed null hypothesis significance test (p > 0.05). Results are expressed as the mean of cell numbers ± standard error of the mean (SEM) from five capillary tubes. The data are normalized so that the control samples are equal to 1. The normalized cell ratio values for individual samples (capillary tubes) are shown as black dots. Source data are provided as a Source Data file.

Fig. S7 Homology models of Td CheW domains and CheA P5. (A) A homology model of the CheW domain of CheW-CheRlike using Thermoanaerobacter tengcongensis CheW (PDB ID: 2QDL) as the template. (B) A homology model of the classical Td CheW using Thermoanaerobacter tengcongensis CheW (PDB ID: 2QDL) as the template. (C) A homology model of CheA P5 using E. coli CheA P5 (PDB ID: 6S1K) as the template.

Fig. S8 (A) Sub-tomogram averaging of Td WT and mutant strains reveal that linked CheA:CheW rings (yellow) run perpendicular to the cell axis via a strict linear orientation of CheA (blue). Dimerization of CheA at the P3 domain (green arrows) links the rings together. Averages from WT Td are illustrated here but apply to all strains. (B) The curvature of the inner membrane of Td at chemotaxis arrays in the reconstructions is 35.8 +/- 6.6 /µm (270 Å radius). The curvature of the CheA:CheW baseplate (red arrow) is 65.6 +/- 19 /µm (152 Å radius). (C) V. cholerae minicells have an inner membrane curvature of 9.15 +/- 4.5 /µm (radius 1092 Å).

Fig. S9 Modeling of CheA:CheW rings to the curvature of the Td baseplate. (A) For single CheA:CheW rings to follow the baseplate curvature, it must bend by an average of 7.6 Å toward the membrane. (B) In order for two linked CheA:CheW rings to run perpendicular to the cell axis, the center of the rings (P3) must bend by 49.2 Å toward the cell membrane.

Fig. S10 Multiple sequence alignments of the CheA P3 domain from several bacteria demonstrates the presence of additional P3 residues in Td and other Spirochetes. (A) CheA P3 alignments of Td and P3 from other bacteria with previously characterized chemotaxis proteins. Td possesses ~50 residues that are not found in the other homologs and are located in between the traditional dimerization helices. (B) Td CheA P3 aligned with other Spirochete P3. The additional residues identified in alignment A are highlighted in blue. Figures were made using Clustal Omega.

Fig. S11 Analysis of non-redundant CheA P3 domains with a 75% sequence identity cut-off (1450 sequences). (A) Conservation scores of the P3 alignment with the 1450 sequences. The traditional P3 helices are largely conserved, but regions located between the helices are non-conserved. (B) Analyses of CheA from different chemotaxis classes reveals that CheA F2 homologs possess the most residues in the non-conserved region of P3. (C) The non- conserved regions are llustrated with the two known CheA P3 structures. Top: Td P3 possesses 71 residues that align to the non-conserved region (PDB ID: 6Y1Y). Bottom: Tm P3 possesses seven residues that align to the non-conserved region (PDB ID: 1B3Q).

Fig. S12 The P3 domain of Td CheA. (A) The CheA P3 dimer contains a cluster of Phe and Tyr residues near the breakages in the helices (black arrows). All Phe and Tyr residues are highlighted in red. (B) The crystal structure of CheA P3 demonstrates asymmetry in the subunits. (C) Repositioning of Y82 in the subunits induces alterations of adjacent residues and may account for subunit asymmetry. (D) Alignment of the P3 structure to a previously determined model of the chemotaxis array in E. coli (PDB ID: 3JA6) indicates that the P3 domain lies within ~15 Å of the receptors (when measuring from peptide back-bone). CheA and CheW in this model are shown in grey.

Fig. S13 Diagrams illustrating construction of the TDE1492::ermB vector (A) for the targeted mutagenesis of TDE1492 (781-1,308 nt) by in-frame replacement of TDE1492 using ermB cassette. These constructs were constructed by two-step PCR followed by DNA cloning. Arrows represent the relative positions and orientations of these primers, which are listed in Table S3. ermB = erythromycin resistance. (B) Characterization of the ΔTDE1492 strain by PCR analysis. The top panel illustrates how the PCR analysis is designed; the bottom panel is the PCR results. Arrows represent the relative positions and orientations of these primers; the numbers are predicted sizes of PCR products generated by the corresponding primers. The primer P7 is located at the 5’-end of TDE1492, P6 at the 3’-end of ermB, P5 at the 5’-end of ermB, P8 at the flanking region of TDE1492, P9 at the middle of TDE1492, and P10 at the 3’- end of TDE1492. The sequences of these primers are listed in Table S3.

Fig. S14 Flowchart of the three major pipelines used to produce the bioinformatics datasets. Steps marked in red represents fetching information from MiST3 database, in green are steps requiring RegArch as a filter, and in blue indicate writing data to file.

Fig. S15 Angular distribution graphs of the sub-tomogram averages for the WT strain (A), the Δ2498 strain (B), the Δ2498 Δ2496 strain (C), and ΔCheR-like strain (D).

Fig. S16 Fourier shell correlation (FSC) graphs of the sub-tomogram averages for the WT strain (A), the Δ2498 strain (B), the Δ2498 Δ2496 strain (C), and ΔCheR-like strain (D). Blue: FSC of masked maps; Green: FSC of unmasked maps; Black: Corrected FSC; Red: Phase randomized FSC.