Spirochetes produce ordered chemoreceptor arrays of unusual composition, arrangement, and symmetry to compensate for a highly curved membrane

Abstract The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology, but most insights have been obtained from only a few model organisms. In all previously described species, chemoreceptors organize with the histidine kinase (CheA) and coupling protein (CheW) into a hexagonal (P6 symmetry) extended array that is considered universal among archaea and bacteria. Here, for the first time, we apply cryo-electron tomography to whole Treponema denticola (Td) cells to investigate the structure of a spirochete (F2) chemotaxis system. The Td chemoreceptor arrays assume a truly unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature present in spirochetes. A two-fold (P2) symmetry of the chemotaxis apparatus in Td emerges from a strict linear organization of the kinase CheA, which generates arrays that run parallel to the cell axis. The arrays have several additional atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered, functional chemosensory apparatus in an extremely curved cell. Furthermore, the previously characterized Td oxygen sensor ODP influences array integrity and its loss substantially orders CheA. These results demonstrate the importance of examining chemotaxis structures of non-model organisms in vivo and suggest a greater diversity of this signaling system than previously thought.

model of the chemotaxis array 4,6,7 . However, emerging research has recently revealed divergent components and arrangements of the chemotaxis apparatus in non-canonical organisms. For example, many bacteria contain multiple copies of CheW, with some fused to other protein modules (i.e. CheV), and in vivo analyses of Vibrio cholerae (Vc) chemotaxis arrays have revealed that CheA and CheW in the rings apparently lack an ordered arrangement 8,9 .
The chemotaxis systems in prokaryotes have been classified into 19 systems based on phylogenomic markers 10 . These classes include 17 'Flagellar' systems (F), one 'Alternative Cellular Function' system (ACF), and one 'Type Four Pilus system' (TFP). The spirochete chemotaxis system belongs to the 'Flagella class 2' (F2) category, which has not been investigated with structural methods 10 . Herein, we examine the in vivo structure of an F2 system in the spirochete Treponema denticola (Td) by cryoelectron tomography (cryo-ET). We imaged chemotaxis arrays and demonstrate the presence of a novel array architecture, that likely owes to the high curvature of the cells. Genetics experiments, bioinformatics analyses, structural investigations, and molecular modeling of Td chemotaxis proteins reveals adaptations that have likely evolved to accommodate formation of an extended chemotaxis array in a highly curved membrane. We demonstrate that a CheR-like fusion domain in a variant CheW is key for maintaining the structural integrity of the arrays. Furthermore, cryo-ET analysis of Td cells lacking the previously characterized oxygen sensor ODP demonstrates significant changes in CheA mobility 11 .

Conservation of F2 chemotaxis proteins F2 systems are the signature systems of Spirochaetia and Brachyspirae
Based on their evolutionary history and gene cluster organization, the spirochete T. denticola has a single F2 chemotaxis system encoded in its genome 10 . In the Microbial Signal Transduction Database version 3 (MiST3) 12 , 306 genomes contain at least 1 CheA of the F2 class (CheA-F2) (Dataset 1). Only three of these genomes were not members of the Spirochaetota phylum and are likely the consequence of lateral gene transfer (see Supplementary Methods).
However, there are 1096 genomes classified as Spirochaetota in GTDB 13 and 804 of these are present in the MiST3 database (Dataset 2) 12 . With these 804 genomes, we mapped the different classes of CheA kinases to the Spirochaetota taxonomy tree ( Figure S1). Based on the topology of this tree, it appears that the major chemosensory systems in the genomes from the Spirochaetota phylum are: F1 (Leptospirae), a transitional hybrid F1/F2 system (Brachyspirae), and F2 (Spirochaetia) (Fig. S1).

CheW-CheR like is only present in complete F2 systems
Intriguingly, the Td genome contains two CheW proteins: a classical CheW protein (TDE1589) and a CheW that possesses a C-terminal CheR-like domain separated by a 28-residue linker (TDE1492, CheW-CheR like ). Typically, CheR is a methyltransferase that, together with the methylesterase CheB, controls the methylation state of the receptors and thus provides an adaptation system 14 . In the CheW-CheR like fusion protein, the linker residues are predicted to form a single alpha helix with flanking disordered regions (Jpred, Fig. S4D). The F2 system's main architectural difference from other systems is the presence of this unusual scaffold protein. Of the 306 genomes with at least one CheA-F2, all of them also contain CheW-CheR like with a few exceptions (See Supplementary Methods). However, all Spirochaetia genomes contain the CheW-CheR like gene (Fig. S1).
The CheW-CheR like has not been found in other chemosensory system classes. In the MiST3 database, there are 274 CheW-CheR like proteins in 273 genomes. Of those, 15 do not contain an F2 chemosensory system but these genomes are not fully assembled (14 at contig and 1 at scaffold level, see Supplementary Methods). Based on these results, we conclude that CheW-CheR like is likely only present in complete F2 systems.
The three F2 CheW domains evolved different sequence patterns F2 systems contain three proteins with a CheW domain: the classical scaffold CheW, CheW-CheR like , and the P5 domain from the histidine kinase CheA. To investigate sequence patterns of these CheW domains, we built a pipeline to produce non-redundant sequence datasets of CheW, CheW-CheR like , and CheA of the class F2 from all genomes with at least one CheA-F2. The final alignment of each group contained the CheW domain portion of 74 CheW proteins, 59 CheW-CheR like proteins, and 73 CheA proteins. The sequences of each group are summarized in sequence logos (Fig. S3A). The logos reveal conserved regions at the ring interfaces. There are two proteins in the F2 system with a CheR domain: the CheR-F2 methyltransferase and the  CheW-CheR like protein. To investigate sequence patterns in these proteins, we produced a final  sequence dataset with the CheR domain of 83 CheR-F2 and 88 CheW-CheR like proteins and summarized them in sequence logos (Fig. S3B). The CheR like domain has a 20% identity to the Td CheR methyl-transferase (TDE0647, LALIGN). The two residues that are responsible for catalytic function to change chemoreceptor methylation levels, R79 and Y218 in Td CheR, are different in the two CheR domains 14 . Both residues are highly conserved in CheR-F2 proteins, but are modified in the CheW-CheR like protein (R79W and Y218F). Furthermore, the conserved region at the C-terminus of CheR is not conserved in CheW-CheR like . Based on these results we speculate that the CheR like domain does not possess methyltransferase activity. Collectively, our analyses suggest that CheR and CheW-CheR like have different biological functions.

The F2 CheR domains evolved different sequence patterns
The structure of the Treponema denticola (Td) chemotaxis array in wild-type cells Cell poles of intact Td cells were imaged by cryo-electron tomography (cryo-ET) and used for threedimensional reconstructions. Top views (cross-sections through the array) and side views (visualizing the long axes of the receptors) of membrane-associated arrays were clearly visible (Fig. 1A, S4A). Sub-volume averaging revealed the conserved receptor trimer-of-dimers in the typical hexagonal arrangement. Remarkably, several novel features of the chemotaxis arrays are apparent (Fig. 1B). Specifically, a density of unknown origin is located in the center of the receptor hexagons and slightly above the plane of the CheA:CheW rings. This density, which will hereafter be referred to as the middle density, extends from two subunits in the rings ( Fig. 2A). Additionally, there are small but distinct puncta of density in between some of the trimer-of-dimer modules (Fig. 1B). However, averages of the arrays at the CheA:CheW layer did not reveal discernible CheA density, indicating either a sparse or disordered distribution of CheA or a highly mobile kinase (Fig. 1C). Lysis of Td cells via lysozyme treatment followed by cryo-ET reveals intact arrays (Fig. S4B).

Fig. 1
Cryo-electron tomography of whole T. denticola (Td) cells reveals the protein arrangement of chemotaxis machinery. (A) Side-views of the membrane-associated chemotaxis apparatus illustrate the location of the receptor layer (Layer 1) and CheA:CheW baseplate (Layer 2). These layers are spaced ~90 Å from one another. (B) Sub-volume averaging of three Td strains reveals the universally-conserved receptor trimer-of-dimer arrangement with 12 nm spacing between opposing trimer-of-dimer modules. Notably, density is apparent in the center of the receptor hexagons (blue arrow) and between some receptor trimer-of-dimer modules (green arrow). In general, the densities in Layer 1 of the wild-type (WT) strain are better resolved. (C) Sub-volume averages at Layer 2 reveal the organization of CheA. Density corresponding to CheA is only apparent in the two Td deletion mutants and CheA is arranged in a linear fashion. In this arrangement, the density between the trimer-of-dimer modules (green arrow) corresponds to the CheA P3 domain.

Arrays in T. denticola deletion mutants
Previous experiments demonstrate that a protein called oxygen-binding diiron protein (ODP) functions as an oxygen sensor for chemotaxis in Td, however the study did not determine if ODP is an integral component of the array 11 . This protein is genetically coupled to a soluble receptor, TDE2496, with unclear signaling capabilities. TDE2496 likely integrates into the membrane-bound arrays based on the observation that no cytoplasmic arrays were observed in the tomograms. Moreover, the Td genome encodes only one CheA homolog, and cytoplasmic receptors often associate with distinct kinases 5,15 . TDE2496 forms trimers-of-dimers in vitro and can modulate CheA activity (Fig. S5A,B). To determine if the presence of ODP (TDE2498) and its cognate receptor (TDE2496) impacts array architecture or integrity, we conducted cryo-ET with two Td gene knock-out strains, Δ2498 and Δ2498Δ2496 11 . Importantly, reverse-transcription PCR confirms that deletion of ODP does not impact transcription of TDE2496 11 . The sub-volume averages of these strains reveal distinct differences in array densities compared to the wild-type (WT) strain ( Fig. 1B,C, Fig. S4A). Namely, the location of CheA at the CheA:CheW layer (Layer 2, Fig 1C) is now clearly visible. Interestingly, CheA arranges in well-ordered linear rows. Placement of CheA necessarily positions the P3 domain in between two of the trimer-of-dimer modules in each hexagon. This position exclusively corresponds to the location of the puncta between receptor trimer-of-dimer modules observed in the WT array, indicating that this density corresponds to the P3 domain (Layer 1, Fig. 1B). Additionally, the receptor densities are less resolved than in the WT strain (Fig. 1B). This is further illustrated by the fact that when the selected cryo-ET particles for WT and mutant strains are averaged using the same size mask, the WT strain generates averages that resolve a larger portion of the array at Layer 1. Thus, the receptors and P3 domains are more ordered across the array in the WT strain whereas the P5-CheW layer is more ordered in the ODP deletion strain (Fig. S5C).

Analyses of the CheW-CheR like protein in T. denticola
To determine the composition of the middle density in the Td arrays, we examined spirochete genomes for unique chemotaxis proteins. The bioinformatics analyses identify CheW-CheR like as a conserved component of the F2 Spirochaetia chemotaxis system. In Td, the CheW-CheR like gene is co-transcribed with the only CheA, CheX, and CheY proteins in the genome (Fig. S4C) 15 . Furthermore, native gel electrophoresis of the purified CheW-CheR like protein demonstrates the presence of a protein dimer, and radioisotope assays that monitor CheA autophosphorylation demonstrate that the CheW-CheR like protein activates CheA kinase activity, similarly to Tm chemotaxis proteins (Fig. 2C,D, Fig. S4E,F). Therefore, we postulated that the middle density may be comprised of two CheR like domains that extend from two CheW-CheR like proteins in the CheA:CheW rings .  The copyright holder for this preprint (which was not peer-reviewed) is the .
A Td strain lacking the CheR like domain (ΔCheR like ) reveals a significant decrease in the prevalence and size of the arrays (Fig. S4A). Due to the small size of the arrays in ΔCheR like , only 194 particles were available for sub-tomogram averaging, but the resulting averages clearly demonstrate that the middle density is no longer present (Fig. 2B, S4A). Like the WT strain, the CheA density below the rings in ΔCheR like is not apparent.

Protein interfaces in the CheA:CheW:CheW-CheR like rings
Bioinformatics analyses demonstrate that all functional Spirochaetota F2 chemotaxis systems possess a CheW-CheR like homolog and at least one classical CheW protein. As only two of the CheW subunits in the hexagonal rings extend to the middle density (which probably arises from the CheW-CheR like protein), and two of the ring positions are occupied by CheA P5, it is likely that the other two positions are occupied by the classical CheW protein. (Fig. 2A, 5A). Within this arrangement three unique interaction interfaces are possible, interface 1 occurs between CheA P5 and the classical CheW (as seen in canonical systems), interface 2 occurs between CheA P5 and the CheW domain of CheW-CheR like , and a third interface (interface 3) occurs between the classical CheW and the CheW domain of CheW-CheR like (Fig. 5A) . To explore the binding interfaces within the Td rings, we analyzed homology models of the classical CheW, the CheW domain of CheW-CheR like , and the CheA P5 domain. The CheW models were generated using a crystal structure of Thermoanaerobacter tengcongensis (Tt) CheW as the template (PDB ID: 2QDL), and the CheA P5 model was generated using a cryo-EM structure of E. coli CheA P5 (PDB ID:6S1K) ( Fig. S6A-C) 16,17 . Three of the four regions with lowest sequence conservation among the three domains are located at interfaces 1-3 (Fig. 3). Alignment of the Td CheW and CheA P5 models to a crystal structure of Tm CheW in complex with Tm CheA P5 (PDB ID: 3UR1) further illustrates that these regions are located at the CheW:P5 ring interfaces (Fig 3) 18 . Mapping the variable regions onto the sequence logos of the F2 Variable regions between the three domains (blue boxes) were determined by sequence alignments of the three domains followed by conservation analysis. These variable regions are located at the rings interface regions. The CheW domains also have variable N-terminal and C-terminal regions that are not complimented in P5 (grey boxes). Variable regions for P5 and the CheW domains are denoted on the homology models by italicized numbers and underlined numbers, respectively.
CheW domains demonstrates that they evolved different sequence patterns in these regions, with the exception of the variable region that is not located at the interaction interface (region 2, Fig. 3, S3).

CheA arrangement and array curvature in T. denticola
Sub-tomogram averaging reveals that Td CheA forms a linear arrangement across the chemotaxis array, linking the CheA:CheW rings into extended 'strands' that are held together by receptor:receptor interactions (Fig. 4A,B). These strands are apparent in the cryo-ET reconstructions and run relatively parallel to the axis of the cells, effectively allowing the strands to remain straight rather than bending to the cell curvature (Fig. 4D). Indeed, the angle between the cell axis and the extended strands is 10.4 +/-8.6°, (n = 26 cells) and no significant difference was found among the three Td strains measured (Table S1C).
The CheA:CheW rings present in a crystal structure (PDB ID: 3UR1) are flat and ~95 Å in diameter 18 . The length across two rings connected by a dimeric CheA is 224 Å (Fig. S8). To determine the extent of buckling that would need to occur in the two CheA:CheW rings if they ran perpendicular to the cell axis, the 224 Å rings were modeled as a chord in a circle with radius 152 Å. Using the equation ℎ = − ! − ! (where h is the height of the circular segment, r is the circle radius, and L is half the chord length (224 Å /2)), the height of the circular segment is 49.2 Å (Fig. S8A). Therefore, the center of the two rings (the P3 domain) would need to buckle by an average of 49.2 Å toward the cell membrane to accommodate the cell curvature. Even with the CheA strands arranged perfectly to the cell axis, this arrangement still necessitates that each single CheA:CheW ring must bend to follow the baseplate curvature. Using the same equation above (where L is 95 Å /2), the height of the circular segment is 7.6 Å (Fig. S8B). Therefore, in Td, the center of a single ring must buckle toward the C membrane by an average of 7.6 Å to align to the measured baseplate curvature.

Spirochetes possess an atypical dimerization domain
The cryo-ET results reveal density corresponding to the P3 domain, which has not been previously reported in in vivo arrays. Sequence alignments of Td CheA with CheA homologs from a variety of model bacteria with previously characterized chemotaxis systems reveal that Td CheA possesses an additional ~50 residues located between the canonical dimerization domain (P3) helices ( Figure  S9A) 7 . CheA homologs from other spirochete genera including Borrelia and Brachyspira also possess additional residues in this region (Fig. S9B). Analysis of non-redundant P3 domains from all CheA classes reveal general sequence conservation in the canonical helices but highly divergent sequences at these additional residues (Fig. S10A). Furthermore, CheA-F2 proteins possess the most residues in this non-conserved region (Fig. S10B,C). X-ray crystallography experiments were used to determine the structure of the isolated Td P3 domain to 1.5 Å (PDB ID: 6Y1Y, Fig. 5B, Table  S2). These experiments revealed that the additional residues adopt the coil-coiled motif of the classic dimerization domain with the exception of a break in one of the helices, producing a discontinuous coiled-coil (Fig. 5B). Interestingly, aromatic residues (Phe, Tyr) cluster near the helix breakages but it's unclear if this arrangement is functionally relevant (Fig. S11A). Asymmetry between the subunits is located exclusively at the additional residues following residue Tyr83, due to differing orientations of Tyr83 in the subunits (Fig. S11B,C). Fitting the new P3 domain into an all-atom chemotaxis array that was generated for previous molecular dynamics simulations (PDB ID: 3JA6) shows that these additional helices are within a ~15 Å from receptors (Fig. S11D) 21 . Additionally, the Td P3 domain possesses a different handedness than Tm P3 observed in previous crystal structures 22,23 .

Discussion
Here, we reveal the protein arrangement of F2 chemotaxis arrays through cryo-ET of intact T. denticola (Td) cells. In this system, three proteins comprise the rings at the receptor tips: CheA, CheW, and a CheW-CheR like protein. Like the Ec system, these proteins in Td are integrated into the array with strict organization 8,19 . However, a new linear arrangement of CheA is present that generates 'strands' of rings interlinked by the CheA dimerization domain (P3). The strands follow the axis of the Td cells, effectively allowing linked rings to align along the path of least curvature and avoid substantial buckling that would otherwise occur. Therefore, it is unlikely that a hexagonal CheA  arrangement present in canonical systems like Ec could form under such curvature constraints. We hypothesize that two of the CheR like domains in CheW-CheR like interact in the center of the rings for additional stability. The deleterious effect on array integrity observed in the ΔCheR like strain is consistent with the expectation that CheR like dimerization plays a key role in array assembly and stabilization. The strict linear arrangement of CheA could be facilitated by the composition of the Td rings; three unique protein interfaces are present in the rings and restrict CheA P5 geometry (i.e. CheA P5 can only occupy these two positions in the six-member ring). Furthermore, the Td CheA P3 domain is clearly discernible in the averaged tomograms, which has not been previously observed in vivo 4,5,19 . As P3 has been previously implicated to directly engage receptors, the elongated P3 domain in spirochetes may have evolved to allow P3:receptor interactions in a highly curved array 21 . Due to the high cell curvature in Td, the receptors are expected to be more splayed compared to cells with less curvature, such as Ec and Vc. The elongated P3 may compensate for this increased distance between the receptors and the P3 domains. As Td cells have the smallest average diameter (0.1 -0.4 µm) of all bacteria with determined chemotaxis architectures thus far, and the novel linear arrangement of CheA produces arrays that can better accommodate extreme cell curvature, we surmise that the array architecture in Td is an adaptation that evolved to produce an extended chemoreceptor apparatus in a highly curved membrane 4,5,7 .
Bioinformatics analyses indicate that the unique protein features seen in Td are largely conserved in Spirochaetia F2 systems. Indeed, in vivo genetics experiments in the Bb have shown that two CheW proteins, a classical CheW (CheW1) and a CheW-CheR like (CheW3), are essential for array formation and chemotactic behavior, and also possess variable regions at the protein interfaces 9 . Bb also possesses two CheA homologs (CheA1-F8 and CheA2-F2) but only one of the homologs (CheA2) contains an elongated P3 domain and is essential for chemotaxis and pathogenicity 24 . Therefore, we predict that a similar chemotaxis arrangement is present in Bb, which has a similar diameter as Td 9 .
Unexpectedly, the placement of CheA in WT Td arrays could not be discerned (with the exception of the P3 domain), but was clearly visible in two Td mutants: one that lacks the previously characterized ODP sensor (Δ2498) and one that lacks both ODP and its cognate receptor (Δ2498Δ2496). As the density corresponding to the P3 domain in the WT strain is clearly discernible, the sparse density corresponding to all other CheA domains (P1, P2, P4, P5) is not attributed to low incorporation of CheA in these arrays. These results indicate that the kinase is highly mobile or more disordered in the WT strain, but is more constrained when ODP is deleted, suggesting that ODP directly affects array structure. However, densities in the three strains do not designate an obvious position for ODP, indicating that ODP may not be an integral component of the array, but rather peripherally interacts with the chemotaxis machinery.
In summary, we illustrate a novel chemotaxis arrangement that has evolved to compliment the spirochetes' high membrane curvature. Therefore, it is likely that the behavior and characteristics of chemoreceptor arrays in general can be influenced by perturbing the shape of the cell membrane. Importantly, previous cryo-ET chemotaxis studies have relied on artificial systems for higher resolution data, but these methods generate arrays with non-native curvature 8,17,20,21 . Collectively, these data exemplify the importance of examining biological structures in native in vivo conditions, as essential cellular features may not be recapitulated under in vitro, ex vivo, and artificial systems. While the use of model organism systems (such as Ec, Bs, Tm) has provided excellent insight into chemotaxis, examining the systems in non-model organisms can lead to new, unexpected advances for understanding the remarkable signaling system of bacterial chemotaxis.

Methods and Materials Bacterial strains, culture conditions, and oligonucleotide primers
Treponema denticola (Td) ATCC 35405 (wild-type) was used in this study. The Td deletion mutants, Δ2498 and Δ2498Δ2986, were generated in a previous study 2 . Cells were grown in tryptone-yeast extract-gelatin-volatile fatty acids-serum (TYGVS) medium at 37°C in an anaerobic chamber in presence of 85% nitrogen, 10% carbon dioxide, and 5% hydrogen 1 . Td mutants were grown with an appropriate antibiotic for selective pressure as needed: erythromycin (50 µg/ml) and gentamycin (20 µg/ml). Escherichia coli 5α strain (New England Biolabs, Ipswich, MA) was used for DNA cloning. The E. coli strains were cultivated in lysogeny broth (LB) supplemented with appropriate concentrations of antibiotics. The oligonucleotide primers for PCR amplifications used in this study are listed in Table  S3. These primers were synthesized by IDT (Integrated DNA Technologies, Coralville, IA). (Fig. S12) was constructed to replace the CheR-like domain (8781-1,308 nt) in TDE1492 with a previously documented erythromycin B resistant cassette (ermB) 3 . The TDE1492::ermB vector was constructed by two-step PCR and DNA cloning. To construct this vector, the 5` end of TDE1492 region and the downstream flanking region were PCR amplified with primers P 1 /P 2 and P 3 /P 4 , respectively, and then fused together with primers P 1 /P 4 , generating Fragment 1. The Fragment 1 was cloned into the pMD19 Tvector (Takara Bio USA, Inc, Mountain View, CA). The ermB cassette was PCR amplified with primers P 5 /P 6 , generating Fragment 2. The Fragment 2 was cloned into the pGEM-T easy vector (Promega, Madison, WI). The Fragment 1 and 2 were digested using NotI and ligated, generating the TDE1492::ermB plasmid. The primers used here are listed in Table S3. To delete TDE1492, the plasmid of TDE1492::ermB was transformed into Td wild-type competent cells via heat shock, as previously described 4 . Erythromycin-resistance colonies that appeared on the plates were screened by PCR for the presence of ermB and absence of TDE1492 (781-1,308 nt) gene. The PCR results showed that the TDE1492 (781-1,308 nt) gene was replaced by ermB cassette as expected (Fig.  S12). One positive clone (ΔTDE1492) was selected for further study.

Bioinformatics scripts and pipelines
We collected all information on the proteins classified as CheA (96,434) and CheW (134,165). To process this dataset we built several scripts and pipelines to produce the tables, figures and datasets used in this analysis (Fig. S13). The scripts are found in Supplementary File 1.

Chemosensory profile of Spirochaetotas
The "spiro-pipeline" selects all genomes from Spirochaetota phylum using gtdb-local package to access GTDB v89, then it filters only the genomes that are also present in MiST3 database. It collects the information on MiST for each genome and appends the complete taxonomy information from GTDB and signal transduction profiles. Finally, the pipeline builds the table with the information in markdown (Dataset 2).

Chemosensory profile of genomes with at least one CheA-F2
The "chea-pipeline" starts from the raw dataset taken from MiST3 database with information on 96,434 CheA genes. Based on MiST3 classification it selects the genomes with at least one CheA-F2 sequences and fetches information about these genomes. At this step, it also checks with the list

5`
3`  12 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 P 7 is located at the 5'-end of TDE1492, P 6 at the 3'-end of ermB, P 5 at the 5'-end of ermB, P 8 at the flanking region of TDE1492, P 9 at the middle of TDE1492, and P 10 at the 3'-end of TDE1492. The sequences of these primers are listed in Table S3.   Table S1B. Statistics of inner membrane curvature for 6Vc mini-cells.

Fig. S13
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.