Selection of sybodies targeting Smc-ScpAB in B. subtilis.

(A) (i) Structural design and randomization scheme of the three synthetic sybody libraries: concave, loop, and convex. Complementarity-determining regions (CDRs) 1, 2, and 3 are shown in yellow, orange, and red, respectively; randomized residues are shown as sticks. Adapted from (Zimmermann et al., 2018). (ii) SMC domain organization. The N- and C-terminal regions encode half an ATPase head, while the middle domain constitutes the hinge dimerization domain. The elongated sequence in-between encodes for long helical sequences that will form an antiparallel intramolecular coiled coil. Hd: head, Hng: hinge, 4N: 4N arm-to-arm contact, J: joint. (iii) SMC complexes harbor juxtaposed arms in their resting state. Upon ATP binding at the heads, the heads dimerize (head engagement), leading to a conformational change of the entire complex in which the arms open. This state is thought to fit a loop of DNA (not shown) in the S compartment formed by the SMC arms, that will be pushed towards the K compartment formed by the heads and kleisin, after ADP release. We hypothesized that SMC-blocking sybodies can stabilize distinct conformation of the bsuSmc-ScpAB complex promoted by ATP hydrolysis and DNA binding, potentially inducing a Δsmc-like phenotype in vivo and enabling biochemical characterization of trapped conformations in vitro. Blue: SMC dimer, Green: kleisin ScpA. The ScpB KITE subunits are not represented for the sake of simplicity. HngDBS: Hinge DNA-binding site, HdDBS: Head DNA-binding site. (B) Framework for sybody selection. In vitro selection starts with ∼1012 sybody variants per library subjected to ribosome display for pre-enrichment, followed by two rounds of phage display. For in vivo selection, 95 randomly selected sybody genes were integrated into the B. subtilis chromosome under the control of a xylose-inducible promoter by allelic replacement at the amyE locus. Growth defects on rich medium were tested on ONA agar plates with or without 0.5% xylose. Example shown: Sb164 (loop library) did not affect growth, whereas Nb31 impaired growth upon induction, suggesting interference with bsuSmc function. (C) Growth assay results for the 95 B. subtilis strains expressing individual xylose-inducible sybodies of the loop library. Bars show colony counts “without” (green) on top of “with” (pink) xylose. Strains are ordered by their original position in the 96-well plate. Fourteen sybodies consistently impaired colony formation under inducing conditions (marked by dotted lines). Sybody numbers indicated above the plots correspond to selected candidates used in subsequent experiments, numbering according to order of first use. Notably, the strain corresponding to the E09 sybody (Sb018), showed an absence of colonies upon sybody induction. However, this sybody candidate gave intermediate phenotypes in later experiments, which is why it was excluded from detailed analysis.

Sybody-induced chromosome segregation defects visualized by ParB-GFP imaging.

(A) i, Top: Schematic illustrating ParB-GFP binding near oriC, enabling visualization of origin positioning. Bottom: Representative images showing ParB-GFP foci in wild-type and Δsmc B. subtilis. Smc WT cells typically display 2–4 foci per cell, whereas Δsmc cells exhibit reduced foci numbers. ii, Number of ParB-GFP foci per µm of cell length in a strain carrying inducible sybody Sb006. A significant decrease in foci is detected from 40 minutes post-induction (pt0-10 = 0.9674; pt0-20 = 0.9033; pt0-30 = 0.3744; pt0-40 = 0.0145; pt0-50 = 0.0004; pt0-60 < 0.001). Based on this, a standardized induction time of ∼35 minutes was used in all subsequent experiments. (B) ParB-GFP foci density (foci/µm) in WT, Δsmc, and sybody-expressing strains after 35 minutes of xylose induction. Violin plots show distribution per condition; solid lines denote the mean, and dotted lines indicate quartiles. (C) Spot assay to assess colony formation of B. subtilis strains bsu or spn variants of Smc-ScpAB and ParB. Top: Schematic shows gene origins (blue: B. subtilis; grey: S. pneumoniae). The leftmost column corresponds to the parental B. subtilis strain 1A700 carrying the S. pneumoniae genes without chloramphenicol resistance (non-growing). The next spots represent the same strains carrying no sybody gene but the Cm resistance (EV for empty vector). Remaining spots are sybody-expressing strains; sybody numbers are indicated above. Cells were grown for 16 h at 37 °C on ONA supplemented with 0.5% xylose and chloramphenicol. Hd: head, Hng: hinge, 4N: 4N arm-to-arm contact, J: joint.

Mapping sybody binding sites on Smc-ScpAB.

(A) Colony formation of B. subtilis strains expressing chimeric Smc-ScpAB complexes comprising S. pneumoniae and B. subtilis sequences. The schematic above the spot assay depicts the species origin of the smc gene, the scpAB operon and parB gene (blue: B. subtilis; grey: S. pneumoniae). “–” indicates no insertion at the amyE locus; “EV” refers to the empty vector control containing only the chloramphenicol resistance cassette that was inserted in amyE; numbered labels correspond to sybodies. Cells were spotted on rich medium (ONA) supplemented with chloramphenicol and xylose and incubated for 16 h at 37 °C. Hd: head, Hng: hinge, 4N: 4N arm-to-arm contact, J: joint. (B) ATP hydrolysis rates of bsuSmc-ScpAB in the presence of sybodies but absence of DNA. Significant effects by one-way ANOVA are indicated by p values. (C) ATPase rates in the presence of 40 bp dsDNA. All sybodies significantly reduced DNA-stimulated ATP hydrolysis. Reported p-values: Sb020 (p = 0.340), Sb194 (p = 0.0049), Sb002 (p = 0.0007), Sb015 (p = 0.0003); all others, p < 0.0001. (D) Schematic summary of sybody binding sites mapped onto the Smc dimer, categorized by their effect on ATPase activity. Sybodies are grouped based on functional impact and mapped to corresponding structural regions: pink/yellow boxes indicate residues 318–339 and 836–857; green boxes mark residues 248–276 and 899–927; and the red box highlights the 4N contact region (approx. residues 290–320).

Proposed models for sybody interactions with bsuSmc-ScpAB during the ATPase cycle.

Model for sybodies of the OpeningUP, ClosingUP and ClosingDOWN group. OpeningUP sybodies, likely prevent complete arm closure. In the presence of DNA, these sybodies may stabilize a suboptimal conformation for head disengagement, resulting in reduced ATPase activity (not shown here). ClosingUP and ClosingDOWN sybodies stabilize a closed-arm conformation. These likely hinder DNA segments from entering the inter-arm space and accessing the hinge-proximal DNA binding site.

Preparation of bsuSmc(C119S, C437S, C826S, E1118Q, R643C)-ScpAB complex and loop extrusion model.

(A) Size-exclusion chromatography profile of biotinylated bsuSmcE(C119S, C437S, C826S, E1118Q, R643C)-ScpAB complex. Peak fractions (elution at ∼13 mL, pooled fractions indicated by dotted lines for a final volume of 1.8 mL) were collected for downstream use; the final concentration was 5.9 µM (dimer). * : A secondary peak appeared at ∼14 mL; its identity was uncertain and was excluded from the peak. SDS-PAGE confirms successful biotinylation of the bsuSmc(C119S, C437S, C826S, E1118Q, R643C)-ScpAB complex. (B) Segment-capture model for bsuSmc-ScpAB, in which ATP binding and hydrolysis drive transitions between open and closed conformations to mediate loop extrusion.

Representative results from in vivo sybody selection based on colony formation.

(A) Representative examples of in vivo transformation assays for nine sybody-expressing B. subtilis strains. Sybody genes were integrated into the amyE locus under xylose-inducible control. Transformants were grown on oxoid nutrient agar (ONA) plates supplemented with chloramphenicol and 0.5% xylose to assess growth defects. Shown are candidates from the concave library: Sb051 (well B08) and Sb078 (D12). Loop library: Sb007 (E05), Sb020 (E12), Sb031 (F05), Sb164 (D12) and NbH07 (Sb194). Convex library: Sb239 (D12) and Sb248 (E10). (B) Growth assay results for the 285 B. subtilis strains expressing individual xylose-inducible sybodies. Bars show each colony counts “without” (green) on top of “with” (pink) xylose. Strains are ordered by their original 96-well plate. Only the loop library yielded sybodies that consistently impaired growth under inducing conditions (dotted line). Sybody numbers indicated above the plots correspond to positive candidates used in subsequent experiments, numbered according to order of use. Notably, the strain corresponding to the E09 M sybody (Sb018), also showed an absence of growth upon sybody induction. However, this sybody candidate gave intermediate phenotypes in later experiments, which is why it was excluded from this study. Results for the loop library are also shown in Figure 1.

Functional impact of sybody expression on chromosome organization, cell length, and growth in B. subtilis.

(A) Average number of ParB-GFP foci per µm of cell length in strains expressing individual sybodies, with and without xylose induction. Most uninduced strains differed significantly from the Smc WT control, likely due to leaky expression from the Pxyl (p < 0.0001), except Sb015 (p > 0.9999), Sb156 (p = 0.2345), and Sb016 (negative control, p > 0.9999). (B) Average cell length across the same conditions, measured in ParB-GFP strains with or without Smc, and with or without each sybody. Bars indicate standard deviation. Tested sybodies caused cell elongation, a characteristic phenotype of impaired Smc activity, as chromosome segregation defects delay cell division, with mean lengths from 4.70 ± 2.16 µm (Sb020) to 8.68 ± 2.99 µm (Sb010), compared to 4.51 ± 1.95 µm in wild-type and 9.96 ± 8.92 µm in Δsmc. (C) Growth curves of B. subtilis strains expressing individual sybodies compared to the WT strain, with and without xylose induction. Each curve represents the mean of two biological replicates. Six sybodies were randomly picked and showed in this figure. Fluorophore-free strains showed wild type-like growth without xylose but strong delays post-induction, followed by partial growth recovery after 12 hours, likely due to xylose depletion or suppressor emergence. In sybody-expressing strains, a clear drop in cell density was observed ∼2.5 hours post-induction.

ATPase hydrolysis rate of the bsuSmc-ScpAB complex with each selected sybody and viability of B. subtilis strains expressing chimeric Smc-ScpAB complexes in the absence of sybody induction.

(A) Spotting assay assessing the growth of B. subtilis strains carrying chimeric Smc-ScpAB operons composed of S. pneumoniae and B. subtilis components. Strains harbor individual sybody constructs integrated at the amyE locus under control of a xylose-inducible promoter but were grown without inducer. “–” indicates no integration at amyE; “EV” corresponds to an empty vector control containing only the chloramphenicol resistance cassette. Numbered labels indicate strains carrying specific sybody constructs. Cells were spotted on rich medium (ONA) containing chloramphenicol and incubated for 16 h at 37 °C. (B) Overall ATPase hydrolysis rate of the bsuSmc-ScpAB complex in presence of each sybody and +/-DNA. Sb06, 021 and 194 triggered a significant difference in ATP hydrolysis between +/-dsDNA conditions. The rest is non-significant.

Hypothetical model for an Opening DOWN class of sybodies, not recovered in this study.

This may indicate that stabilizing an open conformation near the ATPase heads does not interfere with Smc function.