Table S1: Recombination Events Detected Across the BTA Family by RDP

Supplementary

Figure S1Phylogenetic tree of Clostridium botulinum C2 and C3 toxins reconstructed from the similarity sequences by NJ method. Branch lengths are proportional to evolutionary distances. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Dataset used for construction of this tree is available in Supplementary 1.

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Table S1: Recombination events detected across the BTA family by RDP

Daughter / Beginning / Ending / Major parent / Minor parent / Event / Av. P-value / Probability
CLB C[ str. SPA] / 2121 / 2166 / CLB C2-II [ BKT015925] / CD- CdtB / 64 / 1.160X10-03 / 8.7848 E-04
CLB C2-II / 2121 / 2166 / CLB C2-II [ BKT015925] / CD- CdtB / 64 / 1.160X10-03 / 8.7848E-04
CLB C2-II / 93 / 331 / CLB C2-II / CLB C2-II [ BKT015925] / 2 / 1.052x10-02 / 1.0526 E-02
CLB C2-II / 2121 / 2166 / CLB C2-II [ BKT015925] / CD- CdtB / 64 / 1.160x10-03 / 6.0584 E-02
CLB C C2-II / 2160 / 1776 / CLB C[ str. SPA] / CLB D C2-II [str. 1873] / 4 / 2.575X10-08 / 3.0885 E-22
CLB C C2-II / 2086 / 375 / CLB C[ str. SPA] / CLB C2-II / 2 / 2.343X10-03 / 2.3430 E-02
CLB C C2-II / 2121 / 2553 / CLB C2-II [ BKT015925] / CD- CdtB / 64 / 1.160X10-03 / 7.1340 E-04
CLB D C2-II [str. 1873] / 2101 / 1803 / CLB C2-II [ BKT015925] / CLB C[ str. SPA] / 3 / 1.504X10-14 / 2.2564 E-14
CLB D C2-II [str. 1873] / 420 / 1803 / CLB C2-II [ BKT015925] / CLB C2-II / 3 / 1.504X10-14 / 1.6229 E-20
CLB D C2-II [str. 1873] / 1916 / 1984 / CLB C2-II [ BKT015925] / CD- CdtB / 16 / 1.325X10-02 / 8.6710 E-03
CLB D C2-II [str. 1873] / 2084 / 2888 / CLB C2-II [ BKT015925] / CD- CdtB / 16 / 2.628X10-08 / 8.4201 E-09
PBA_ADP-RT [DSM29 / 48 / 83 / BAC-[ Rock4-2 ADP-RT] / CLB-Ia / 1 / 1.622X10-02 / 1.6228 E-02
PBA- dendritiformis _C3[ADP-RT] / 48 / 83 / BAC-[ Rock4-2 ADP-RT] / CLB-Ia / 1 / 4.438X10-02 / 0.044381917645917
Staup ADP-RT / 3 / 67 / Stau[21333] / Stau mono-ADP-RT [C3] / 6 / 2.885X10-03 / 2.7156 E-05
Staup ADP-RT / 281 / 342 / Stau[21333] / Stau mono-ADP-RT [C3] / 16 / 1.027X10-02 / 1.7710 E-02
Staup ADP-RT / 282 / 320 / Stau[21333] / CLB-C2I / 16 / 1.027X10-02 / 7.3794 E-03
Staup ADP-RT / 287 / 338 / Stau[21333] / CLB D C3 [str. 1873] / 16 / 1.027X10-02 / 5.5984 E-04
Staup ADP-RT / 299 / 342 / Stau[21333] / BAC-[ Rock4-2 ADP-RT] / 16 / 1.027X10-02 / 9.7286 E-04

CLB C -C2-II : Clostridium botulinum C2 toxin (component-II)

CLB C2-II : Clostridium botulinum C2 toxin, component II

CLB C2-II [ BKT015925] : Clostridium botulinum BKT015925 C2 toxin, component II

CLB D C2-II [str. 1873] : Clostridium botulinum D str. 1873 C2 toxin, component II

CLB D C3 [str. 1873]: Clostridium phage D-1873 partial mono-ADP-ribosyltransferase C3

CLB C[ str. SPA] : Clostridium botulinum C str. Stockholm protective antigen

Stau[21333] : Staphylococcus aureus subsp. aureus 21333 epidermal cell differentiation inhibitor

Staup ADP-RT: Staphylococcus aureus partial ADP-ribosyltransferase

Stau mono-ADP-RT [C3]: Staphylococcus aureus Mono-ADP-ribosyltransferase C3 precursor (Exoenzyme C3)

CLB C [str. Eklund m ADP ribosyltransferaeC3 : Clostridium botulinum C str. Eklund mono-ADP-ribosyltransferae C3

CD - [R20291] : Clostridium difficile R20291 adp-ribosyltransferase binding component

CD-C2 [CD69] : Clostridium difficile CD69 C2 toxin domain protein

CD- CdtB : [Clostridium] difficileCdtB

BAC-[ADP-ribosyltransferase] : Bacillus cereus partial ADP-ribosyltransferase

BAC-[ Rock4-2 ADP-RT]: Bacillus cereus Rock4-2 ADP-ribosyltransferase

CLB-C2I: Clostridium botulinum C2 toxin (component I)

PBA_ADP-RT [DSM29]: Paenibacillusalvei DSM 29 ADP-ribosyltransferase

PBA- dendritiformis _C3[ADP-RT]: Paenibacillusdendritiformis C454 mono-ADP-ribosyltransferase C3

CLB-Ia : Clostridium perfringens iota toxin component Ia

Figure S2 Estimates of network parameters for detected recombination events in binary toxin A family

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Figure S3Correlation of evolution rate on mutability (A) and functional mutable sites identified (B) in C2I structure

Thirty sites are identified in the C2I at mutable grade of 5 and 12 sites at a mutable grade of 9. The rate of evolution is noted to be low in the motif regions, suggested that these residues contribute a crucial role in its molecular function.

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Table S2 Amino acid mutations that stabilize the overall structure and pathogenecity of C2I

Mutant / CUPSAT / mCSM / Wild type / Mutant type / SDM / Virulence
SASA / ΔΔG
(kcal/mol) / SA (%) / HB / SA (%) / HB / ΔΔG
(kcal/mol)
R265A / 0.34 / 48.7 / 57 / USHB / 57 / NHB / 0.35 / NV
R299Y / 0.66 / -0.386 / 32 / NHB / 42 / NHB / 0.69 / NV
R299N / 0.22 / -0.332 / 32 / NHB / 27 / NHB / -2.39 / V
S348V / 5.98 / -0.408 / 49 / NHB / 49 / NHB / 2.24 / V
S348L / 5.61 / -0.315 / 49 / NHB / 42 / NHB / 1.48 / NV
S348I / 6.6 / -0.315 / 49 / NHB / 51 / NHB / 3.02 / V
S348M / 5.91 / 0.035 / 49 / NHB / 27 / NHB / 2.33 / V
S348W / 0.46 / -0.976 / 49 / NHB / 38 / SHB / 1.44 / NV
S348F / 4.8 / -1.051 / 49 / NHB / 31 / NHB / 2.74 / V
S348Q / 3.88 / -0.738 / 49 / NHB / 33 / USHB / 1.54 / NV
S348K / 5.17 / -0.649 / 49 / NHB / 39 / USHB / 0.65 / NV
S348Y / 2.72 / -0.73 / 49 / NHB / 29 / NHB / 2.66 / V
S348C / 10.41 / -0.489 / 49 / NHB / 49 / SHB / 0.9 / NV
S348R / 6.66 / -0.445 / 49 / NHB / 37 / SHB / 1.6 / NV
T349V / 1.41 / -0.831 / 2.0 / NHB / 1.0 / NHB / 2.95 / V
T349I / 2.05 / -0.591 / 2.0 / NHB / 1.3 / NHB / 2.88 / V
T349F / 1.24 / -1.332 / 2.0 / NHB / 1.1 / NHB / 1.93 / NV
T349Y / 1.43 / -0.985 / 2.0 / NHB / 3.0 / NHB / 0.99 / NV
T349H / 0.89 / -1.773 / 2.0 / NHB / 1.0 / NHB / -0.47 / NV
S350V / 2.23 / -0.441 / 16 / SHB / 14 / NHB / 3.37 / V
S350M / 2.62 / -0.15 / 16 / SHB / 15 / NHB / 2.92 / V
S350W / 4.14 / 1.1 / 16 / SHB / 11 / SHB / 0.93 / NV
S350T / 1.64 / 1.1 / 16 / SHB / 17 / NHB / 0.04 / NV
S350F / 2.88 / 1.1 / 16 / SHB / 10 / NHB / 3.41 / V
S350Y / 4.44 / 1.1 / 16 / SHB / 12 / SHB / 2.09 / V
S350R / 3.38 / 1.1 / 16 / SHB / 22 / NHB / -0.28 / NV
S350H / 4.42 / 1.1 / 16 / SHB / 16 / SHB / 1.8 / NV
E389K / 0.33 / 12.3 / 14 / SHB / 12 / USHB / -1.94 / NV
F398Y / 0.63 / 6.1 / 6.0 / NHB / 2.0 / NHB / -1.65 / NV

SASA: Solvent accessible surface area; HB: Hydrogen bonding; NHB: No hydrogen bonding; SHB: Saturated hydrogen bonding; USHB: Unsaturated hydrogen bonding; V: Virulence; NV: Non-virulence

Table S3 Amino acid mutations that stabilize the overall structure and pathogenecity of C2II

Mutant / CUPSAT / mCSM / Wild type / Mutant type / SDM / Virulence
SASA / ΔΔG
(kcal/mol) / SA (%) / HB / SA (%) / HB / ΔΔG
(kcal/mol)
E307K / 29 / 0.309 / 29 / USHB / 28 / SHB / 0.18 / V
E307S / 29 / -0.1 / 29 / USHB / 35 / NHB / -0.62 / NV
E307C / 29 / 0.079 / 29 / USHB / 30 / USHB / 0.79 / NV
E399A / 58 / -0.725 / 58 / SHB / 48 / NHB / 1.3 / NV
E399S / 58 / -0.399 / 58 / SHB / 54 / NHB / -0.34 / NV
E399C / 58 / -0.092 / 58 / SHB / 47 / SHB / -0.68 / NV
F456A / 76 / -1.445 / 76 / NHB / 55 / NHB / -1.79 / NV
F456V / 76 / -0.982 / 76 / NHB / 53 / NHB / -0.30 / NV
F456L / 76 / -0.775 / 76 / NHB / 50 / NHB / -0.41 / NV
F456I / 76 / -0.775 / 76 / NHB / 51 / NHB / -0.12 / NV
F456M / 76 / -0.597 / 76 / NHB / 58 / NHB / 0.13 / NV
F456S / 76 / -1.205 / 76 / NHB / 53 / NHB / -1.96 / NV
F456K / 76 / -0.505 / 76 / NHB / 56 / SHB / -2.10 / V
F456Y / 76 / -0.379 / 76 / NHB / 70 / NHB / -0.41 / NV
F456C / 76 / -0.592 / 76 / NHB / 52 / SHB / 1.92 / NV
F456H / 76 / -0.085 / 76 / NHB / 47 / SHB / -0.57 / NV
G305K / 117 / -0.678 / 117 / NHB / 86 / SHB / 1.17 / NV
G305A / 117 / -0.231 / 117 / NHB / 90 / NHB / 2.23 / V
G305M / 117 / -0.562 / 117 / NHB / 79 / NHB / 1.69 / NV
G305D / 117 / -0.672 / 117 / NHB / 91 / USHB / 2.20 / V
G305H / 117 / -0.411 / 117 / NHB / 85 / SHB / 2.21 / V

SASA: Solvent accessible surface area; HB: Hydrogen bonding; NHB: No hydrogen bonding; SHB: Saturated hydrogen bonding; USHB: Unsaturated hydrogen bonding; V: Virulence; NV: Non-virulence

FigureS4 Conformational dynamic simulations for comparison of the protein structural backbone disparity (near-native shifts) of C2 toxin and its avirulent mutants

Figure S5 showing the structural fluctuation at the residue where mutated in Clostridium botulinum C2 toxin

FigureS6 Molecular dynamic simulation for comparison of structural fluctuation (RMSF) of C2 toxin and its avirulent mutants

FigureS7 Antibody epitope prediction for comparison of immunogenic determinants in C2 toxin and its avirulent mutants

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