Fig. S1. Map of the replication region of pBMB0228. Relevant restriction sites and ORFs are shown. Putative rep genes, double-strand origins (dso) and single-strand origin (sso) are indicated. Five derivatives consisting of fragments subcloned from pBMB0249 are shown, together with their segregational stabilities in B. thuringiensis strain BMB171. The orientation of arrows indicates the direction of transcription.

Fig. S2. Comparison of Mob regions from different plasmids of Gram-positive bacteria.

A. Comparison of oriT from plasmids of the pMV158 superfamily. Arrow indicates the nick site.

B. Motifs conserved among Mob proteins from the pMV158 superfamily. The motifs HxxR (motif I), NY(D/E)L (motif II) and HxDExxPHuH (motif III) are shown. Conserved amino acids at a given residue are indicated by identical colour shadings.

Fig. S3. Plasmid extraction of L colony of BMB171 (pBMB0228::Erm) (lane 1 and 2) and YBT1518 (pBMB0228::Erm) (lane 3 and 4) after growing for 6 generations (lane 1 and 3) and 60 generations (lane 2 and 4); Lane M, λDNA/HindIII markers.

Fig. S4. Resolution of pBMB0228::Erm during conjugation. Lane 1, donor strain BMB171 (containing single pBMB0228::Erm); lane 2, BMB171 (containing pBMB0228::Erm, pBMB02281::Erm and pBMB02282) used as reference with pBMB02281::Erm; lane 3, recipient strain B. cereus UW85R; lanes 4–7, four independent transconjugants with B. cereus UW85R as recipient; lanes 8–11, four independent transconjugants with B. thuringiensis BMB171R as recipient.

Fig. S5. The examination of transformants for the presence of the cointegrate or of a resolved plasmid containing the selective marker. Total plasmid extracted from 60 generation cultures and retransformed into BMB71, with selection for antibiotic resistance.

A. Overall experimental design used to compare the resolution frequency of these mutant plasmids.

B. Transfer the single colonies of BMB171 (pBMBM12) from Spc- or Kan-resistant plates on Spc- and Kan- resistant plates to determine whether pBMBM12 can resolve into two constituent plasmids.

C. Plasmids extracted from individual transformants: C, cointegrates; R, resolved plasmids.

D. PCR analysis of these transformants using primer pair repL1-repL2 (a and c) and primer pair rep1-rep2 (b and d). The 60 generation cultures were BMB171(pBMB0228::Erm) (lanes 1–11), BMB171(pBMBM12) (lanes 12–23), BMB171(pBMBM2) (lanes 24–35) and BMB171(pBMBM1) (lanes 36–46). Lanes numbered in black indicate transformants that contain the cointegrate. Lanes numbered in red indicate transformants that contain a resolved plasmid with the selective marker. Lane M1, λDNA/HindIII markers. Lane M2, Trans2K Plus DNA Marker. Lanes marked ck are a positive control for PCR, with YBT-1518 plasmid DNA as template.

Fig. S6. Plasmid profiles of donor, recipient and transconjugant strains. Lanes 1–4, four independent transconjugants from mating 4Q7 (pBMB02281::Erm + pAW63::Tn5401) with BMB171R (pBMB02282::Spc). Lane 5, donor strain 4Q7 (pBMB02281::Erm + pAW63::Tn5401). Lane 6, recipient strain BMB171R (pBMB02282::Spc). Lanes 7–10, four independent transconjugants from mating 4Q7 (pBMB02282::Spc + pAW63::Tn5401) with BMB171R (pBMB02281::Erm). Lane 11, donor strain 4Q7 (pBMB02282::Spc + pAW63::Tn5401). Lane 12, recipient strain BMB171R (pBMB02281::Erm). Lane 13, AW48 used to indicate the conjugative plasmid pAW63::Tn5401. pBMB171 is a endogenous plasmid in BMB171R which was not cured (He et al., 2010).

Fig. S7. pAW63::Tn5401 cannot mediate the oriT site-specific recombination.

A. Plating single colonies of BMB171 (pBMBT10 + pBMBmob1) and BMB171 (pBMBT10 + pAW63::Tn5401) onto Spc-containing plates after growing for 60 generations.

B. Observation of the derivatives of strain BMB171 containing pBMBT10, with pBMBmob1 or pAW63::Tn5401. PC, phase contrast; GFP, fluorescence microscopy. Recombination assays were performed as described in Experimental procedures. Recombination frequency was estimated as the number of Spc-sensitive colonies and combined with observations of the number of fluorescent cells under phase contrast and fluorescence microscopy. Each experiment was performed in triplicate.

Table S1. Open reading frames identified in pBMB0228.

Table S2. The resolution of pBMB0228 was mediated by Mob02281 or Mob02282.

Table S3. Mobilization efficiency of pBMB02281::Erm and pBMB02282::Spc into recipient cells.

Table S4. Bacterial strains and plasmids used in this study.

Table S5. Primers used in this study.

Appendix S1. Plasmid construction.

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