Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae

Abstract

Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements-in particular bacteriophages and plasmids-the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements¹. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic^2-4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shaped the evolution of the most successful lineage of pandemic V. cholerae.

Extended Data Fig. 1. Characterisation of plasmids from an environmental Californian V. cholerae population.

a-b, Agarose gels showing plasmid DNA preparations from either a collection of pandemic and clinical strains with environmental strain Sa5Y as control (a) or the environmental non-O1/non-O139 Californian V. cholerae isolates (b). The letters above the gels in (b) denote the phylogenetic clade based on comparative genome hybridization. gDNA, genomic DNA; pDNA, plasmid DNA. Strains SL4G and SO5Y contain a pSa5Y-like plasmid; strains SA7G and E7G contain an identical plasmid with a replication initiation protein (Rep) based origin. Strains SA7G and E7G additionally contain large plasmids pSA7G1, pSA7G2 and pE7G of ^~ 80kb that are not resolved on the gels. c, Copy number of pSa5Y in exponentially growing and stationary cells, as determined by quantitative PCR. Bar charts show mean ± sd from three independent experiments (individual dots). d, Schematic comparing the pB1067 minimal origin region (dashed lines) with the homologous region of pSa5Y. Sequences encoding equivalents of the experimentally verified RNA I and RNA II from pB1067 are indicated by arrows along with a predicted transcriptional terminator (inverted triangles). e, Clustal Omega alignment comparing the pB1067 minimal origin region and the pSa5Y origin region. f, Validation of the pSa5Y origin of replication (ori). The putative pSa5Y ori and ori sequences from plasmids pB1067 (pB1067 ori), pBAD (ColE1 ori) and pACYC177 (p15A ori) were cloned into a conditionally replicating plasmid (pMJ174) containing the pir-dependent R6K ori. The resulting plasmids were introduced into pir- and pir+ E. coli strains and spotted on LB+Kan plates. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 2. Conservation of the plasmid stability phenotype dependent on VC1771-70 and VC0492-90.

a, pSa5Y-Kan stability in representative wild-type (WT) 7PET strains and their ΔVC1771 derivatives after growth for approx. 50 generations without antibiotic selection. b, Comparison of VC1769-1772 transcript levels between strains A1552, classical O395 and ATCC25872 in exponentially growing cells as determined by qRT-PCR. c, Phylogenetic tree based on the protein sequence of VC1771 showing that all classical strains examined (green) encode the defective VC1771[K891] variant, whereas all 7PET strains examined (blue) encode the active VC1771[E891] variant. One lineage of 7PET strains encodes an additional S29P substitution. Strains tested in this work are highlighted (*). d, Effect of different VC1771 variants on pSa5Y-Amp stability in the classical strain O395. e, pSa5Y-Amp stability in strain A1552 encoding a classical variant of VC1771, in the presence and absence of VC0490-93. f, Retention of plasmids with various origins of replication in strain A1552 and its ΔVC1770 and ΔVC0490 derivatives. The different origins of replication (see Methods) were cloned into a neutral plasmid backbone containing a conditional origin of replication inactive in the tested strains. Plasmid stability in (d-f) was evaluated after growth for approx. 50 generations without antibiotic selection. g, Morphology of exponentially growing cells carrying pBAD, grown in the absence and presence of ampicillin (100 μg/mL), in strain A1552 and its ΔVC1770-72, ΔVC0490-93 and ΔVC1770-72 ΔVC0490-93 derivatives. Images are representative of the results of three independent experiments. Scale bar = 5 μm. h, Comparison of transcript levels of VC1770-72 in A1552 and ΔVC0490-93 (top) and VC0490-93 in A1552 and ΔVC1770-72 (bottom) in exponentially growing cells as determined by qRT-PCR. Bar charts show mean ± sd from three independent experiments (individual dots).

Extended Data Fig. 3. Production of DdmABC leads to plasmid-dependent toxicity in V. cholerae and E. coli

a, Growth of V. cholerae A1552 derivatives carrying arabinose-inducible ddmABC (TnddmABC) or TnddmABC encoding DdmA and DdmC variants was evaluated on plates, either without additions or supplemented with 0.02% or 0.2% arabinose, in the absence and presence of plasmid pSa5Y-Amp, as indicated. b, Growth of E. coli MG1655 derivatives carrying TnddmABC was evaluated on plates in the absence and presence of plasmid pSa5Y-Kan as in (a). Note the transition in appearance of MG1655-TnddmABC carrying pSa5Y + 0.2% arabinose, switching from dark to pale colonies. c, Cells of V. cholerae strain Δddm (ΔddmDE ΔddmABC) TnddmABC were imaged following growth for approx. 10 generations in either the absence or presence of 0.02 or 0.2% arabinose, and the absence and presence of pSa5Y-Amp, as indicated. DNA was stained with DAPI. Anucleate cells are indicated with black arrowheads; cells with abnormal nucleoids are indicated with white arrowheads. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm.

Extended Data Fig. 4. Validation of plasmid stability by ParB/parS labelling in individual cells.

a, Visualisation of plasmid pSa5Y in a strain constitutively expressing yGFP-ParB^MT1 by incorporating the MT1 parS site into the plasmid (parS+). b, pSa5Y-parS^MT1 retention and localisation in single cells of the indicated strains following growth for approx. 50 generations without antibiotic selection. c, Comparison of pSa5Y-parS^MT1 stability for the same cultures as in (b) determined using either a plating assay or by quantifying the fraction of plasmid-containing cells by microscopy. d, pSa5Y-parS^MT1 localisation in single cells of the indicated strains following growth for approx. 10 generations without antibiotic selection. e-f, Quantification of the fraction of plasmid containing cells (e) and the fraction of cells containing plasmid clusters (f) for the cultures shown in (d), mean values are shown above the bars. Bar charts show mean ± sd from three independent experiments (individual dots). All strains carry a chromosomally integrated yGFP-ParB^MT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm.

Extended Data Fig. 5. Plasmid elimination by DdmDE is rapid and does not depend on cell division while cell division inhibition enhances plasmid-dependent toxicity by DdmABC.

Time-course showing the effect of DdmDE (a) and DdmABC (b) production on pSa5Y-parS^MT1 plasmid retention and localisation after addition of cephalexin (Ceph, 5 μg/ml) to block cell division. Arabinose (+Ara) and/or Cephalexin (+Ceph) were added to exponentially growing cells of Δddm (ΔddmDE ΔddmABC) and imaged after 1, 2 and 3 hours of growth, as indicated. Expression from TnddmDE and TnddmABC was induced with 0.2 or 0.02% arabinose, respectively. Arrowheads in panel (a) highlight examples of plasmid-free cells already detectable after only 1h of TnddmDE induction. Open arrowheads in panel (b) indicate examples of cells undergoing plasmolysis due to enhanced DdmABC toxicity. All strains carry a chromosomally integrated yGFP-ParB^MT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 5 μm.

Extended Data Fig. 6. Individual production of DdmD and DdmE or DdmA, DdmB and DdmC does not lead to plasmid and phage-related phenotypes.

a, Effect of individual production of DdmD or DdmE on pSa5Y-parS^MT1 plasmid retention in V. cholerae Δddm (ΔddmDE ΔddmABC). Cells were imaged after growth for approx. 10 generations without antibiotic selection, in the absence (No Ara) and presence (+ Ara) of 0.2% arabinose. b, Stability of a conditionally replicating plasmid carrying either an empty transposon or a transposon with inducible ddmDE, ddmD or ddmE in E. coli strain S17-1λpir. Cultures were evaluated after growth for approx. 10 generations without antibiotic selection, in the absence (grey bars) and presence (green bars) of 0.2% arabinose. Bar charts show mean ± sd from three independent experiments (individual dots). The inset shows the plasmid extraction yield from the same cultures. For gel source data, see Supplementary Figure 1. c, Effect of individual production of DdmA, DdmB or DdmC on pSa5Y-parS^MT1 plasmid localisation in V. cholerae Δddm (ΔddmDE ΔddmABC). Cells were imaged after growth for approx. 10 generations without antibiotic selection, in the absence (No Ara) and presence (+ Ara) of 0.02% arabinose. d, Fold protection against coliphages P1 and λ in E. coli strain MG1655 conferred by either DdmABC or by DdmA, DdmB and DdmC produced individually. Transposon constructs TnddmABC, TnddmA, TnddmB and TnddmC were integrated in the chromosome and their expression induced by inclusion of 0.2% arabinose in the medium. Fold protection was determined by plaque assays. Strains in panels (a) and (c) carry a chromosomally integrated yGFP-ParB^MT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm.

Extended Data Fig. 7. Structural modelling of DdmABC and DdmDE.

Cartoon representations showing the predicted structures of V. cholerae strain A1552 DdmABC and DdmDE, highlighting identified functional domains. Side chains of residues predicted to be involved in either nuclease activity or nucleotide binding and hydrolysis are shown as red sticks: DdmA, active site residues of the PD-(D/E)×K superfamily nuclease motif (⁴²D...⁵⁵QDK⁵⁷); DdmC, the predicted ATP-binding site formed by the N-terminal Walker A motif (³⁴GSSKSGKS⁴¹) and the abnormal C-terminal Walker B motif (⁵⁶⁰YIIYDQ⁵⁶⁵) and DdmD, superfamily II helicase motifs I (⁵²GIGKT⁵⁶), II (²⁷²DEID²⁷⁵), III (⁵⁴³SATA⁵⁴⁶) and VI (⁸⁵¹QAVGRAGR⁸⁵⁸) that comprise the predicted ATP-binding site, and active site residues of the PD-(D/E)×K superfamily nuclease motif (¹⁰⁸⁵D...¹¹⁰⁰DSK¹¹⁰²). Active site residues of the PD-(D/E)×K motif are shown in bold. Residues targeted by site-directed mutagenesis are underlined. The position of the DdmD residue E891, which in 6^th pandemic classical V. cholerae strains is typically K891, is shown as a green sphere, located adjacent to the arginine finger (Motif VI). Structural modelling was done using RoseTTAFold (DdmABC) and AlphaFold2 (DdmDE). Images were prepared using PYMOL.

Extended Data Fig. 8. DdmD, DdmA and DdmC variants remain non-functional even when overproduced.

a, pSa5Y-Amp plasmid retention in strains producing DdmD variants from TnddmDE in a ΔddmDE or Δddm (ΔddmDE ΔddmABC) background, as indicated, after growth for approx. 50 generations without antibiotic selection. b, Western blots showing protein levels of DdmD variants and DdmE. The predicted molecular masses of DdmD and DdmE are 136 and 79.1 kDa, respectively. DdmDE production in (a-b) was induced by 0.2% arabinose. c, Effect of DdmD variants production on pSa5Y-parS^MT1 plasmid retention in a Δddm background after growth for approx. 10 generations without antibiotic selection, in the absence (No Ara) and presence of 0.2% arabinose (+ Ara). d, pSa5Y-Amp plasmid retention in strains producing DdmA and DdmC variants from TnddmABC in a ΔddmABC or Δddm (ΔddmDE ΔddmABC) background, as indicated, after growth for approx. 50 generations without antibiotic selection. e, Western blots showing protein levels of DdmA and DdmC variants. The predicted molecular masses of DdmA and DdmC are 44.5 and 74.6 kDa, respectively. DdmABC production in (d-e) was induced by 0.02% arabinose. f, Effect of DdmA and DdmC variants production on pSa5Y-parS^MT1 plasmid localisation in a Δddm background after growth for approx. 10 generations without antibiotic selection, in the absence (No Ara) and presence of 0.02% arabinose (+ Ara). Bar charts show mean ± sd from three independent experiments (individual dots). Strains in panels (c) and (f) carry a chromosomally integrated yGFP-ParB^MT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm. For Western blotting source data, see Supplementary Figure 1.

Extended Data Fig. 9. Distribution of DdmDE and DdmABC homologues.

a-b, Taxonomic distribution of putative homologues of DdmD (a) and DdmE (b) derived from homology searching using PHMMER. The numbers in parentheses at each node indicate the relative number of search hits, and the blue bars represent the distribution of significant hits, respectively, within each taxonomic group. A full list of significant matches is provided in Supplementary Table 2. c, Schematics showing examples of intact two-gene ddmDE operons within the Lactobacillaceae. Operons were identified by examining the genomic loci of PHMMER hits to DdmD within each taxonomic group. The numbers in parentheses below each gene represent the size of the encoded protein (aa). d, Taxonomic distribution of putative homologues of DdmC derived from homology search using PHMMER, as described in (a). 685/728 (94%) of the significant matches to DdmC contain the same C-terminal DUF3732. e, Schematics showing examples of intact three-gene ddmABC operons caried on plasmids of the Rhizobiaceae. The presence of ddmABC on a plasmid is based either on the sequence annotation or on the proximity (*) to plasmid-specific genes (repABC and tra).

Extended Data Fig. 10. Plasmid acquisition and stability in V. cholerae 7PET.

a, Transformation frequencies of indicated strains with plasmid pSa5Y-Kan (left) and pBAD-Kan (right) introduced by natural transformation on chitin. b, Transformation frequencies of indicated strains with plasmid pSa5Y-Kan introduced by electroporation. c, Conjugative transfer frequencies of indicated strains with plasmid pSa5Y-oriT. d, Stability of IncC plasmid pVCR94 and its derivatives with deletions in genes encoding the host defence evasion locus (hde), a homologue of the partitioning protein ParM, and a toxin-antitoxin system (TA) in strain A1552. e, Stability of plasmid pSa5Y-Kan either alone or in a strain carrying plasmid pVCR94. f, Stability of plasmid pVCR94 evaluated in the indicated strains, and the expression of TnddmABC and TnddmDE induced with either 0.02% or 0.2% arabinose, as indicated. Plasmid retention in panels (d-f) was evaluated after growth for approx. 50 generations without antibiotic selection. g, Effect of DdmABC production on growth evaluated after culturing for 8h in liquid media (equivalent to approx. 10 generations) without antibiotic selection, in the absence and presence of 0.2% arabinose, with either no plasmid, pSa5Y-Amp or pVCR94, as indicated. h, Co-culture experiments evaluating competition between A1552 (WT) and Δddm strains carrying either no plasmid, pVCR94 or pW6G, competed against A1552ΔlacZ. i, Co-culture experiments evaluating competition over time between A1552ΔlacZ and either A1552 (WT) or A1552Δddm (Δddm), carrying either no plasmid, pVCR94 or pW6G, after growth for approx. 10, 20, 30, 40 and 50 generations without antibiotic selection. j, Co-culture experiments evaluating competition between A1552 (WT), A1552-SXT, A1552-Vch Ind5 and A1552 carrying pVCR94, competed against A1552ΔlacZ. For panels h and j the competitive index of the indicated strains was evaluated after growth for approx. 50 generations without antibiotic selection. Data represent mean ± sd from three independent experiments (individual dots). Significant differences were determined by one-way ANOVA with either Dunnett’s (a-c) or Tukey’s (h and j) post-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns – not significant. k, Proposed model of elimination of foreign elements by DdmABC and DdmDE. (i) Small multi-copy number plasmids are recognised by DdmE, which in turn leads to plasmid targeting and degradation by the helicase-nuclease DdmD. This process is enhanced by DdmABC, potentially due to its ability to cluster plasmids facilitating recognition by DdmE and/or it may directly participate in plasmid degradation via the nuclease DdmA. (ii-iii) Foreign DNA is recognised by the SMC-like protein DdmC, switching DdmABC to an active state, which in turn leads to activation of DdmA nuclease activity. (ii) Bacteriophage infection and immediate DNA replication leads to strong activation of DdmA, causing acute toxicity via host DNA damage. This results in abortive infection, protecting the population by preventing the release of mature virions. DdmA may also degrade bacteriophage DNA. (iii) Large low copy number plasmids lead to chronic but low-level activation of DdmA, resulting in host DNA damage, that imparts a competitive defect compared to plasmid-free cells. This Abi-like mechanism counter-selects against plasmid carrying cells, leading to their elimination from the population. Created with BioRender.com.

Fig. 1. Plasmids are unstable in V. cholerae O1 El Tor.

a, Time-course of pBAD and pSa5Y (pSa5Y-Amp) stability in 7PET strain A1552. Strains were grown without (-Amp) and with (+Amp) selection, with sampling after growth for 0 and approx. 10, 30 and 50 generations. b, Morphology of exponentially growing cells carrying either pBAD or pSa5Y without and with ampicillin (100 μg/ml), as indicated. Images are representative of three independent experiments. Scale bar = 5 μm. c, Retention of pSa5Y (pSa5Y-Kan) in various O1, O139 and non-O1/non-O139 (environmental isolates and toxigenic O37 strain ATCC25872) strains. d, Retention of plasmids with various origins of replication in A1552 and three non-O1/non-O139 (SL5Y, SP6G, SA3G) environmental isolates. The different origins of replication (see Methods) were cloned into a neutral plasmid backbone containing a conditional origin of replication inactive in the tested strains. Plasmid stability in c-d was determined after growth for approx. 50 generations without antibiotic selection. Bar charts show mean ± sd from three independent experiments (individual dots).

Fig. 2. V. cholerae O1 El Tor contains two plasmid defence systems on pathogenicity islands.

a, Schematic showing Vibrio pathogenicity island 2 (VPI-2) highlighting the region missing in strain MO10.b, pSa5Y-Amp stability in strains A1552 (O1), MO10 (O139), an A1552 transformant with the O1 serogroup exchanged to O139 (O1>O139) and transformants with the O1 serogroup cluster partially exchanged by the O139 region, with (AIIIHSGC#4) and without (AIIIHSGC#5) co-transfer of the truncated version of VPI-2 (ΔVC1760-VC1788). c, Effect of deletion of VPI-2 segments missing in MO10 (Δ#1-#5, marked in a) on plasmid stability. d, Effect of individual deletions of VC1770-VC1772 on plasmid stability. e, Expression of VC1770 and VC1771 complements the respective deletion but does not complement the deletion of VC1770-72. f, Locations of CTXΦ, VPI-1/-2, and VSP-I/-II on chromosome I of A1552. g, Schematic showing Vibrio seventh pandemic island (VSP-II) and positions of the deleted segments. h, pSa5Y-Amp stability in strain A1552 deleted for VPI-1, VSP-I and VSP-II. i, Effect of deletion of VSP-II segments (Δ#1-#5, marked in g) on plasmid stability. j, Effect of individual deletions of VC0490-VC0493 on plasmid stability. k, Expression of VC0490 and VC0492 complements the respective deletion but does not complement the deletion of VC0490-VC0493. Genes were expressed from a chromosomally integrated transposon (Tn) using the arabinose-inducible P_BAD promoter. Unless noted otherwise, plasmid retention was evaluated in derivatives of strain A1552, after growth for approx. 50 generations without antibiotic selection, in either LB or for panels (e) and (k) LB + 0.2% arabinose. Bar charts show mean ± sd from three independent experiments (individual dots).

Fig. 3. Mode of action of DdmDE and DdmABC systems.

a, Effect of DdmDE (TnddmDE) and DdmABC (TnddmABC) production on pSa5Y-Amp stability in V. cholerae, in the presence and absence of the other system, after growth for approx. 50 generations without antibiotic selection. Δddm – ΔddmDEAddmABC. b, Stability of a neutral plasmid backbone utilising various different origins of replication in E. coli strain MG1655 bearing either Tn-empty, TnddmDE or TnddmABC, after growth for approx. 10 generations without antibiotic selection. c, Fold protection against five coliphages conferred by either DdmDE, DdmABC or DdmABC variant production in E. coli, as determined by plaque assays. d, Growth kinetics of E. coli cultures carrying either Tn-empty (No system) or TnddmABC (+DdmABC) with either no phage or infected at time 0 with phage P1 at MOI 0.2 and 10. Inset shows accelerated lysis of DdmABC-producing culture. Data representative of three independent experiments.e, Effect of DdmDE and DdmABC production on pSa5Y-parS^MT1 localisation and retention in V. cholerae, in the presence and absence of the other system, after growth for approx. 10 generations without antibiotic selection. Charts below images show quantified plasmid retention. Images representative of three independent experiments. Scale bar = 2.5 μm. f-i, Schematics showing conserved domains and features identified in DdmDE (f) and DdmABC (h). Residues targeted by site-directed mutagenesis are highlighted in red. Numbers at boundaries indicate protein size (aa). Retention of pSa5Y-Amp in V. cholerae strains encoding variants of DdmD (g), DdmA and DdmC (i) was evaluated after growth for approx. 50 generations without antibiotic selection. Bar charts show mean ± sd from three independent experiments (individual dots). ND, not determined. Genes in panels (a-e) were expressed from a chromosomally integrated transposon (Tn) using the arabinose-inducible P_BAD promoter. Growth media were supplemented with 0.2% arabinose, except for TnddmABC in the presence of plasmids, which used 0.02% arabinose due to toxicity.

Fig. 4. Impact of DdmDE and DdmABC systems on V. cholerae 7PET ecology.

a, Retention of small (<10kb) and large (pSA7G1, 79.4kb; pW6G, 306.5kb; pVCR94, 171.8kb) plasmids native to V. cholerae, V. fischeri (pES213) or E. coli (pBAD) evaluated in A1552 (WT) and Δddm (ΔddmDE ΔddmABC) after growth for approx. 50 generations without antibiotic selection. Plasmids contain an added kanamycin resistance cassette for quantification, except pVCR94, for which natural ampicillin resistance was used. b, Conjugation assay showing the transfer frequency of the large plasmids into A1552 and derivative strains. c-d, Co-culture experiments evaluating competition between the indicated strains after growth for approx. 50 generations without antibiotic selection. c, Competitive index of strains A1552 (WT), ΔddmDE and ΔddmABC carrying either no plasmid, pVCR94 or pW6G, competed against A1552ΔlacZ. d, Competitive index of strains A1552 (WT), A1552-SXT, A1552-VchInd5 and A1552 carrying pVCR94, competed against A1552ΔlacZ carrying pVCR94. Data represent mean ± sd from three independent experiments (individual dots). Significant differences were determined by one-way ANOVA with either Dunnett’s (b and c) or Tukey’s (d) post-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns – not significant.