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Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae

Nature volume 604, pages 323–329 (2022)Cite this article

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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 pandemic2,3,4. 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.

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Fig. 1: Plasmids are unstable in V. cholerae O1 El Tor.
Fig. 2: V. cholerae O1 El Tor contains two plasmid defence systems on pathogenicity islands.
Fig. 3: Mode of action of the DdmDE and DdmABC systems.
Fig. 4: Effect of the DdmDE and DdmABC systems on V. cholerae 7PET ecology.

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Data availability

The data that support the findings of this study are provided within the manuscript and its associated Supplementary Information. Full details and links to the publicly available databases used in bioinformatic analyses are provided in the Methods and the associated references. The DNA sequences of newly identified plasmids from environmental V. cholerae isolates have been deposited in NCBI’s GenBank database with the following accession numbers: pSa5Y (CP089143), pSO5Y (CP089144), pSA7G3 (CP089145), pSL4G (CP089146) and pE7G2 (CP089147). Source data are provided with this paper.

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Acknowledgements

We thank V. Tripathi and A.‐C. Portmann for preliminary work on the characterization of pSa5Y and S. Stutzmann for technical support and, together with C. Stoudmann, help with streaking colonies. We further thank B. Correia for advice on structural predictions, S. Gruber for discussions on SMC-like proteins, F. Le Roux for discussions on MRB-based plasmids, E. Rocha for comments on homologue distribution and various members of the Blokesch laboratory for discussions. We also acknowledge the gift of strains from A. Boehm (Stanford University), J. J. Mekalanos (Harvard Medical School) and A. B. Dalia (Indiana University); plasmids from V. Burrus (Université de Sherbrooke), D. K. Chattoraj (National Cancer Institute), F. Le Roux (Ifremer) and E. V. Stabb (University of Illinois Chicago); and phages from K. D. Seed (University of California, Berkeley). This work was supported by an ERC Consolidator grant (724630-CholeraIndex) from the European Research Council and a project grant from the Swiss National Research Foundation (310030_185022) to M.B. and by EPFL intramural funding. M.B. is a Howard Hughes Medical Institute International Research Scholar (grant 55008726).

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Author notes

  1. These authors contributed equally: Milena Jaskólska, David W. Adams

Authors and Affiliations

  1. Laboratory of Molecular Microbiology, Global Health Institute, School of Life Sciences, Station 19, EPFL-SV-UPBLO, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Milena Jaskólska, David W. Adams & Melanie Blokesch

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  1. Milena Jaskólska
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Contributions

M.J., D.W.A. and M.B. conceived the project, designed the experiments, constructed strains and plasmids and analysed the results. M.J. and D.W.A. performed all experiments in collaboration, except for phage assays, which were done by M.J., and microscopy, which was done by D.W.A. D.W.A. performed bioinformatic analyses. D.W.A. and M.J. wrote the manuscript with input from M.B. M.B. acquired funding and supervised the project.

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Correspondence to David W. Adams or Melanie Blokesch.

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Competing interests

M.B., M.J. and D.W.A. are inventors on patent application EP21183501.2 filed with the European Patent Office.

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Extended data figures and tables

Extended Data Fig. 1 Characterization 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 hybridization62. 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 ~80 kb that are not resolved on the gels79. c, Copy number of pSa5Y in exponentially growing and stationary cells, as determined by qPCR. 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 Fig. 1.

Source data

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 quantitative reverse transcription PCR (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).

Source data

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-ParBMT1 by incorporating the MT1 parS site into the plasmid (parS+). b, pSa5Y-parSMT1 retention and localisation in single cells of the indicated strains following growth for approx. 50 generations without antibiotic selection. c, Comparison of pSa5Y-parSMT1 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-parSMT1 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-ParBMT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm.

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

Time-course showing the effect of DdmDE (a) and DdmABC (b) production on pSa5Y-parSMT1 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 h 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 1 h 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-ParBMT1 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-parSMT1 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 Fig. 1. c, Effect of individual production of DdmA, DdmB or DdmC on pSa5Y-parSMT1 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-ParBMT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm.

Source data

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)xK 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)xK superfamily nuclease motif (1085D…1100DSK1102). Active site residues of the PD-(D/E)xK motif are shown in bold. Residues targeted by site-directed mutagenesis are underlined. The position of the DdmD residue E891, which in 6th 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-parSMT1 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-parSMT1 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-ParBMT1 fusion. All images are representative of the results of three independent experiments. Scale bars = 2.5 μm. For Western blotting source data, see Supplementary Fig. 1.

Source data

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-VchInd5 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.

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Supplementary information

Supplementary Information

This file contains Supplementary Tables 1 and 4–8, the legends for Supplementary Tables 2 and 3, the legends for the supplementary videos and supplementary references.

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Supplementary Fig. 1

This file contains uncropped agarose gel images, scans of uncropped western blot membranes and photographs of phage plaque enumeration for the main and extended data figures.

Supplementary Table 2

Putative homologues of DdmDE and DdmABC detected with PHMMER. The table lists the significant matches to each protein sequence of interest as well as the alignment between the query and target sequence.

Supplementary Table 3

Results of remote homology searches using HHpred. The table lists all matching database sequences to each protein sequence of interest, followed by a list of query–template alignments.

Supplementary Video 1

Imaging of pSa5Y using a ParB/parS fluorescent reporter system. Exponentially growing cells of stain A1552-yGFP–ParBMT1 carrying pSa5Y-parSMT1 were imaged automatically by time-lapse microscopy at 1-second intervals for 10 seconds. The video is a composite of three separate examples and is displayed at two frames per second. Scale bar, 2.5 μm; time (s), as indicated.

Supplementary Video 2

Overexpression of ddmABC clusters pSa5Y into static foci. Cells of strain A1552-yGFP–ParBMT1 ΔddmDE ΔddmABC TnddmABC carrying pSa5Y-parSMT1 were cultured in the absence (No Ara) or presence (+Ara) of 0.02% arabinose, as indicated, and were imaged automatically by time-lapse microscopy at 0.6-second intervals for 20 seconds. The videos are displayed at five frames per second. Scale bar, 2.5 μm; time (s), as indicated.

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Jaskólska, M., Adams, D.W. & Blokesch, M. Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae. Nature 604, 323–329 (2022). https://doi.org/10.1038/s41586-022-04546-y

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