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Compensatory mutations improve general permissiveness to antibiotic resistance plasmids

Nature Ecology & Evolution volume 1, pages 1354–1363 (2017)Cite this article

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Abstract

Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multidrug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental–modelling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase β-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase–plasmid interactions that can be rapidly ameliorated.

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Fig. 1: Plasmid persistence profiles measured at 100 gen. intervals during the 600 gen. evolution experiment for replicate populations A to C.
Fig. 2: The mutations responsible for increased plasmid persistence are located in the host chromosome.
Fig. 3: Evolution of plasmid cost into a benefit rather than a change in segregational loss frequency facilitated improved plasmid persistence.
Fig. 4: Pseudomonas sp. nov. H2 evolved to be more permissive towards both related and unrelated plasmids.
Fig. 5: SNPs identified in the chromosome of each sequenced clone, as compared with the Rif-sensitive reference strain Pseudomonas sp. nov. H2.
Fig. 6: At least two mutations were required for full plasmid persistence, one plasmid-adaptive and one environment-adaptive mutation.

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References

  1. Gogarten, J. P. & Townsend, J. P. Horizontal gene transfer, genome innovation and evolution. Nat. Rev. Microbiol. 3, 679–687 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Schwarz, S. & Johnson, A. P. Transferable resistance to colistin: a new but old threat. J. Antimcrob. Chemother. 71, 2066–2070 (2016).

    Article  Google Scholar 

  3. Baker, K. S. et al. The Murray collection of Enterobacteriacae: a unique resource. Genome Med. 7, 97–114 (2015).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Thomas, C. M. The Horizontal Gene Pool — Bacterial Plasmids and Gene Spread (Harwood Academic, Amsterdam, 2000).

    Book  Google Scholar 

  5. Datta, N. & Hughes, V. M. Plasmids of the same Inc groups in enterobacteria before and after the medical use of antibiotics. Nature 306, 616–617 (1983).

    Article  CAS  PubMed  Google Scholar 

  6. Popowska, M. & Krawczyk-Balska, A. Broad-host-range IncP-1 plasmids and their resistance potential. Front. Microbiol. 4, 1–8 (2013).

    Article  Google Scholar 

  7. De Gelder, L., Ponciano, J. M., Joyce, P. & Top, E. M. Stability of a promiscuous plasmid in different hosts: no guarantee for a long-term relationship. Microbiology 153, 452–463 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Ebersbach, G. & Gerdes, K. Plasmid segregation mechanisms. Annu. Rev. Genet. 39, 453–479 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Stewart, F. M. & Levin, B. R. The population biology of bacterial plasmids: a priori conditions for the existence of conjugationally transmitted factors. Genetics 87, 209–228 (1977).

    PubMed Central  CAS  PubMed  Google Scholar 

  10. Harrison, E., Guymer, D., Spiers, A. J., Paterson, S. & Brockhurst, M. A. Parallel compensatory evolution stabilizes plasmids across the parasitism–mutualism continuum. Curr. Biol. 25, 2034–2039 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. San Millan, A., Toll-Riera, M., Qi, Q. & MacLean, R. C. Interactions between horizontally acquired genes create a fitness cost in Pseudomonas aeruginosa. Nat. Commun. 6, 6845 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Shintani, M. et al. Response of the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7 plasmid pCAR1. Environ. Microbiol. 12, 1413–1426 (2010).

    CAS  PubMed  Google Scholar 

  13. De Gelder, L., Williams, J. J., Ponciano, J. M., Sota, M. & Top, E. M. Adaptive plasmid evolution results in host-range expansion of a broad-host-range plasmid. Genetics 178, 2179–2190 (2008).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Sota, M. et al. Shifts in the host range of a promiscuous plasmid through parallel evolution of its replication initiation protein. ISME J. 4, 1568–1580 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Bouma, J. E. & Lenski, R. E. Evolution of a bacteria/plasmid association. Nature 335, 351–352 (1988).

    Article  CAS  PubMed  Google Scholar 

  16. San Millan, A. et al. Positive selection and compensatory adaptation interact to stabilize non-transmissible plasmids. Nat. Commun. 5, 5208 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Dahlberg, C. & Chao, L. Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12. Genetics 165, 1641–1649 (2003).

    PubMed Central  CAS  PubMed  Google Scholar 

  18. Loftie-Eaton, W. et al. Evolutionary paths that expand plasmid host-range: implications for spread of antibiotic resistance. Mol. Biol. Evol. 33, 885–897 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Maestro, B., Sanz, J. M., Díaz-Orejas, R. & Fernández-Tresguerres, E. Modulation of pPS10 host range by plasmid-encoded RepA initiator protein. J. Bacteriol. 185, 1367–1375 (2003).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Yano, H. et al. Evolved plasmid–host interactions reduce plasmid interference cost. Mol. Microbiol. 101, 743–756 (2016).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Heuer, H., Fox, R. E. & Top, E. M. Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host. FEMS Microbiol. Ecol. 59, 738–748 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Pansegrau, W. et al. Complete nucleotide sequence of Birmingham IncPα plasmids: compilation and analysis. J. Mol. Biol. 239, 626–663 (1994).

    Article  Google Scholar 

  23. Saunders, J. R. & Grinsted, J. Properties of RP4, an R factor which originated in Pseudomonas aeruginosa S8. J. Bacteriol. 112, 690–696 (1972).

    PubMed Central  CAS  PubMed  Google Scholar 

  24. Baquero, F., Coque, T. M. & de la Cruz, F. Ecology and evolution as targets: the need for novel eco-evo drugs and strategies to fight antibiotic resistance. Antimicrob. Agents Chemother. 55, 3649–3660 (2001).

    Article  CAS  Google Scholar 

  25. Ponciano, J. M., De Gelder, L., Top, E. M. & Joyce, P. The population biology of bacterial plasmids: a hidden Markov model approach. Genetics 176, 957–968 (2007).

    Article  PubMed Central  PubMed  Google Scholar 

  26. Lenski, R. E., Simpson, S. C. & Nguyen, T. T. Genetic analysis of a plasmid-encoded, host genotype-specific enhancement of bacterial fitness. J. Bacteriol. 176, 3140–3147 (1994).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Heuer, H., Ebers, J., Weinert, N. & Smalla, K. Variation in permissiveness for broad-host-range plasmids among genetically indistinguishable isolates of Dickeya sp. from a small field plot. FEMS Microbiol. Ecol. 73, 190–196 (2010).

    CAS  PubMed  Google Scholar 

  28. Klümper, U. et al. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community. ISME J. 9, 934–945 (2014).

    Article  PubMed Central  CAS  Google Scholar 

  29. Kelley, A. L., Mezulis, S., Tayes, M. C., Wass, N. M. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Yachdav, G. et al. PredictProtein - an open resource for online prediction of protein structural and functional features. Nucleic Acids Res. 42, 337–343 (2014).

    Article  CAS  Google Scholar 

  31. Byrd, A. K. & Raney, K. D. Superfamily 2 helicases. Front. Biosci. 17, 2070–2088 (2013).

    Article  CAS  Google Scholar 

  32. Merrikh, H., Zhang., Y., Grossman, A. D. & Wang, J. D. Replication–transcription conflicts in bacteria. Nat. Rev. Microbiol. 10, 449–458 (2013).

    Article  CAS  Google Scholar 

  33. Merrikh, C. N., Brewer, B. J. & Merrikh, H. The B. subtilis accessory helicase PcrA facilitates DNA replication through transcription units. PLoS Genet. 11, e1005289 (2015).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Fairman-Williams, M. E., Guenther, U. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Baharoglu, Z., Bikard, D. & Mazel, D. Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation. PLoS Genet. 6, e1001165 (2010).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Ingmer, H., Miller, C. & Cohen, S. N. The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response. Mol. Microbiol. 42, 519–526 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Boubakri, H., de Septenville, A. L., Viguera, E. & Michel, B. The helicases DinG, Rep and UvrD cooperate to promote replication across transcriptional units in vivo. EMBO J. 29, 145–157 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Epshtein, V. et al. UvrD facilitates DNA repair by pulling RNA polymerase backwards. Nature 505, 372–377 (2014).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Guy, C. P. et al. Rep provides a second motor at the replisome to promote duplication of protein-bound DNA. Mol. Cell 36, 654–666 (2009).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Borukhou, S. & Nudler, E. RNA polymerase holoenzyme: structure, function and biological implications. Curr. Opin. Microbiol. 6, 93–100 (2003).

    Article  CAS  Google Scholar 

  41. Kuznedelov, K. et al. A role for interaction of the RNA polymerase flap domain with σ subunit in promoter recognition. Science 295, 855–857 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Brandis, G., Wrande, M., Liljas, L. & Hughes, D. Fitness-compensatory mutations in rifampicin-resistant RNA polymerase. Mol. Microbiol. 85, 142–151 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Reynolds, M. G. Compensatory evolution in rifampin-resistant Escherichia coli. Genetics 156, 1471–1481 (2000).

    PubMed Central  CAS  PubMed  Google Scholar 

  44. Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Laboratory Press, New York, 2001).

  45. Joyce, P. et al. Modeling the impact of periodic bottlenecks, unidirectional mutation, and observational error in experimental evolution. J. Math. Biol. 50, 645–662 (2005).

    Article  PubMed  Google Scholar 

  46. Simonsen, L., Gordon, D. M., Stewart, F. M. & Levin, B. R. Estimating the rate of plasmid transfer: an end-point method. J. Gen. Microbiol. 136, 2319–2325 (1990).

    Article  CAS  PubMed  Google Scholar 

  47. Deatherage, D. E. & Barrick, J. E. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol. Biol. 1151, 165–188 (2014).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Krzywinski, M. I. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Institute of Allergy and Infectious Diseases grant R01 AI084918 of the National Institutes of Health (NIH). The genome resequencing was done by the IBEST Genomics Research Core and made possible thanks to an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the NIH under grant number P30 GM103324. K.B. was in part supported by a University of Idaho Department of Biological Sciences undergraduate research grant and by an NIGMS INBRE award, grant number P20 GM103408. H.Q. was supported by a National Science Foundation REU Site award, 1460696. We thank the laboratory of C. Marx for providing us with vector pPS04 and we thank H. Merrikh for useful suggestions.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Idaho, PO Box 443051, Moscow, ID, 83844, USA

    Wesley Loftie-Eaton, Kelsie Bashford, Kieran Dong, Jack Millstein & Eva M. Top

  2. Institute for Bioinformatics and Evolutionary Studies (IBEST), University of Idaho, PO Box 443051, Moscow, ID, 83844, USA

    Wesley Loftie-Eaton, Jack Millstein, Samuel Hunter & Eva M. Top

  3. Point Loma Nazarene University, San Diego, CA, 92106, USA

    Hannah Quinn

  4. Department of Microbiology, Health Sciences Building, University of Washington, Seattle, WA, 98195, USA

    Maureen K. Thomason & Houra Merrikh

  5. Department of Biology, University of Florida, PO Box 118525, Gainesville, FL, 32611, USA

    Jose M. Ponciano

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Contributions

W.L. and E.M.T conceived the project and wrote the manuscript. W.L., K.B., H.Q., K.D., M.K.T. and J.M. performed the experiments. W.L. and J.M.P. performed the statistical analyses. W.L. and S.H. performed the bioinformatic analysis. H.M. facilitated part of the work and helped with data interpretation.

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Correspondence to Eva M. Top.

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Loftie-Eaton, W., Bashford, K., Quinn, H. et al. Compensatory mutations improve general permissiveness to antibiotic resistance plasmids. Nat Ecol Evol 1, 1354–1363 (2017). https://doi.org/10.1038/s41559-017-0243-2

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