Measuring Plasmid Stability in Gram-Negative Bacteria

  • Damián Lobato-MárquezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2075)


In this chapter, a highly sensitive method to measure plasmid stability in Gram-negative bacteria is described. This procedure is based on the counterselection of plasmid-containing cells using an aph-parE cassette. When bacteria carrying the aph-parE module in the plasmid of interest are grown in media containing rhamnose as the only carbon source, the PparE promoter is induced, ParE is synthesized, and plasmid-containing cells are eliminated; bacteria that have lost the plasmid survive. The absence of the kanamycin resistance marker (aph) can be used to confirm the loss of the plasmid in rhamnose grown bacteria.

Key words

Plasmid stability aph-parE Rhamnose Gram-negative Toxin–antitoxin Salmonella 



I thank Alexandra Willis, Gloria del Solar Dongil, and Ramon Díaz Orejas for their critical review of the manuscript.

The protocol described in this chapter was originally described in Lobato-Márquez et al. [8].

D.L.-M. is funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. H2020-MSCA-IF-2016-752022.


  1. 1.
    Gerdes K, Larsen JE, Molin S (1985) Stable inheritance of plasmid R1 requires two different loci. J Bacteriol 161:292–298PubMedPubMedCentralGoogle Scholar
  2. 2.
    del Solar GH, Puyet A, Espinosa M (1987) Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Nucleic Acids Res 15:5561–5580CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bochner BR, Huang HC, Schieven GL, Ames BN (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–933PubMedPubMedCentralGoogle Scholar
  4. 4.
    Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1111PubMedPubMedCentralGoogle Scholar
  5. 5.
    García-Quintanilla M, Prieto AI, Barnes L, Ramos-Morales F, Casadesus J (2006) Bile-induced curing of the virulence plasmid in Salmonella enterica serovar Typhimurium. J Bacteriol 188:7963–7965CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Reyrat JM, Pelicic V, Gicquel B, Rappuoli R (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 66:4011–4017PubMedPubMedCentralGoogle Scholar
  7. 7.
    Li XT, Thomason LC, Sawitzke JA, Costantino N, Court DL (2013) Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res 41:e204CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lobato-Márquez D, Molina-García L, Moreno-Córdoba I, García-del Portillo F, Díaz-Orejas R (2016) Stabilization of the virulence plasmid pSLT of Salmonella Typhimurium by three maintenance systems and its evaluation by using a new stability test. Front Mol Biosci 3:66Google Scholar
  9. 9.
    Lobato-Márquez D, Molina-García L (2017) Evaluation of plasmid stability by negative selection in Gram-negative bacteria. BioProtocol 7(9)Google Scholar
  10. 10.
    Maisonneuve E, Shakespeare LJ, Jorgensen MG, Gerdes K (2011) Bacterial persistence by RNA endonucleases. Proc Natl Acad Sci U S A 108:13206–13211CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lobato-Márquez D, Moreno-Córdoba I, Figueroa V, Díaz-Orejas R, García-del Portillo F (2015) Distinct type I and type II toxin-antitoxin modules control Salmonella lifestyle inside eukaryotic cells. Sci Rep 5:9374CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jiang Y, Pogliano J, Helinski DR, Konieczny I (2002) ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase. Mol Microbiol 44:971–979CrossRefPubMedGoogle Scholar
  13. 13.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Infection Biology, Faculty of Infectious and Tropical DiseasesLondon School of Hygiene and Tropical MedicineLondonUK

Personalised recommendations