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Protein–Protein Interaction: Bacterial Two-Hybrid

  • Gouzel Karimova
  • Emilie Gauliard
  • Marilyne Davi
  • Scot P. Ouellette
  • Daniel Ladant
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

The bacterial two-hybrid (BACTH, for “Bacterial Adenylate Cyclase-Based Two-Hybrid”) system is a simple and fast genetic approach to detecting and characterizing protein–protein interactions in vivo. This system is based on the interaction-mediated reconstitution of a cyclic adenosine monophosphate (cAMP) signaling cascade in Escherichia coli. As BACTH uses a diffusible cAMP messenger molecule, the physical association between the two interacting chimeric proteins can be spatially separated from the transcription activation readout, and therefore it is possible to analyze protein–protein interactions that occur either in the cytosol or at the inner membrane level as well as those that involve DNA-binding proteins. Moreover, proteins of bacterial origin can be studied in an environment similar (or identical) to their native one. The BACTH system may thus permit a simultaneous functional analysis of proteins of interest—provided the hybrid proteins retain their activity and their association state. This chapter describes the principle of the BACTH genetic system and the general procedures to study protein–protein interactions in vivo in E. coli.

Key words

Two-hybrid system Protein interaction assay Membrane protein cAMP signaling Chimeric proteins 

Notes

Acknowledgments

This work was supported by Institut Pasteur and the Centre National de la Recherche Scientifique (CNRS UMR 3528, Biologie Structurale et Agents Infectieux). E.G. was supported by Ph.D. funding from the Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.

References

  1. 1.
    Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246CrossRefGoogle Scholar
  2. 2.
    Stynen B, Tournu H, Tavernier J, Van Dijck P (2012) Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. Microbiol Mol Biol Rev 76:331–382CrossRefGoogle Scholar
  3. 3.
    Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95:5752–5756CrossRefGoogle Scholar
  4. 4.
    Karimova G, Dautin N, Ladant D (2005) Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J Bacteriol 187:2233–2243CrossRefGoogle Scholar
  5. 5.
    Jack RL, Buchanan G, Dubini A, Hatzixanthis K, Palmer T, Sargent F (2004) Coordinating assembly and export of complex bacterial proteins. EMBO J 23:3962–3972CrossRefGoogle Scholar
  6. 6.
    Paschos A, den Hartigh A, Smith MA, Atluri VL, Sivanesan D, Tsolis RM, Baron C (2011) An in vivo high-throughput screening approach targeting the type IV secretion system component VirB8 identified inhibitors of Brucella abortus 2308 proliferation. Infect Immun 79:1033–1043CrossRefGoogle Scholar
  7. 7.
    Cisneros DA, Bond PJ, Pugsley AP, Campos M, Francetic O (2012) Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation. EMBO J 31:1041–1053CrossRefGoogle Scholar
  8. 8.
    Georgiadou M, Castagnini M, Karimova G, Ladant D, Pelicic V (2012) Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly. Mol Microbiol 84:857–873CrossRefGoogle Scholar
  9. 9.
    Zoued A, Durand E, Brunet YR, Spinelli S, Douzi B, Guzzo M, Flaugnatti N, Legrand P, Journet L, Fronzes R et al (2016) Priming and polymerization of a bacterial contractile tail structure. Nature 531:59–63CrossRefGoogle Scholar
  10. 10.
    Karimova G, Ullmann A, Ladant D (2000) A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli. Methods Enzymol 328:59–73CrossRefGoogle Scholar
  11. 11.
    Ladant D, Ullmann A (1999) Bordatella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol 7:172–176CrossRefGoogle Scholar
  12. 12.
    Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH (2004) Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol 14:10–20CrossRefGoogle Scholar
  13. 13.
    Karimova G, Ullmann A, Ladant D (2001) Protein-protein interaction between Bacillus stearothermophilus tyrosyl-tRNA synthetase subdomains revealed by a bacterial two-hybrid system. J Mol Microbiol Biotechnol 3(1):73–82PubMedGoogle Scholar
  14. 14.
    Fransen M, Brees C, Ghys K, Amery L, Mannaerts GP, Ladant D, Van Veldhoven PP (2002) Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay. Mol Cell Proteomics 1:243–252CrossRefGoogle Scholar
  15. 15.
    Dautin N, Karimova G, Ladant D (2003) Human immunodeficiency virus (HIV) type 1 transframe protein can restore activity to a dimerization-deficient HIV protease variant. J Virol 77:8216–8226CrossRefGoogle Scholar
  16. 16.
    Battesti A, Bouveret E (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. Methods 58:325–334CrossRefGoogle Scholar
  17. 17.
    Ouellette SP, Gauliard E, Antosova Z, Ladant D (2014) A Gateway((R)) -compatible bacterial adenylate cyclase-based two-hybrid system. Environ Microbiol Rep 6:259–267CrossRefGoogle Scholar
  18. 18.
    Sambrook J, Russell DW (2006) The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  19. 19.
    Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795CrossRefGoogle Scholar
  20. 20.
    Karimova G, Robichon C, Ladant D (2009) Characterization of YmgF, a 72-residue inner membrane protein that associates with the Escherichia coli cell division machinery. J Bacteriol 191:333–346CrossRefGoogle Scholar
  21. 21.
    Karimova G, Davi M, Ladant D (2012) The beta-lactam resistance protein Blr, a small membrane polypeptide, is a component of the Escherichia coli cell division machinery. J Bacteriol 194:5576–5588CrossRefGoogle Scholar
  22. 22.
    Griffith KL, Wolf REJ (2002) Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. Biochem Biophys Res Commun 290:397–402CrossRefGoogle Scholar
  23. 23.
    Ouellette SP, Rueden KJ, Gauliard E, Persons L, de Boer PA, Ladant D (2014) Analysis of MreB interactors in Chlamydia reveals a RodZ homolog but fails to detect an interaction with MraY. Front Microbiol 5:279CrossRefGoogle Scholar
  24. 24.
    Robichon C, Karimova G, Beckwith J, Ladant D (2011) Role of leucine zipper motifs in association of the Escherichia coli cell division proteins FtsL and FtsB. J Bacteriol 193:4988–4992CrossRefGoogle Scholar
  25. 25.
    Battesti A, Bouveret E (2008) Improvement of bacterial two-hybrid vectors for detection of fusion proteins and transfer to pBAD-tandem affinity purification, calmodulin binding peptide, or 6-histidine tag vectors. Proteomics 8:4768–4771CrossRefGoogle Scholar
  26. 26.
    Ouellette SP, Karimova G, Subtil A, Ladant D (2012) Chlamydia co-opts the rod shape-determining proteins MreB and Pbp2 for cell division. Mol Microbiol 85:164–178CrossRefGoogle Scholar
  27. 27.
    Dautin N, Karimova G, Ullmann A, Ladant D (2000) Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli. J Bacteriol 182:7060–7066CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Gouzel Karimova
    • 1
  • Emilie Gauliard
    • 1
    • 2
  • Marilyne Davi
    • 1
  • Scot P. Ouellette
    • 3
  • Daniel Ladant
    • 1
  1. 1.Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et ChimieInstitut Pasteur, CNRS, UMR 3528ParisFrance
  2. 2.Université Paris Diderot, Sorbonne Paris Cité, Cellule PasteurParisFrance
  3. 3.Division of Basic Biomedical Sciences, Sanford School of MedicineUniversity of South DakotaVermillionUSA

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