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Spectrophotometric Assays to Quantify the Activity of T4SS ATPases

  • Elena CabezónEmail author
  • Ignacio Arechaga
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2075)

Abstract

Biogenesis of T4SS apparatus and substrate transport require energy. Conjugative T4SS have three ATPases that enable DNA processing and transport of the nucleoprotein complex to the recipient cell. In the conjugative plasmid R388, these ATPases are named TrwB, TrwK, and TrwD. Here, three different spectrophotometric assays to measure the enzymatic properties of these ATPases are described. The choice of the assay will depend on the specific requirements of each enzyme.

Key words

ATPase activity Molecular motor Coupled assay Spectrophotometric assay 

Notes

Acknowledgments

This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) grants BFU2016-78521-R (to E.C. and I.A.) and BFU2014-61823-EXP (to E.C.).

References

  1. 1.
    Waters VL (1999) Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance. Front Biosci 4:D433–D456CrossRefPubMedGoogle Scholar
  2. 2.
    de la Cruz F, Davies J (2000) Horizontal gene transfer and the origin of species: lessons from bacteria. Trends Microbiol 8:128–133CrossRefPubMedGoogle Scholar
  3. 3.
    Cabezón E, Ripoll-Rozada J, Peña A, de la Cruz F, Arechaga I (2015) Towards an integrated model of bacterial conjugation. FEMS Microbiol Rev 39:81–95PubMedGoogle Scholar
  4. 4.
    Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, Lu F, Redzej A, Fronzes R, Orlova EV, Waksman G (2014) Structure of type IV secretion systems. Nature 508:550–553CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Atmakuri K, Cascales E, Christie PJ (2004) Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol Microbiol 54:1199–1211CrossRefPubMedGoogle Scholar
  6. 6.
    Kornberg A, Pricer WE Jr (1951) Enzymatic phosphorylation of adenosine and 2,6-diaminopurine riboside. J Biol Chem 193:481–495PubMedGoogle Scholar
  7. 7.
    Tato I, Zunzunegui S, de la Cruz F, Cabezón E (2005) TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase. Proc Natl Acad Sci U S A 102:8156–8161CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Matilla I, Alfonso C, Rivas G, Bolt E, de la Cruz F, Cabezón E (2010) The conjugative DNA translocase TrwB is a structure- specific DNA binding protein. J Biol Chem 285:17537–17544CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Arechaga I, Zunzunegui S, Peña A, Fernández-Alonso MC, Rivas G, de la Cruz F (2008) ATPase activity and oligomeric state of TrwK, the VirB4 homologue of plasmid R388 type IV secretion system. J Bacteriol 190:5472–5547CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Peña A, Ripoll-Rozada J, Zunzunegui S, Cabezón E, de la Cruz F, Arechaga I (2011) Auto-inhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems. J Biol Chem 286:17376–17382CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ripoll-Rozada J, Peña A, Rivas S, Moro F, de la Cruz F, Cabezón E, Arechaga I (2012) Regulation of the type IV secretion ATPase by magnesium. J Biol Chem 287:17408–17414CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89:4884–4887CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Taussky HH, Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202:675–685PubMedGoogle Scholar
  14. 14.
    Baykov AA, Evtushenko OA, Avaeva SM (1988) A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem 171:266–270CrossRefPubMedGoogle Scholar
  15. 15.
    Geladopoulos TP, Sotiroudis TG, Evangelopoulos AE (1991) A malachite green colorimetric assay for protein phosphatase activity. Anal Biochem 192:112–116CrossRefPubMedGoogle Scholar
  16. 16.
    Cogan EB, Birrell GB, Griffith OH (1999) A robotic-based automated assay for inorganic and organic phosphates. Anal Biochem 271:27–35CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Departamento de Biología Molecular, Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC)Universidad de Cantabria-CSICSantanderSpain

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