, Volume 22, Issue 3, pp 461–471 | Cite as

Functional dissection of the three N-terminal general secretory pathway domains and the Walker motifs of the traffic ATPase PilF from Thermus thermophilus

  • Kerstin Kruse
  • Ralf Salzer
  • Friederike Joos
  • Beate Averhoff
Original Paper


The traffic ATPase PilF of Thermus thermophilus powers pilus assembly as well as uptake of DNA. PilF differs from other traffic ATPases by a triplicated general secretory pathway II, protein E, N-terminal domain (GSPIIABC). We investigated the in vivo and in vitro roles of the GSPII domains, the Walker A motif and a catalytic glutamate by analyzing a set of PilF deletion derivatives and pilF mutants. Here, we report that PilF variants devoid of the first two or all three GSPII domains do not form stable hexamers indicating a role of the triplicated GSPII domain in complex formation and/or stability. A pilFΔGSPIIC mutant was significantly impaired in piliation which leads to the conclusion that the GSPIIC domain plays a vital role in pilus assembly. Interestingly, the pilFΔGSPIIC mutant was hypertransformable. This suggests that GSPIIC strongly affects transformation efficiency. A pilF∆GSPIIA mutant exhibited wild-type piliation but reduced pilus-mediated twitching motility, suggesting that GSPIIA plays a role in pilus dynamics. Furthermore, we report that pilF mutants with a defect in the ATP binding Walker A motif or in the catalytic glutamate residue are defective in piliation and natural transformation. These findings show that both, ATP binding and hydrolysis, are essential for the dual function of PilF in natural transformation and pilus assembly.


ATPase Natural competence Type IV pili Cell motility Membrane transport Thermophile 



General secretory pathway


Type IV pilus


Type II secretion system


Gel filtration




Polyacrylamide gel electrophoresis


Sodium dodecyl sulfate



This work was supported by a grant from the Deutsche Forschungsgemeinschaft (AV 9/6-2). We thank Bernd Ludwig (Goethe University, Frankfurt, Germany) for the kind gift of plasmid pDM12.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

792_2018_1008_MOESM1_ESM.pdf (798 kb)
Supplementary material 1 (PDF 798 kb)


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Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Kerstin Kruse
    • 1
  • Ralf Salzer
    • 1
  • Friederike Joos
    • 2
  • Beate Averhoff
    • 1
  1. 1.Molecular Microbiology & Bioenergetics, Institute of Molecular BiosciencesGoethe University FrankfurtFrankfurt am MainGermany
  2. 2.Department of Structural BiologyMax-Planck Institute of BiophysicsFrankfurt am MainGermany

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