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

Extremophiles : Life Under Extreme Conditions
Kerstin KruseBeate Averhoff

Abstract

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 t...Continue Reading

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Citations

May 3, 2018·The Biochemical Journal·Andreas Sukmana, Zhaomin Yang
Sep 21, 2018·Scientific Reports·Richard CollinsJeremy P Derrick
Dec 24, 2019·The Biochemical Journal·Keane J Dye, Zhaomin Yang
May 8, 2020·Nature Communications·Alexander NeuhausVicki A M Gold
Aug 17, 2021·PLoS Pathogens·Edgar E LlontopChuck S Farah

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Methods Mentioned

BETA
size-exclusion chromatography
PCR
electrophoresis
gel filtration
gel
electron microscopy

Software Mentioned

ImageJ
ComEC

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