Structure of a type IV pilus machinery in the open and closed state

Gold, Vicki A. M.; Salzer, Ralf; Averhoff, Beate; Kühlbrandt, Werner

doi:10.7554/elife.07380

Cited by 113 publications

(120 citation statements)

References 57 publications

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“…PilQ was found to undergo substantial conformational changes between the closed and the open, pilus-extruding state (18). SP-EM analyses of purified PilQ complexes revealed that the structure comprises six stacked rings (N0-N5) and a cone structure (20), consistent with the in situ data of the entire T4P machinery (18).…”

supporting

confidence: 63%

“…SP-EM analyses of purified PilQ complexes revealed that the structure comprises six stacked rings (N0-N5) and a cone structure (20), consistent with the in situ data of the entire T4P machinery (18). Structural analyses of PilQ complexes formed by different PilQ variants led to the identification of an unusual ␣␣␤␣␤␤␣ fold as the N0 ring-forming domain.…”

mentioning

confidence: 71%

“…Based on its position, the lower part of the cone is likely to be gate 1, as identified by cryo-ET and subtomogram averaging (Fig. 4, B and C) (18). In all ring variants, the secretin domain and gate 1 were clearly visible (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we reported the first in situ structure of an entire T4P machinery by electron cryotomography (cryo-ET) using the thermophilic bacterium Thermus thermophilus HB27 as a model organism (18). The central membrane-embedded part of the structure is formed by the secretin PilQ, which plays a dual role in T4P extrusion and natural transformation (9, 18 -20).…”

mentioning

confidence: 99%

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Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus

Salzer

D’Imprima

Gold

et al. 2016

Journal of Biological Chemistry

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Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique ␤␤␤␣␤ fold is essential for the formation of gate 2. Furthermore, we identified four ␤␣␤␤␣ domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wildtype cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.

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supporting

confidence: 63%

mentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus

Salzer

D’Imprima

Gold

et al. 2016

Journal of Biological Chemistry

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“…The role of the T4P retraction ATPase may be in remodeling T4P to DNA uptake complexes. Alternatively, the T4P may be essential for opening the outer membrane secretin pore formed by PilQ (19,48), to bind DNA at the extracellular side and enable threading into the pore.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

Hepp

Maier

2016

Proc. Natl. Acad. Sci. U.S.A.

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Horizontal gene transfer can speed up adaptive evolution and support chromosomal DNA repair. A particularly widespread mechanism of gene transfer is transformation. The initial step to transformation, namely the uptake of DNA from the environment, is supported by the type IV pilus system in most species. However, the molecular mechanism of DNA uptake remains elusive. Here, we used singlemolecule techniques for characterizing the force-dependent velocity of DNA uptake by Neisseria gonorrhoeae. We found that the DNA uptake velocity depends on the concentration of the periplasmic DNA-binding protein ComE, indicating that ComE is directly involved in the uptake process. The velocity-force relation of DNA uptake is in very good agreement with a translocation ratchet model where binding of chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of uptake. The model yields a speed of DNA uptake of 900 bp·s −1 and a reversal force of 17 pN. Moreover, by comparing the velocityforce relation of DNA uptake and type IV pilus retraction, we can exclude pilus retraction as a mechanism for DNA uptake. In conclusion, our data strongly support the model of a translocation ratchet with ComE acting as a ratcheting chaperone.

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The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermus thermophilus

2018

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A major driving force for the adaptation of bacteria to changing environments is the uptake of naked DNA from the environment by natural transformation, which allows the acquisition of new capabilities. Uptake of the high molecular weight DNA is mediated by a complex transport machinery that spans the entire cell periphery. This DNA translocator catalyzes the binding and splitting of double‐stranded DNA and translocation of single‐stranded DNA into the cytoplasm, where it is recombined with the chromosome. The thermophilic bacterium Thermus thermophilus exhibits the highest transformation frequencies reported and is a model system to analyze the structure and function of this macromolecular transport machinery. Transport activity is powered by the traffic ATPase PilF, a soluble protein that forms hexameric complexes. Here, we demonstrate that PilF physically binds to an inner membrane assembly platform of the DNA translocator, comprising PilMNO, via the ATP‐binding protein PilM. Binding to PilMNO or PilMN stimulates the ATPase activity of PilF ~ 2‐fold, whereas there is no stimulation when binding to PilM or PilN alone. A PilM K26A variant defective in ATP binding still binds PilF and, together with PilN, stimulates PilF‐mediated ATPase activity. PilF is unique in having three conserved GSPII (general secretory pathway II) domains (A–C) at its N terminus. Deletion analyses revealed that none of the GSPII domains is essential for binding PilMN, but GSPIIC is essential for PilMN‐mediated stimulation of ATP hydrolysis by PilF. Our data suggest that PilM is a coupling protein that physically and functionally connects the soluble motor ATPase PilF to the DNA translocator via the PilMNO assembly platform.

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Structure of a type IV pilus machinery in the open and closed state

Cited by 113 publications

References 57 publications

Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus

Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermus thermophilus

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