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. 2014 Oct 31;289(44):30343-30354.
doi: 10.1074/jbc.M114.598656. Epub 2014 Sep 8.

Zinc and ATP binding of the hexameric AAA-ATPase PilF from Thermus thermophilus: role in complex stability, piliation, adhesion, twitching motility, and natural transformation

Ralf Salzer  1 , Martin Herzberg  2 , Dietrich H Nies  2 , Friederike Joos  3 , Barbara Rathmann  4 , Yvonne Thielmann  4 , Beate Averhoff  5
Affiliations

Zinc and ATP binding of the hexameric AAA-ATPase PilF from Thermus thermophilus: role in complex stability, piliation, adhesion, twitching motility, and natural transformation

Ralf Salzer et al. J Biol Chem. .

Abstract

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.

Keywords: ATPases Associated with Diverse Cellular Activities (AAA); Cell Motility; DNA Transformation; Type IV Pili; Zinc.

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Figures

FIGURE 1.
FIGURE 1.
Organization of the PilF domains (A) and heat stability of PilF variants (B). The general secretory pathway domains are abbreviated with GSPII. The Walker A and Walker B motifs are indicated. The conserved tetracysteine motif is enlarged. The purified complexes of the PilF variants were incubated for 0, 2.5, or 5 min at 70 °C, and then the samples were cooled on ice. 35 μg of the treated samples was separated by clear-native PAGE, and the proteins were stained with Coomassie Blue (B).
FIGURE 2.
FIGURE 2.
Determination of the zinc content of PilF variants via ICP-MS. 0.1 and 0.25 mg of purified PilF complexes were used for the determination of the zinc content by ICP-MS. Results were calculated from three biological independent experiments.
FIGURE 3.
FIGURE 3.
Thermofluor assays of the purified PilF wild-type complex. Thermofluor assays were performed at pH 8.5 (50 mm Bicine buffer with 200 mm NaCl). 20 μl of purified PilF complex (0.1 μg/μl) were used in thermofluor assays. The melting curve of PilF wild-type complex was measured in Rotor Gene Q5 Plex HRM thermo cycler (A). SYPRO® Orange was used to measure fluorescence in the HRM channel with the excitation wavelength at 460 nm and the emission wavelength at 510 nm. The first derivative of the melting curve is shown in B. Two transition temperatures were observed at 80.8 ± 0.8 °C and 90.5 ± 0.0 °C. The effect of cysteine mutations on AMP-PNP- and ADP-mediated stabilization of PilF complexes is shown in C. Unfolding of the PilF complexes was measured by thermofluor assays. The bars indicate the first melting transitions of different PilF complexes in the absence or in the presence of 5 mm AMP-PNP and 2.5 mm ADP, respectively. Black, wild type; white, C1A; light gray, C2A; middle gray, HC2A; dark gray, C3A; vertical stripes, C4A; diagonal stripes, 2CysA; horizontal stripes, 3CysA; black and white boxes, 4CysA.
FIGURE 4.
FIGURE 4.
Dissociation temperature of PilF wild-type protein in presence of different concentrations of AMP-PNP (A) or ADP (B). Calculation of the dissociation constant by subtracting the melting transition without AMP-PNP or ADP from the melting transitions with different amounts of AMP-PNP (■) or ADP (▾) (C). The values are plotted against the nucleotide concentration. Dissociation constants are calculated by Graph Pad Prism 4.
FIGURE 5.
FIGURE 5.
Effect of the cysteine mutations in PilF on twitching motility of T. thermophilus at 64 °C. Cells were grown for 3 days on MM containing 1% BSA under humid conditions. After growth plates were stained with Coomassie, cells were removed, and pictures were inverted. The scale bar corresponds to 0.5 cm.
FIGURE 6.
FIGURE 6.
Effect of growth temperature on adhesion of pilF mutants. T. thermophilus cells were incubated in TM+ medium at 64 °C (A) or at 55 °C (B) in 96-well plates for 3 days. Adherence was calculated by the absorbance of crystal violet (570 nm) mediated by the adhered cells deviated by the amount of planktonic cells (600 nm). The mean was built from three independent cultures and in triplicate.
FIGURE 7.
FIGURE 7.
Effect of the growth temperature on twitching motility (A) and piliation (B) of T. thermophilus pilF mutants. Cells were grown at 55 °C for 5 days on MM containing 1% BSA. Plates were stained with Coomassie Blue, and the cells were removed after staining. Pictures were inverted. For piliation analyses the cells were transferred to copper grids and covered with 1.5-nm platinum/carbon in an angle of 25° and at minimum 250 cells per variant, and temperature was analyzed.
FIGURE 7.
FIGURE 7.
Effect of the growth temperature on twitching motility (A) and piliation (B) of T. thermophilus pilF mutants. Cells were grown at 55 °C for 5 days on MM containing 1% BSA. Plates were stained with Coomassie Blue, and the cells were removed after staining. Pictures were inverted. For piliation analyses the cells were transferred to copper grids and covered with 1.5-nm platinum/carbon in an angle of 25° and at minimum 250 cells per variant, and temperature was analyzed.
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