Abstract
Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.
Highlights
Of the various genes involved in the biogenesis of type IV pili and type II secretion systems, putative NTPases, including PulE of K. oxytoca [8], PilB of P. aeruginosa [9], OutE of E. chrysanthemi [6], and EpsE of V. cholerae [10], collectively known as the PulE-VirB11 family, are highly conserved
PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis
R64 and ColIb-P9 thin pili belong to the type IV pilus family based on the amino acid sequence similarities between various pil products and proteins related to the formation of other type IV pili
Summary
Bacterial Strains and Plasmids—The bacterial strains used in this work were E. coli JM83 ⌬(lac-proAB) rpsL thi ara 80 dlacZ⌬M15 [29], NF83 recA56 ara ⌬(lac-proAB) rpsL 80 dlacZ⌬M15 [30], BL21 (DE3) dcm ompT hsdS gal (DE3) [31], and TN102 Nalr [23]. Reactions were carried out at 30 °C for 30 min in 40 l of buffer A (50 mM MES (pH 6.5), 5 mM MgCl2, 25 mM KCl, and 1 mM DTT) containing 1 mM [␥-32P]ATP (0.9 mCi/mmol) and 2 M His tag PilQ protein. Reactions were carried out at 37 °C in a mixture (1 ml) containing 50 mM MES buffer (pH 6.5), 5 mM MgCl2, 25 mM KCl, 1 mM DTT, 3 mM phosphoenolpyruvate, 0.25 mM NADH, 5 units of pyruvate kinase, 10 units of lactate dehydrogenase, 6 g of His tag PilQ protein, and various concentrations of ATP, and decrease in absorbance at 340 nm was measured using a UV160 spectrophotometer (Shimazu). The purified wild-type and mutant His tag PilQ proteins (500 g) were applied to a Superose 6 gel filtration column (Amersham Pharmacia Biotech) equilibrated with buffer B (20 mM Tris-HCl (pH 7.0), 100 mM NaCl, and 5 mM MgCl2) with or without 1 mM ATP. The samples were applied to SDSPAGE (15% gel), and tryptic fragments of PilQ protein were detected by Western blot analysis using anti-PilQ antibody
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