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INTRODUCTION |
Type IV pili have been identified in various Gram-negative
pathogens such as Neisseria gonorrhoeae, Vibrio
cholerae, Pseudomonas aeruginosa, and enteropathogenic
Escherichia coli (1). The contribution of type IV pili to
virulence lies primarily in their ability to promote the attachment of
the pathogens to various receptors of host cells during colonization.
Type IV pili are also required for bacterial locomotion known as
twitching motility (2) as well as for the social gliding motility of
myxobacteria (3). Proteins with extensive sequence similarity to type
IV pilins were also found to be the essential components of protein secretion systems of Gram-negative bacteria and the DNA uptake systems
of Gram-positive bacteria (4). Gram-negative bacteria such as
Klebsiella oxytoca, Erwinia chrysanthemi,
P. aeruginosa, and Xanthomonas campestris have
protein secretion systems, called type II secretion systems or the main
terminal branch of the general secretory pathway, which export
extracellular proteins including pullulanase, pectinase, cellulase, and
toxins outside of the outer membrane (5-7). The genes involved in
piliation and protein export are related through amino acid sequence
similarity among the structural subunits and conserved assembly
proteins (4).
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. They are also found in type IV secretion systems including
the conjugation systems of IncP plasmids RP4 and R751, and IncW plasmid
R388 (11), the T-DNA transfer systems of agrobacterial Ti and Ri
plasmids (12), and the Ptl protein secretion system of Bordetella
pertussis (13). The encoded proteins contain several conserved
motifs: Walker box A and B involved in ATP binding (14), an Asp box, and a His box (see Fig. 4A). In addition to these motifs, a
group of PulE-VirB11 family NTPases, related to type IV pilus
biogenesis systems and type II secretion pathways, contain two
CXXC motifs and an Arg box (8). Many of the PulE-VirB11
family NTPases have been shown to be essential for piliation,
extracellular protein secretion, transformation, and conjugal transfer
(15). Several PulE-VirB11 family NTPases have been purified and
characterized (10, 16-18). Genetic studies have also been performed
for the PulE-VirB11 family NTPases (8, 19-21). In addition, TrwD and TrbB proteins of plasmids R388 and RP4, respectively, and HP0525 protein in the cag pathogenicity island of
Helicobacter pylori have been shown to exhibit NTPase
activity (17, 22).
IncI1 plasmids R64 and ColIb-P9 form two types of sex pili, a thick
rigid pilus and a thin flexible pilus. The thin pilus is required only
for conjugation in liquid media (23). A
16-kb1 DNA segment, including
the traA-D and pilI-V genes, of the
54-kb R64 transfer region is required for the formation of thin pilus (24). 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. The products of the traBC and
pilK-V genes were shown to be essential for the formation of
thin pilus on the cell surface (25, 26). R64 and ColIb-P9 thin pili
function in the formation of donor-recipient cell aggregates in liquid
matings. Specificity of recipient cells is determined by seven
C-terminal segments of PilV adhesins, which are switched through
shufflon multiple inversion (27, 28). The R64 pilQ product
shares amino acid sequence similarity with PulE-VirB11 family
NTPases (24).
In this work, the R64 pilQ gene product, essential for thin
pilus formation, was overproduced and purified. Purified PilQ protein
formed homooctamers and displayed ATPase activity. Several amino acid
substitutions were introduced into the pilQ gene within conserved motifs. Effects of pilQ mutations on thin pilus
formation, ATPase activity, and multimer stability were examined.
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EXPERIMENTAL PROCEDURES |
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).
Plasmid vector pUC119 (32) was used for cloning. pET28b and pET11a (31)
were used for overexpression of the pilQ gene. pKK641A' (28)
carried traABCD and pil genes for R64 thin pilus formation. pKK641A' pilQ1 (25) carried the pilQ1
frameshift mutation. pKK661 (28) was a pHSG576-derivative plasmid
carrying genes for remaining R64 transfer functions including
oriT. pKK702a (25) was a pUC119-derivative plasmid carrying
R64 pilQ gene.
Media--
Luria-Bertani (LB) medium was prepared as previously
described (33). The solid medium contained 1.5% agar. Antibiotics were added to liquid and solid media at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 25 µg/ml; kanamycin, 50 µg/ml; and nalidixic acid, 20 µg/ml.
Conjugal Transfer--
E. coli NF83 or BL21 (DE3)
cells harboring pKK661, pKK641A' pilQ1 and pilQ
plasmid, and E. coli TN102 cells were used as donor and
recipient cells, respectively. Donor cells were grown to the log phase
and mixed with recipient cells in the stationary phase. After standing
for 90 min at 37 °C, the mixture was plated at various dilutions
onto LB plates containing chloramphenicol and nalidixic acid. Since
pKK661, pKK641A' pilQ1 and pilQ plasmid complement each other, donor cells harboring pKK661, pKK641A' pilQ1 and pilQ plasmid transmitted pKK661
carrying oriT into recipient cells (23). Transfer frequency
is presented as the ratio (expressed as percentage) of the number of
transconjugants to that of donor cells. In the case of BL21 (DE3)
harboring pEQ11, LB medium containing 0, 10, 50, or 100 µM IPTG was used.
Construction of Plasmids--
Construction of plasmids and other
methods of DNA manipulation were performed as described previously
(33).
To construct the T7 promoter overexpression system of the
pilQ gene, an NdeI site was introduced at the
initiation codon of the pilQ gene by PCR using appropriate
primers and pKK702a DNA as a template. The
NdeI-BamHI fragment of the PCR product was inserted into the NdeI-BamHI site of pET28b to
give pEQ28. The XbaI-BamHI fragment of pEQ28 was
inserted into the XbaI-BamHI sites of pUC119 and
pET11a to give pKK702h and pEQ11, respectively.
The pilQ mutants were constructed by the PCR-mediated
site-directed mutagenesis method using pKK702h as a template (34). The
resultant mutants are as follows: F209S (TTT to TCT), I219T (ATA to
ACA), T234I (ACC to ATC), K238Q (AAA to CAA), D263N (GAT to AAT), D304N
(GAT to AAT), H328A (CAC to GCC), C375A (TGT to GCT), C423A (TGT to
GCT), R432A (AGA to GCA), and I490V (ATC to GTC). To construct the
340 deletion mutant, the NdeI-AccI fragment of
pKK702h was introduced into the HincII site of pUC119 by
blunt end ligation after treatment with Klenow fragment. To construct mutant pilQ overexpression plasmids, the
XbaI-BamHI fragments of pKK702h with mutant
pilQ genes were inserted into the
XbaI-BamHI site of pET28b.
Purification of PilQ Protein--
An overnight culture of BL21
(DE3) cells harboring pEQ28 was diluted 50-fold in LB medium (500 ml)
containing kanamycin and incubated at 37 °C with shaking. When the
A600 of the culture reached ~0.5, IPTG was
added to a final concentration of 1 mM. After 1 h, the
induced cells were harvested by centrifugation and used for the
purification of PilQ protein.
All purification steps were carried out at 4 °C. IPTG-induced cells
were resuspended in 50 ml of TS buffer (20 mM Tris-HCl (pH
8.0) and 100 mM NaCl) and disrupted by a French pressure
cell. Cell extract was centrifuged at 100,000 × g for
30 min. The supernatant was applied to a Talon Co2+
affinity column (CLONTECH). After washing the
column with TS buffer containing 10 mM imidazole, bound
proteins were eluted with TS buffer containing 300 mM
imidazole. The PilQ-containing fraction was dialyzed against 20 mM Tris-HCl (pH 7.5) and loaded onto a MonoQ ion exchange
column (Amersham Pharmacia Biotech) equilibrated with 20 mM
Tris-HCl (pH 7.5). The column was washed with 20 mM
Tris-HCl (pH 7.5), and then bound protein was eluted with a linear
gradient (from 0 to 1.0 M) of NaCl in 20 mM
Tris-HCl (pH 7.5). The PilQ-enriched fractions were applied to a
Sephacryl S-200 gel filtration column (Amersham Pharmacia Biotech)
equilibrated with buffer G (20 mM Tris-HCl (pH 7.5) and 100 mM NaCl). Peak fractions of PilQ protein were concentrated;
dialyzed against a solution containing 20 mM Tris-HCl (pH
7.5), 1 mM EDTA, 1 mM DTT, 100 mM
NaCl, and 30% glycerol; and stored at
80 °C.
Determination of ATPase Activity--
ATPase activity of His tag
PilQ protein was estimated by the thin layer chromatography (TLC)
method (35). Reactions were carried out at 30 °C for 30 min in 40 µl 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 µM His tag PilQ protein. Aliquots (1 µl) from
reaction mixtures were spotted onto polyethyleneimine-cellulose plates
(Merck) and developed in 1 M formic acid and 0.5 M LiCl. The spots of free phosphate and ATP were visualized
and quantitated by a BAS 2000 image analyzer (Fuji Film). The effects
of pH on ATPase activity were examined using MES buffer for pH 5.0-7.0 and Tris buffer for pH 7.5-9.0.
To determine the kinetics of PilQ ATPase activity, a spectrophotometric
method (36) was performed with slight modifications. 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 µg of His tag PilQ
protein, and various concentrations of ATP, and decrease in absorbance at 340 nm was measured using a UV160 spectrophotometer (Shimazu). Km and Vmax values were
calculated from Eadie-Scatchard plots.
Preparation of Anti-PilQ Protein Antibody--
Four doses of the
purified His tag PilQ protein were injected subcutaneously into the
back of a New Zealand White rabbit in an emulsion containing complete
Freund's adjuvant. Anti-PilQ antibody was purified by affinity
chromatography using PilQ-conjugated ECH Sepharose 4B (Amersham
Pharmacia Biotech).
Preparation of Thin Pilus Fraction--
Crude thin pilus
fraction was prepared as described previously (37). E. coli
cells harboring pKK641A' pilQ1 and pilQ plasmids with or without mutations were grown to late log phase with shaking at
37 °C. The culture medium was centrifuged at 140,000 × g for 1 h. The pellet is referred to as the thin pilus fraction.
Subcellular Fractionation--
Cells harboring pKK641A' or
pKK702a were lysed by EDTA-lysozyme and fractionated to periplasm,
cytoplasmic, and crude membrane fraction as described previously (38).
Crude membrane fraction was separated to outer membrane and inner
membrane by the three-step sucrose gradient procedure (39). Amounts of
PilQ protein in all fractions were determined by Western blot analysis
using anti-PilQ antibody.
Gel Filtration Chromatography--
Size and stability of PilQ
multimer were analyzed by gel filtration chromatography. The purified
wild-type and mutant His tag PilQ proteins (500 µg) 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. Protein concentration and ATPase activity
were determined by a protein assay kit (Bio-Rad) and by the TLC method, respectively. The following proteins were used as molecular mass standards: ferritin (450 kDa), catalase (240 kDa), and bovine serum
albumin (68 kDa).
Trypsin Digestion of Wild-type and Mutant PilQ Proteins--
To
analyze ATP-induced conformational change of the PilQ protein, trypsin
sensitivity of His tag PilQ protein was compared in the presence and
absence of 1 mM ATP. Two µg of the purified His tag PilQ
protein with or without mutations was digested on ice with 0.3 µg of
trypsin in 20 µl of buffer A with or without 1 mM ATP.
After 30 min, the reaction was stopped by the addition of 1 µl of 4 mM phenylmethylsulfonyl fluoride and 5 µl of 20% trichloroacetic acid. The precipitates of acid-denatured proteins were
collected by centrifugation, washed with acetone, and dissolved in SDS
sample buffer. The samples were applied to SDS-PAGE (15% gel), and
then tryptic fragments of PilQ protein were detected by Western blot
analysis using anti-PilQ antibody.
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RESULTS |
Overexpression and Purification of PilQ Protein--
To study the
characteristics of the R64 pilQ product, the pilQ
gene was cloned into pET28b to give pEQ28 (Fig.
1A). pEQ28 carried a modified
pilQ gene in which a stretch of six histidine residues was
attached at its N terminus. To examine the effects of the PilQ
N-terminal His tag on PilQ activity, the complementation activity of
pEQ28 for the pilQ1 frameshift mutation in R64 liquid matings was estimated. For that purpose, pEQ11, an Ampr
derivative of pEQ28, was constructed. E. coli strain BL21
(DE3) harboring pKK661, pKK641A' pilQ1 and pEQ11, and strain
TN102 were used as donor and recipient cells, respectively (Table
I). The transfer frequency of pKK661 from
donor cells harboring pKK661 and pKK641A' pilQ1 and was less
than 0.0001%. When pKK702a carrying the wild-type pilQ gene
on pUC119 was introduced into donor cells, the transfer frequency was
increased to 0.5-1.0%. When pEQ11 was introduced into donor cells,
the transfer frequency was also increased to wild-type levels in the
presence of 0-100 µM IPTG. These results suggest that
the presence of the His tag at the N terminus of PilQ protein does not
affect PilQ activity. Therefore, His tag PilQ protein was purified and
used for further analyses to characterize its enzymatic and biochemical
features.

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Fig. 1.
Purification of wild-type and mutant R64 PilQ
proteins. A, nucleotide and amino acid sequences around
the translation initiation site of pilQ overexpression
plasmid pEQ28. Vector and R64 pilQ sequences are described
in lowercase and uppercase letters,
respectively. The ribosomal binding site of T7 gene 10 (rbs) is underlined. The start codon of the R64
pilQ gene is marked by a bent arrow.
The His tag sequence is underlined. B,
purification of wild-type and mutant R64 PilQ proteins. E. coli BL21 (DE3) cells harboring pEQ28 with or without mutations
were treated with 1 mM IPTG for 1 h. A soluble
fraction was prepared from the induced cells (lane
S). The soluble fraction was applied to a Co2+
affinity column, and bound protein was eluted with 300 mM
imidazole (lane E). For further purification, the
eluate fraction was successively subjected to MonoQ ion exchange and
S-200 gel filtration chromatography. Peak fractions of PilQ protein
were pooled and concentrated (lane P). PilQ
mutant proteins were purified by the same procedure. After SDS-PAGE
(12%), proteins were stained with Coomassie Brilliant Blue. Molecular
size markers (lane M), with the size in kDa, are
indicated at the left side.
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Table I
Effects of overexpression of pilQ gene with His tag on the
complementation of the pilQ1 mutation
Transfer frequency of pKK661 from E. coli BL21 (DE3) donor
cells harboring pKK661, pKK641A' pilQ1, and complementation
plasmids in liquid mating was estimated in the presence of various
concentrations of IPTG, and is indicated as a percentage of
transconjugants relative to donor cells.
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Upon IPTG induction, E. coli BL21 (DE3) cells harboring
pEQ28 overproduced a 58-kDa protein (data not shown). Since this
protein was not produced in the absence of induction and the apparent molecular mass of the overproduced protein is consistent with the
calculated value of 59,995 Da, the 58-kDa protein is most likely to be
His tag PilQ protein. After ultracentrifugation of cell extract, most
overproduced PilQ protein was recovered in the soluble fraction (Fig.
1B, lane S). The PilQ protein was
purified by Co2+ affinity chromatography (Fig.
1B, lane E). The peak fraction was
further purified by MonoQ anion exchange and Sephacryl S-200 gel
filtration chromatography (Fig. 1B, lane
P). SDS-PAGE followed by silver staining indicated that the
purity of His tag PilQ protein was over 98% (data not shown).
Approximately 2.5 mg of purified His tag PilQ protein was obtained from
a 500-ml culture.
The purified His tag PilQ protein was used to immunize a rabbit. The
purified anti-PilQ antibodies specifically reacted against a 58-kDa
PilQ protein present in whole cell lysate from E. coli cells
harboring pKK641A' or pKK702a.
Subcellular Localization of PilQ Protein--
Subcellular
fractionation experiments were performed to determine localization of
PilQ protein within cells. E. coli cells harboring pKK641A'
or pKK702a were separated into cytoplasmic, periplasmic, inner
membrane, and outer membrane fractions. Proteins in every fraction were
separated by SDS-PAGE and analyzed for PilQ protein by Western blotting
using anti-PilQ antibody. PilQ protein was recovered almost exclusively
in the cytoplasmic fraction of cells harboring pKK641A' or pKK702a
(Fig. 2). These results indicate that the
PilQ protein is a cytoplasmic protein.

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Fig. 2.
Localization of R64 PilQ protein.
Subcellular fractionation of cells harboring pKK641A' (upper
panel) or pKK702a (lower panel) were carried
out as detailed under "Experimental Procedures." Each fraction was
analyzed by SDS-PAGE (12%), followed by Western blot analysis using
anti-PilQ antibody. Lane W, whole cell lysate;
lane C, cytoplasm; lane P,
periplasm; lane I, inner membrane;
lane O, outer membrane.
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Detection of ATPase Activity--
To detect the ATPase activity of
the purified His tag PilQ protein, thin layer chromatography (TLC)
analysis was performed. When the purified PilQ protein was incubated
with [
-32P]ATP, release of a Pi
radiochromatographic spot was observed on a TLC plate in a time- and
PilQ concentration-dependent manner (data not shown),
indicating that the PilQ protein contains ATPase activity.
The effects of different reaction conditions on His tag PilQ ATPase
activity were examined (Table II). When
PilQ ATPase reactions were carried out under different pH conditions
using MES (pH 5-7) and Tris (pH 7.5-9) buffers, maximum ATPase
activity was observed around pH 6.5, while either increasing or
decreasing the pH from 6.5 significantly lowered the ATPase activity.
The PilQ ATPase required divalent cations since EDTA, a divalent cation
chelator, strongly inhibited ATPase activity. The optimal concentration of Mg2+ required for ATPase activity was ~5
mM, whereas a similar concentration of Ca2+
resulted in lower activity. The optimal salt concentration was ~25
mM for both KCl and NaCl, while KCl produced slightly
higher ATPase activity than NaCl. Therefore, further ATPase assays were performed with 5 mM MgCl2, 25 mM
KCl and at pH 6.5. Kinetics of ATP-hydrolyzing activity of PilQ protein
was estimated by the spectrophotometric method. As derived from the
Eadie-Scatchard plot (data not shown), the apparent
Km for ATP was 0.26 mM, and the
Vmax value was 0.71 nmol/min/mg of protein.
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Table II
Characteristics of the ATPase activity of PilQ protein
The effects of different reaction conditions on ATPase activity of His
tag PilQ protein were examined. ATPase activity was determined as a
release of Pi from [ -32P]ATP and is given in
nanomoles of released Pi/min/mg of protein. Reactions were
carried out at 30 °C for 30 min in a mixture containing 50 mM buffer, 5 mM MgCl2, 25 mM KCl, 1 mM DTT, 1 mM
[ -32P]ATP, and 2 µM His tag PilQ protein
whenever otherwise indicated.
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Inhibition of ATP Hydrolysis with NTPs or dNTPs--
To examine
whether His tag PilQ protein is able to bind to NTPs or dNTPs other
than ATP, the inhibition of PilQ ATPase by NTPs or dNTPs was estimated.
A standard ATPase assay with [
-32P]ATP was performed
in the presence of 5-fold excess of unlabeled NTPs or dNTPs. Unlabeled
ATP apparently inhibited PilQ ATPase by 79% as expected (Fig.
3). PilQ ATPase inhibition by dATP and dGTP were 55% and 30%, respectively, while inhibition by GTP, CTP,
UTP, dCTP, and dTTP was less than 20%. These results indicate that
PilQ protein exhibits the highest affinity to ATP followed by dATP and
dGTP. PilQ affinity to other NTPs or dNTPs is low. It is of great
interest whether these NTPs or dNTPs are hydrolyzed by PilQ protein.
[
-32P]dATP hydrolysis by PilQ protein was demonstrated
by TLC analysis (data not shown).

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Fig. 3.
Distinct substrate affinity of R64 PilQ
protein. For detection of distinct substrate affinity, a standard
ATPase assay with [ -32P]ATP as substrate was carried
out with 5-fold molar excess of unlabeled NTPs or dNTPs. Effects of
inhibitors were expressed as residual [ -32P]ATP
hydrolysis activity. The rate of ATP hydrolysis without NTPs or dNTPs
was defined as 100%.
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Construction of the Mutant pilQ Genes--
In the previous
sections, we have demonstrated that R64 PilQ protein contains ATPase
activity. Hence, it was of interest to test whether the ATPase activity
of PilQ protein is required for R64 thin pilus biogenesis. For that
purpose, several amino acid substitutions were introduced within the
conserved motifs of the His tag pilQ genes, including the
NTP-binding motifs, by the PCR method. In
summary, 11 missense and 1 deletion
mutants were obtained (Fig. 4A, Table
III).

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Fig. 4.
Characteristics and positions of
pilQ mutations. A, schematic
representation of the C-terminal half of PilQ. At the top,
several conserved motifs of R64 PilQ sequence are shown; Walker box A,
Walker box B, Asp box, His box, two CXXC motifs, and Arg
box. Highly conserved amino acids within these motifs were replaced by
amino acids indicated by downward arrows. The
number of amino acid residues in PilQ protein is indicated
above the bar. B, intracellular mutant
PilQ production (upper panel) and extracellular
pilin production (lower panel) in various
pilQ mutants. Thin pili were precipitated by
ultracentrifugation of culture medium in which cells harboring pKK641A'
pilQ1 and indicated pilQ mutant plasmid had grown
(thin pilus fraction). Whole cell lysates and thin pilus fractions were
analyzed by SDS-PAGE (12%), and then PilQ and pilin proteins were
detected by Western blot analysis using anti-PilQ and anti-pilin
antibody, respectively. The positions of PilQ and pilin proteins are
indicated at the left.
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Table III
Characteristics of the products of various mutant pilQ genes
The pilQ activity was estimated as transfer frequency of
pKK661 from E. coli NF83 donor cells harboring pKK661,
pKK641A' pilQ1 and pKK702h with or without His tag
pilQ mutations in liquid matings, and is shown as a
percentage of transconjugants relative to donor cells. Transdominance
of mutant His tag pilQ genes was estimated as transfer
frequency of pKK661 from donor cells harboring pKK661, pKK641A', and
pKK702h with or without His tag pilQ mutations. Mutant His
tag PilQ proteins were purified from induced E. coli BL21
(DE3) cells harboring pEQ28 with the indicated mutations. ATPase
activity of the purified mutant His tag PilQ proteins was determined by
TLC method. The relative ATPase activity of the mutant PilQ proteins to
that of the wild type is indicated as a percentage in parentheses.
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Intracellular production of mutant His tag PilQ protein was analyzed by
Western blotting using anti-PilQ antibody (Fig. 4B, top panel). E. coli cells carrying
missense pilQ genes produced mutant PilQ proteins at the
same levels as that of the wild-type PilQ protein. Extracellular thin
pilus production was analyzed using anti-pilin antibody (Fig.
4B, bottom panel). The culture media
of E. coli cells harboring pKK641A' pilQ1 and
mutant pilQ plasmids were ultracentrifuged to recover thin
pili detached from the cells. Cells harboring pilQ F209S,
T234I, K238Q, D263N, H328A, C375A, and C423A mutants produced very little or
no thin pili, whereas cells harboring the remaining missense
pilQ mutants produced similar amounts of thin pili as the
wild type.
The activity of mutant PilQ proteins for thin pilus biogenesis was also
determined as transfer frequency in liquid matings. The effects of the
addition of the mutant pilQ plasmids on the transfer
frequency in liquid mating from donor cells harboring pKK661 and
pKK641A' pilQ1 were estimated. E. coli NF83 donor
cells harboring pKK661, pKK641A' pilQ1 and pKK702h
transmitted pKK661 into the recipient cells by conjugation at a
frequency of 1.6%, while those harboring only pKK661 and pKK641A'
pilQ1 did not (Table III, pilQ activity).
Different levels of recovery in the transfer frequency were observed by
introducing various mutant pilQ genes into donor cells.
Transfer frequencies were recovered to the wild-type levels by the
pilQ I219T, D304N, R432A, and
I490V mutants. For the pilQ F209S,
T234I, K238Q, H328A, C375A,
and C423A mutants, various levels of recovery in transfer
frequencies were observed. For the pilQ D263N and
340 mutants, no recovery was observed. These results are
consistent with the results of extracellular thin pilus production
(Fig. 4B) and indicate that the pilQ F209S, T234I, K238Q, D263N, H328A,
C375A, C423A, and
340 mutant are defective in R64 thin pilus biogenesis and subsequently in R64 liquid matings.
Transdominance of the Mutant pilQ Genes over Wild-type pilQ
Gene--
Addition of pKK702h (multicopy wild-type His tag
pilQ) to donor cells harboring pKK661 and pKK641A' had little
effect on the transfer frequency (Table III, Transdominance). The
effects of introduction of multiple copies of mutant pilQ
genes to donor cells harboring pKK661 and pKK641A' on the transfer
frequency were studied to test whether the His tag pilQ
mutants were dominant negative over the wild-type pilQ gene.
The introduction of pilQ T234I, K238Q,
D263N, and H328A mutants into donor cells
harboring pKK661 and pKK641A' decreased the transfer frequency,
indicating the transdominant character of these mutants. All of the
four mutants exhibited reduced or no pilQ activity during
liquid matings.
ATPase Activity of Mutant PilQ Proteins--
The products of four
mutant His tag pilQ genes (pilQ T234I,
K238Q, D263N, and H328A), which
exhibited reduced transfer frequency and transdominant character in
liquid matings, were overproduced and purified by the same procedure as
for wild-type His tag PilQ protein (Fig. 1B). ATPase
activity of mutant PilQ proteins was determined. All of the mutant PilQ
proteins exhibited reduced ATPase activity (Table III, ATPase
activity). The ATPase activity of PilQ D263N was particularly low.
Residual transfer frequencies in these mutants were roughly dependent
on their residual ATPase activity. These results suggest that reduced
ATPase activity in the pilQ T234I, K238Q,
D263N, and H328A mutants results in their reduced
transfer frequency. Thus, it may be concluded that the ATPase activity
of PilQ protein is required for R64 thin pilus biogenesis and
consequently for R64 liquid matings. The products of the other mutant
pilQ genes (pilQ F209S, C375A,
C423A, and
340) with reduced transfer
frequency were also overproduced. However, these mutant proteins could
not be purified due to their insolubility.
ATP Induced Conformational Change in Mutant PilQ Proteins--
The
PilQ T234I, K238Q, D263N, and H328A mutant proteins exhibited reduced
ATPasae activity. It is important to test whether these mutant proteins
are defective in ATP binding. Conformational changes of wild-type and
mutant PilQ proteins caused by ATP binding were examined by trypsin
digestion. By trypsin digestion of wild-type PilQ protein, only two
major fragments (36 and 29 kDa) were produced in the absence of ATP,
while two additional minor fragments (32 and 23 kDa) were produced in
the presence of 1 mM ATP (Fig.
5). Conformational change of PilQ protein
by ATP-binding may result in distinct proteolytic fragment patterns.
Distinct proteolytic patterns in the absence and presence of ATP were
detected for PilQ T234I, D263N, and H328A proteins. However, for PilQ
K238Q, the Walker box A mutant, the 29-kDa fragment was produced even in the absence of ATP. These results suggest that PilQ K238Q mutant is
unable to bind to ATP, whereas PilQ T234I, D263N, and H328A mutants are
able to bind to ATP despite residual ATPase activity.

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Fig. 5.
Protease digestion of mutant PilQ
proteins. Two µg of purified wild-type and mutant PilQ proteins
were digested on ice with 0.3 µg of trypsin for 30 min in the
presence (+) or absence ( ) of 1 mM ATP. Trypsin was
inactivated by the addition of 4 mM phenylmethylsulfonyl
fluoride and 20% trichloroacetic acid. The samples were subjected to
SDS-PAGE (15%), and then PilQ fragments were analyzed by Western
blotting using anti-PilQ antibody. Lane N
represents wild-type PilQ protein without trypsin. Intact PilQ protein
and minor proteolytic fragments produced only in the presence of ATP
are marked by arrow with PilQ and asterisk,
respectively, at the right side. Molecular size markers are
indicated at the left.
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Formation and Stability of the PilQ Multimer--
Formation of
hexameric ring structures of RP4 TrbB protein and H. pylori
HP0525 protein, members of PulE-VirB11 family NTPases, have been
reported in the presence of dATP and Mg2+ (40). In the
Sephacryl S-200 gel filtration chromatography during PilQ purification,
the wild-type and mutant His tag PilQ proteins were eluted at a
position corresponding to a multimer larger than a monomer. The precise
size and stability of PilQ multimers were determined by Superose 6 gel filtration chromatography in the presence or absence of 1 mM ATP. The wild-type His tag PilQ protein was eluted at a
position corresponding to 460-kDa irrespective of the presence or
absence of 1 mM ATP (Fig.
6A). Since the molecular mass
of the PilQ monomer was shown to be 58 kDa, wild-type PilQ protein most
likely forms a homooctamer in the presence and absence of 1 mM ATP. These results also indicate that the PilQ octamer
is stable in the presence or absence of 1 mM ATP. The
ATPase activity of each fraction roughly corresponds to its protein
concentration. Three mutant PilQ proteins (T234I, K238Q, and H328A)
formed stable multimers with reduced ATPase activity in the presence
and absence of 1 mM ATP (data not shown). In contrast, PilQ
D263N multimer was unstable especially in the absence of ATP, since
PilQ D263N protein was eluted as a mixture of multimers and monomers
(Fig. 6B). The PilQ D263N monomer exhibited weak ATPase
activity, while its multimer did not. When concentrated PilQ D263N
monomer fraction was rechromatographed on Superose 6, PilQ D263N
protein was again eluted as a mixture of multimers and monomers,
indicating that PilQ D263N protein is in equilibrium with multimer and
monomer forms (data not shown). These results indicate that the region
containing the PilQ D263N mutation is involved in stable multimer
formation.

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Fig. 6.
Instability of the PilQ D263N multimer.
Purified PilQ wild-type (A) and PilQ D263N (B)
proteins were analyzed by Superose 6 gel filtration chromatography with
(left graph) or without (right
graph) 1 mM ATP. Fractions were tested for
ATPase activity and subjected to SDS-PAGE (12%). Separated proteins
were stained with Coomassie Brilliant Blue (upper
panel). In all graphs, filled circles
represent the ATPase activity (left scale), and
open circles the amount of protein
(right scale). Elution points of molecular size
markers are indicated by downward arrowheads
above the panels: 1, ferritin (450 kDa); 2, catalase (240 kDa); 3, bovine serum
albumin (68 kDa).
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DISCUSSION |
In the present work, we have overproduced the R64 pilQ
gene encoding a PulE-VirB11 family NTPase with an N-terminal His tag. His tag PilQ protein was purified to near homogeneity (>98%). Purified PilQ protein displayed ATPase activity with a
Vmax of 0.71 nmol/min/mg of protein and a
Km of 0.26 mM at pH 6.5. The Vmax and Km values of
PilQ ATPase are similar to those reported for PulE-VirB11 family
NTPases such as R388 TrwD, RP4 TrbB, and H. pylori cag
HP0525 (17, 22).
PilQ protein was shown to be located in the cytoplasmic fraction
irrespective of the presence or absence of the other Pil proteins.
These results differ from those of K. oxytoca PulE protein, which was shown to be located in the inner membrane (8). R388 TrwD was
located in the cytoplasmic and outer membrane fraction (17). PilQ
ATPase may function at or near the inner membrane, since it is involved
in the formation of thin pilus, a cell envelope-associated appendage.
Association of PilQ and the other Pil components may be weak.
Optimum pH for PilQ ATPase was different from those of
R388 TrwD, RP4 TrbB, and H. pylori cag HP0525 (17, 22). The
pH optimum of PilQ was located at approximately 6.5, while that of TrwD
and HP0525 ATPase and TrbB dATPase was located at approximately 9.0-9.5. Differences in substrate affinity were found among PilQ, TrbB, HP0525, and TrwD NTPases. PilQ, HP0525, and TrwD exhibited the
highest activity for ATP, while TrbB exhibited maximum activity for
dATP. Preferred substrates for PilQ are ATP, dATP, and dGTP, whereas
those for TrbB are dATP, GTP, and ATP, and those for HP0525 are ATP,
dATP, CTP, and dCTP. Furthermore, TrbB protein displayed distinct
optimum pH for ATPase and GTPase activities (22). The biological
significance of differences in substrate affinity remains to be elucidated.
PilQ protein formed homooctamers either in the presence or absence of
ATP. PilQ homooctamer formation was observed even in the absence of
Mg2+ (data not shown). Formation of homohexameric ring
structure was reported for TrbB, HP0525, and TrwD (22, 40). TrbB
hexamer was formed in the presence of Mg2+ and dATP or
dADP, while TrbB tetramer was formed in the absence of Mg2+
and dATP. In contrast, HP0525 hexamer was formed irrespective of the
presence or absence of Mg2+ and dATP. The requirement of
NTPs for multimer formation of various PurE-VirB11 NTPases may depend
on differences in their protein structure.
Mutation analyses have been performed on various PulE-VirB11 family
NTPases (8, 19-21). In the present report, we have introduced several
amino acid substitutions into the pilQ gene within the Walker box A, B, Asp box, His box, Arg box, and CXXC motifs.
Two Walker box A mutants, PilQ T234I and PilQ K283Q, failed to produce thin pilus and exhibited low transfer frequencies. Purified mutant proteins displayed reduced ATPase activities. PilQ K283Q affected ATPase activity more severely than PilQ T234I. A trypsin digestion assay revealed that PilQ K238Q lost ATP binding activity, while PilQ
T234I retained it. The failure of ATP binding of PilQ K238Q may be due
to the loss of positive charge required for the direct interaction with
and
phosphates of ATP resulting from the substitution of lysine
by glutamine. Similar Walker box A mutants of PulE and TrbB also
displayed severe effects on pullulanase secretion and conjugation,
respectively (8, 22). Walker box B mutant PilQ D304N had no effects on
thin pilus formation and transfer frequency.
Asp box mutant PilQ D263N exhibited the most severe effects on thin
pilus biogenesis and liquid matings. The purified PilQ D263N protein
displayed very low ATPase activity. The PilQ D263N octamer was
unstable, especially in the absence of ATP. His box mutant PilQ H328A
failed to produce thin pilus and exhibited low transfer frequency. The
purified PilQ H328A protein displayed 43% ATPase activity.
Four mutant genes (pilQ T234I, K238Q,
D263N, and H328A) exhibited dominant negative
character over the wild-type allele. The dominant negative character of
these mutants suggests that mixed octamers can be formed from the
mutant and wild-type PilQ proteins. RP4 TrbB, H. pylori
HP0525, and R388 TrwD form hexameric ring structures. Krause et
al. (40) postulated that such ring structures may aid a repetitive
step/process of NTP hydrolysis. Incorporation of mutant monomers into
PilQ octamers may inhibit the function of the octamer as a whole.
Hence, the observed transdominance of PilQ mutants over the wild-type
allele supports the above postulation. In this regard, the finding that
the monomer form of the PilQ D263N mutant exhibited residual ATPase
activity, is of particular interest.
The four mutant proteins (PilQ T234I, K238Q, D263N, and H328A)
displayed varying levels of residual ATPase activities (2-43% of
wild-type). In contrast, the amounts of thin pilus produced by these
mutants were very low as were their transfer frequencies (less than
1.5% of the wild-type). This situation is different in RP4 TrbB
mutants (22). Some TrbB mutants exhibiting very low dATPase activity
still exhibited wild-type levels of conjugation activity. Dependence of
the R64 thin pilus biogenesis system on PilQ is stronger than that of
RP4 system on TrbB.
Since the two CXXC motifs and the Arg box are notably
conserved among PulE-VirB11 family NTPases of type IV pilus biogenesis systems and type II secretion systems, mutations were introduced into
these motifs in PilQ. Two CXXC motif mutants, PilQ C375A and
C423A, failed to produce thin pili and exhibited low transfer frequencies. Overexpression of these mutants resulted in insoluble PilQ
mutant proteins. Insolubility of these mutants may be due to the
failure of correct folding. Cysteine residues in PilQ CXXC motifs may be responsible for intramolecular disulfide bond formation, as was suggested for K. oxytoca PulE (21). Lack of
transdominance may be related to the insolubility or abnormal
conformation of these mutant proteins, as they were unable to form
mixed octamers with wild-type protein. Arg box mutant PilQ R432A
carried pilQ activities similar to the wild type, although
this arginine residue in the Arg box is highly conserved in many
PulE-VirB11 family NTPases of type IV biogenesis systems and type II
secretion systems.
Four additional mutants, PilQ F209S, I219T, I490V, and
340, have
been constructed. PilQ F209S and
340 failed to produce thin pili and
exhibited low or no transfer frequencies, respectively, whereas PilQ
I219T and I490V were normal. Insolubility and lack of transdominance
suggest incorrect folding of PilQ F209S and
340.
In conclusion, ATPase activity and multimer formation of PilQ
protein are essential for R64 thin pilus biogenesis. However, further
investigation is required to reveal the precise role(s) of R64 PilQ
ATPase in the biogenesis of R64 thin pilus.