(Received for publication, August 8, 1995; and in revised form, August 28, 1995)
From the
The pilA gene of Neisseria gonorrhoeae encodes
the response regulator of a two-component regulatory system that
controls pilin gene expression. Examination of the primary sequence of
PilA indicates that the protein contains at least two functional
domains. The N-terminal region has a proposed helix-turn-helix motif
thought to be involved in DNA binding. This region also contains the
residues that are presumed to form the acidic pocket involved in
phosphorylation by PilB, the sensor kinase of the system. The C
terminus of the protein has extensive homology to the G (GTP-binding)
domains of the eukaryotic signal recognition particle (SRP) 54-kDa
protein and the subunit of the SRP receptor, or docking protein.
This homology also extends to similar regions of the bacterial SRP
homologs Ffh and FtsY. Here, we demonstrate that purified PilA has
significant GTPase activity, and that this activity has an absolute
requirement for MgCl
and is sensitive to KCl and low pH. We
also show that PilA has a strict specificity for GTP, and that GTP
hydrolysis follows first order kinetics, with a maximum velocity (V
) of 1900 pmol of P
produced per
min per mg of protein and a K
for GTP of
9.6 µM at 37 °C.
Neisseria gonorrhoeae (GC) pili undergo phase (on-off)
variation at a fairly high frequency in vitro (10 to 10
per cell per generation; (1) ).
Mechanisms for phase variation are numerous and occur at multiple
levels. One mechanism involves intra- and intergenic recombination
between silent pilin sequences (pilS) and the pilin expression
locus, pilE(1, 2) . Recombination of pilS sequences into pilE can result in a P
(piliated) to P
(nonpiliated) or P
to P
switch if the recombination event alters
the reading frame, or if parts of the transcriptional or translational
machinery are removed(3) . Alternatively, recombination can
lead to production of altered pilin proteins such as L-pilin
that are not assembled into a functional pilus(4) . Phase
variation also occurs at the post-translational level. Incorrect
processing of propilin can result in the production of a secreted form
of pilin, S-pilin, that is not efficiently assembled into
pili(5) . PilC, a pilus-associated protein that has been
implicated in pilus assembly, has also been proposed to contribute to
pilin phase variation. PilC expression is subject to phase variation
via a slipped-strand misrepair of a tract of guanine residues in the
signal-peptide encoding region of the gene(6) . In addition,
other pilus assembly genes may also have a role in pilin phase
variation(7, 8) .
Transcriptional regulation of pilE also contributes to pilin phase variation. Taha et al.(9) have shown that gonococcal pilin expression is transcriptionally controlled by a regulatory system encoded by the pilA and pilB genes. PilA and PilB have homology to the two-component family of prokaryotic proteins which transduce environmental signals to cytoplasmic regulators via phosphorylation(10) . PilA is the response regulator of this system and is essential in the gonococcus, and PilB is the sensor kinase. The signal to which PilB responds is presently unknown.
PilA is a unique response regulator in that it is a transcriptional activator in the absence of PilB and a repressor in its presence(11) . We have previously demonstrated that PilA is a DNA-binding protein that binds specifically to a region 5` to the pilE1 promoter in a complex manner that may involve looping of the DNA(12) . The PilA in these experiments was purified from a strain lacking pilB and was presumed to be unphosphorylated. Taha and Giorgini (13) have shown, using enriched extracts from Escherichia coli harboring plasmids with pilA alone or pilA and pilB, that the presence of PilB may increase PilA DNA binding.
PilA is a 417-amino acid protein with at
least two functional domains. The amino-terminal portion is predicted
to contain a DNA binding motif (helix-turn-helix; Fig. 1) as
well as the acidic pocket presumed to be the site of phosphorylation by
PilB(13) . The carboxyl-terminal part of PilA has significant
homology to the N-terminal G (GTP-binding) domains of the 54-kDa
subunit of the eukaryotic signal recognition particle
(SRP)()(11) . Recently, homologs of this protein
(SRP54) have been identified in bacteria and are designated Ffh
(fifty-four homolog; Refs. 14, 15, 16, and 17). The C terminus of PilA
has 61% amino acid similarity, and 31% identity, with the G domain of E. coli Ffh (Fig. 1).
Figure 1: Schematic alignment of PilA, E. coli FtsY, and E. coli Ffh. Shaded regions denote the putative GTP-binding domains. Black boxes indicate conserved residues presumed to form the GTP-binding site. D indicates presumed DNA-binding (helix-turn-helix) motif of PilA(11) . G (GTP-binding) and M (protein- and RNA-binding) domains as defined by homology to the eukaryotic SRP54 proteins are indicated below. Percent identity and similarity compared to PilA are indicated for the shaded G regions. AA, amino acid residues.
The eukaryotic SRP is important for targeting and insertion of the signal sequence of exported proteins into the endoplasmic reticulum (ER) membrane. The SRP54 protein is associated with the 7 S RNA in the complex and binds to the signal sequence of the nascent protein as it emerges from the ribosome(18) . The complex then travels to the ER membrane where it binds a docking protein which also has a GTP binding activity. The bacterial homolog Ffh is associated with a 4.5 S RNA in an SRP-like complex and functions in targeting proteins to the bacterial cytoplasmic membrane in a manner similar to its mammalian counterpart(19) . The prokaryotic docking protein has been identified as FtsY(20) , which also has significant sequence homology to PilA (Fig. 1). The SRP54 proteins have two domains: an ``M'' domain, which interacts with the signal peptide of nascent proteins as well as to the 7 S (4.5 S in E. coli) RNA, and a ``G'' domain, which has the GTPase activity required for the interaction of the complex with the docking protein(19) .
Taha et al.(21) examined the
effect of PilA on the export and maturation of alkaline phosphatase and
-lactamase in E. coli and of opacity proteins in N.
gonorrhoeae. They concluded that PilA was not likely to be part of
an SRP-like complex in gonococci. This is not surprising, as the
similarities between PilA and the SRP54 proteins lie exclusively in the
G domains (Fig. 1). The extensive homology suggested to us that
PilA might have a GTPase activity which, while not involved in protein
translocation, may be part of the function of PilA as a transcriptional
regulator. In this report we present evidence that purified
preparations of PilA have significant GTPase activity and characterize
this activity biochemically.
SRP54 and the bacterial homolog Ffh hydrolyze GTP as they
dissociate from the docking protein (SRP receptor) during protein
translocation(19) . Analysis of the PilA amino acid sequence
reveals a segment of 295 amino acids that is similar to the G
(GTP-binding) domains of the SRP54 and SRP receptor family of proteins (Fig. 1; (11) ). It was therefore of interest to
determine whether PilA was capable of hydrolyzing GTP. Purified PilA
was incubated with GTP labeled with P at the
or
position, and radiolabeled P
was measured. In
reactions containing [
-
P]GTP, radiolabeled
P
increased linearly with increasing PilA concentrations,
but not in reactions with [
-
P]GTP (Fig. 2). This result suggests that PilA has GTP-hydrolyzing
activity and that the cleavage likely occurs between the
and
phosphates.
Figure 2:
GTP hydrolysis is PilA-specific and occurs
between the - and
-phosphates. Reactions were carried out for
60 min at 37 °C under standard conditions in the presence of 100
µM [
-
P]GTP or
[
-
P]GTP as indicated.
, PilA +
[
-
P]GTP;
, TT (extract from a
non-PilA containing strain) + [
-
P]GTP;
, PilA + [
-
P]ATP. Activity is
expressed as picomoles of P
released per min as a function
of protein concentration, after correcting for background P
released in samples containing no protein. Assays were performed
in triplicate, and values are from one representative experiment.
Standard deviations were less than 15%.
To demonstrate that the GTPase activity measured
in these experiments was PilA-specific, protein extracts prepared from
an E. coli strain bearing the PilA expression plasmid without
the pilA gene were subjected to the same purification
procedure and assayed for GTPase activity (TT; Fig. 2). Release
of [P]P
in the presence of varying
concentrations of this extract was not significantly greater than the
background from parallel samples containing no protein. These results
indicated that the GTP hydrolyzing activity is PilA-dependent.
Buffer components for the GTP hydrolysis reaction were varied to
determine the requirements for the enzymatic activity of PilA (Table 1). Reactions carried out in the absence of MgCl yielded P
levels equal to those from parallel samples
containing no protein, indicating an absolute requirement for
MgCl
for GTP hydrolysis. The omission of dithiothreitol
from the reactions resulted in an
30% reduction of GTPase
activity. We have previously observed aggregation of purified PilA in
the absence of reducing agents (data not shown), and it is likely that
the aggregated form is less active. The addition of Triton X-100 (final
concentration 0.2%) also reduced the GTPase activity of PilA
30%.
This was interesting, as our previous results indicated that 0.2%
Triton X-100 slightly improved the DNA binding activity of PilA in a
gel shift assay(12) . The omission of glycerol (final
concentration 10%) from the reactions resulted in a dramatic reduction
in GTPase activity at low protein concentrations (Table 1). At
high protein concentrations (4.1 µM), the absence of
glycerol resulted in a 2.5-fold increase in GTPase activity over that
observed at lower protein concentrations (0.8 µM) under
similar conditions. This may indicate that glycerol has a stabilizing
effect on the purified protein.
Variations of the pH of the reaction
had only a small effect on GTPase activity. Lowering the pH from 7.5 to
6.8 reduced the GTPase activity of PilA by 15% (Table 1).
Increasing the pH from 7.5 to 9.5 had no effect on the GTPase activity.
Lower pH (6.8) also seemed to reduce the stability of GTP. In the
absence of protein there was a much higher background of
[
P]P
after a 1-h incubation compared
to similar samples at higher pH (data not shown).
KCl concentration had a dramatic effect on the GTPase activity of PilA (Table 1). Activity was highest in the absence of KCl and decreased more than 5-fold in 500 mM KCl. Substitution of KCl with potassium glutamate (10 mM or 100 mM) had similar effects on the GTPase activity of PilA (data not shown). Interestingly, the DNA binding activity of PilA is also optimal under conditions of low ionic strength (10 mM potassium glutamate(12) ).
The
nucleotide specificity of PilA was also determined.
[-
P]ATP was incubated with varying
concentrations of PilA under conditions optimal for GTP hydrolysis. The
amount of radiolabeled P
was
2-fold greater than the
background observed in samples lacking PilA, but was not stimulated by
increasing PilA concentrations (0.5-5.0 µg). The ability of
various nucleotide analogs to inhibit GTP hydrolysis was then
determined. Addition of the guanine nucleotides GTP, GDP, and GTP
S
at a 10-fold excess over the substrate concentration reduced PilA
GTPase activity by as much as 95% (Table 2). In contrast, none of
the adenine nucleotides tested (ATP, ADP, and cAMP) was able to inhibit
the activity significantly. Varying the time between the addition of
competing nucleotide and the radiolabeled substrate (which was always
added last) had no effect on the inhibition by guanine analogs or the
lack of inhibition by adenine analogs. These results demonstrate that
PilA specifically hydrolyzes GTP, and that this activity is not
affected by preincubation with unlabeled nucleotides as has been
observed for FtsZ-catalyzed GTPase activity (23) .
The
activity of PilA is directly proportional to protein concentration (Fig. 2), indicating that GTP hydrolysis is a first order
reaction. Examination of the velocity of the reaction as a function of
substrate concentration revealed that the reaction follows
Michaelis-Menten kinetics. A double reciprocal plot of the data is
shown in Fig. 3. From this plot we determined a value of 1840
pmol min mg
of protein for the
maximum velocity (V
) of the reaction, and a
value of 9.2 µM for the Michaelis-Menten constant (K
). K
and V
were calculated from these same data using a derivation of the
Michaelis-Menten equation(24) . Using the following equations,
where a = substrate (GTP) concentration and v = velocity (activity) at a given substrate concentration, a K
of 9.6 µM and a V
of 1900 pmol min
mg
were
determined.
Figure 3:
Double reciprocal plot of PilA GTPase
activity. GTP concentration was varied from 10 µM to 500
µM. Reactions were carried out for 60 min with 2.04
µM PilA. Activity was determined as picomoles of
P released per min per mg of protein. The y intercept = V
-1, and the x intercept is K
-1. V
= 1883 pmol
min
mg
. K
= 9.2 µM.
Finally, a direct linear plot analysis gave similar values for K and V
(data not shown).
Other prokaryotic GTP-hydrolyzing proteins that have been
characterized include FtsZ from E. coli and Bacillus
subtilis(23, 25) , Obg from B.
subtilis(26) , Era from E. coli(27) , and
the E. coli SRP54 homolog Ffh(20) . It is interesting
that each of these proteins is required for viability, as is PilA in N. gonorrhoeae(11) . The kinetic parameters for some
of these proteins have been determined, and PilA compares favorably to
these. The V of Obg is 75 pmol min
mg
(28) ,
30-fold less than the
values determined for E. coli FtsZ (2100 pmol min
mg
; (23) ) and PilA (1900 pmol
min
mg
). It should be noted,
however, that the GTPase activity of FtsZ is believed to involve
cooperativity(21) , thus the specific activity of FtsZ varies
considerably. The K
for GTP of Obg is 5.4
µM, similar to that observed for PilA (
10
µM). The intracellular level of GTP in E. coli is
nearly 1 mM(29) , and, assuming that GTP levels are
similar in the gonococcus, the K
of PilA for GTP
is well within the physiological range.
There are significant
differences in the activities of these proteins which may reflect their
differing intracellular functions. Obg, which is involved in Bacillus sporulation, and Era, a membrane protein of unknown
function, are both phosphorylated during GTP
hydrolysis(28, 30) . In contrast, FtsZ and Ffh do not
appear to be phosphorylated by GTP in the reaction; GTP hydrolysis by
these proteins results in release of the free P (and GDP).
Attempts to isolate a phosphorylated PilA intermediate by the methods
used for Obg and Era were unsuccessful, suggesting that PilA is not
autophosphorylated in the GTPase reaction (data not shown).
Obg and Era are believed to play a role in regulation of cellular processes. Phosphorylated and unphosphorylated forms of these proteins may have different activities, and the ratios between the two forms are likely to be important in vivo. It is thought that Obg senses GTP levels as part of the regulatory cascade controlling Bacillus sporulation(31) , as fluctuations in GTP levels in the cell are believed to trigger this phenomenon(32) . Era has been implicated in playing a role in adaptation to thermal stress and in the control of cell division(33, 34) . Thus, these proteins may be sensing GTP levels in the cell as a signal for a changing environmental condition. It is possible that changing intracellular GTP levels may also be a signal for PilA-mediated regulation of gene expression.
FtsZ and Ffh apparently use the phosphate bond energy released during GTP hydrolysis for their primary functions. FtsZ polymerizes at the cell division septum in a GTP-dependent manner and is believed to act as a cytoskeletal element that orchestrates invagination of the bacterial membrane in the process of cell division (25, 35) . Ffh transfers the nascent peptide from the SRP complex to the docking protein during protein translocation, also in a GTP-dependent manner(20) . It is conceivable that PilA may hydrolyze GTP to provide energy for the activation of transcription.
The sequences of the GTP-binding regions of the prokaryotic GTPases can be divided into 3 groups. The GTP-binding sites of Obg and Era have high homology to the GTP-binding sites of eukaryotic G-proteins and E. coli translation elongation factors(26, 36) . FtsZ contains a 7-amino acid segment homologous to a region of tubulin believed to bind guanine nucleotides(35) . The GTP-binding regions of Ffh and PilA share homology with the G domains of the SRP54 and SRP receptor proteins(11, 15) . Interestingly, conservation of GTP-binding domains in these proteins is not necessarily correlated with conservation of function. Obg and Era are apparently involved in signal transduction, like their eukaryotic G-protein counterparts. FtsZ plays a structural role, like its eukaryotic homolog, tubulin. In contrast, PilA has a strong sequence similarity to proteins involved in protein translocation, but is more similar in function to the ATP-hydrolyzing transcriptional regulator NtrC(37) .
NtrC is
the response regulator of the two-component system that controls the
expression of a number of genes in response to nitrogen availability in
bacteria via the alternative factor,
(38) . NtrC activation of transcription is
dependent on phosphorylation by NtrB, the sensor kinase of the system (39) . NtrC ATPase activity is phosphorylation- and
DNA-dependent and is coupled to open complex
formation(37, 40, 41) . It is possible that
GTP hydrolysis by PilA may be coupled to activation of transcription in
a similar manner.
We have previously shown that PilA binds to the N. gonorrhoeae pilE promoter in a sequence-specific manner(10) . This interaction involves multiple regions of the DNA and may involve DNA-bending or loop formation. In this work, we show that, in addition to its DNA binding activity, PilA has a GTP hydrolyzing activity. This GTP-specific activity suggests some interesting possibilities for understanding the PilA-PilB regulatory system. Taha et al.(11) showed that, in E. coli, PilA and PilB together repress a pilE-CAT (chloramphenicol-acetyltransferase) promoter fusion while PilA alone activates transcription of this fusion. These data indicate that PilB modulates the transcriptional activity of PilA. The exact nature of the PilA-PilB interaction is unclear at present, but is likely to involve phosphorylation(11, 40) . PilB modulation of PilA transcriptional activity could occur by a number of mechanisms. PilB could inhibit binding of PilA to promoter DNA. This is unlikely, as Taha et al.(11) have observed increased DNA-binding in enriched extracts of an E. coli strain expressing pilA and pilB. Alternatively, PilB may reduce the transcriptional activation activity of PilA by inhibiting its GTPase activity. This would assume that GTP hydrolysis is coupled to PilA transcription activation, which has not yet been determined. PilB effects on the GTPase activity of PilA are also unknown. Finally, PilB may affect the interaction between PilA and RNA polymerase to inhibit transcription independent of effects on GTPase activity. As PilA is the first example of a DNA-binding protein with a GTP-specific hydrolyzing activity, it will be very interesting to examine the relationship between these two activities and their effects on gene expression in Neisseria.