Stability and DNA Binding of the Phd Protein of the Phage P1
Plasmid Addiction System*
Ehud
Gazit
and
Robert T.
Sauer§
From the Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
 |
ABSTRACT |
The plasmid addiction module of bacteriophage P1
encodes two proteins, Doc, a toxin that is stable to proteolytic
degradation, and Phd, the toxin's antidote that is proteolytically
unstable. Phd has been shown to autoregulate its expression by specific DNA binding. Here, we investigate the secondary structure and thermal
stability of Phd, the effect of operator DNA binding on the structure
and stability of Phd, and the stoichiometry, affinity, and
cooperativity of Phd binding to operator subsites and intact operator
DNA. Phd folds as a monomer at low temperatures or in the presence of
osmolytes but exists predominantly in an unfolded conformation at
37 °C. The native state of Phd is stabilized by operator binding.
Two Phd monomers bind to each operator subsite, and four monomers bind
to the intact operator. The subsite binding reaction shows a
second-order dependence on protein concentration and monomer-bound DNA
species are unpopulated, suggesting that two Phd molecules bind
cooperatively to each operator subsite. In intact operator binding
experiments, both dimer-bound and tetramer-bound DNA species are
populated, and binding occurs at protein concentrations similar to
those required for subsite binding, suggesting that there is no
significant dimer-dimer cooperativity.
 |
INTRODUCTION |
The stable and efficient maintenance of low-copy plasmids within
bacterial cells is ensured, in part, by addiction mechanisms mediated
by specific proteins (1, 2). For example, when bacteriophage P1
lysogenizes Escherichia coli as a low copy plasmid, the rate
of spontaneous plasmid loss is only about one per 105
generations (3). Two proteins comprise the plasmid addiction system of
bacteriophage P1: Doc (death on
cure), a 126-residue toxic protein, and Phd
(prevent host death), a 73-residue
antidote (4). This system functions to kill cells that have been cured of the plasmid. The addiction mechanism depends on significant differences in the proteolytic stability of Doc, which is resistant to
proteolysis, and Phd, which is degraded in a manner dependent on the
host-encoded ClpXP protease complex (5). In P1 lysogens, a
concentration of Phd sufficient to suppress Doc toxicity is maintained
by a continuous synthesis of Phd molecules de novo. In a
bacterial cell that has lost the P1 genome, existing Phd is degraded
and because there is no further synthesis of new Phd, levels fall, and
the cytoplasmically inherited Doc kills the P1-free daughter cells.
Post-segregational killing of plasmid-cured cells is also used by other
low-copy number plasmids, including the F (6), RK2 (7), and R1 (8)
plasmids of E. coli, and similar systems in
Streptomyces lividans and Klebsiella oxytoca (for
review, see Refs. 1 and 9). In each case studied, a long-lived toxin and short-lived antidote are part of the addiction mechanism. Interestingly, a functionally similar two-protein module is encoded by
the mazE and mazF genes of E. coli
(10), which are regulated by the cellular level of ppGpp, an indicator
of amino acid starvation. Functional homologues of the Phd and Doc
proteins are also found upstream of the Ecoprrl type IC DNA
restriction and modification operon (11). These results suggest that
addiction system homologues may function in the response to starvation
and DNA restriction.
The Phd and Doc genes are expressed from the same operon (4), and Phd
negatively regulates its own expression as well as that of Doc (12),
ensuring a relatively low level of expression of both proteins in P1
lysogens. The DNA site bound by Phd is 23 base pairs in length and
includes two 10-base pair subsites that are roughly palindromic and
separated by 3 base pairs (12) (see Sequence
1).
In footprinting experiments, Phd
occupies the left operator subsite at roughly 10-fold lower
concentrations than the right subsite (12). Studies in vivo
show that Phd alone is sufficient to repress transcription from the
plasmid addiction operon (12).
In this work, we characterize the secondary structure and thermal
stability of the purified Phd protein, study the effect of operator DNA
binding on the stability of Phd, and determine the stoichiometry,
mechanism, and cooperativity of Phd binding to the intact operator DNA
and to operator subsites. The results are discussed in the context of
the plasmid addiction mechanism. Phd shows some functional and sequence
similarities to Arc, MetJ, and other members of the ribbon-helix-helix
family of DNA-binding proteins (12-14), but the studies reported here
suggest that Phd has a different structure from Arc.
 |
MATERIALS AND METHODS |
Protein Purification--
E. coli transformed with
plasmid pHAL20 (encoding Phd under tac-promotor control)
(12) was grown in LB broth containing 100 µg/ml ampicillin (LB-amp)
at 37 °C to an A600 of approximately 0.7, and
protein expression was induced by the addition of 100 µg/ml
isopropyl-1-thio-
-D-galactopyranoside for 30 min. Cells were harvested by centrifugation and stored frozen at
80 °C prior to purification. The cells were thawed at 4 °C, resuspended in buffer A (50 mM NaCl, 50 mM Tris-HCl (pH 7.4),
1 mM EDTA) with 50 µM phenylmethylsulfonyl
fluoride, and lysed by sonication. Insoluble material was removed by
centrifugation for 10 min at 13,000 × g, followed by a
0.22-µm filtration. The supernatant was applied to a Mono S HR 5/5
FPLC column (Amersham Pharmacia Biotech) equilibrated in buffer A, and
proteins were eluted with a gradient from 50 to 1000 mM
NaCl. A peak that included Phd eluted at approximately 400 mM NaCl. This peak was applied to a reverse-phase semipreparative C18 column (Vydac) and was resolved by
using a 0-80% acetonitrile gradient in water and 0.1%
trifluoroacetic acid. At this point, Phd was greater than 95% pure as
assessed by Coomassie staining of SDS-polyacrylamide gels.
matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectrometry performed using a Voyager-DE STR Biospectrometry
Workstation confirmed the molecular mass expected for purified Phd with
no modifications (8128 daltons).
Biophysical Studies--
Circular dichroism (CD) spectra were
obtained by using an AVIV 60DS spectrapolarimeter equipped with a
temperature-controlled sample holder and a 10-mm path length cuvette.
Mean residue ellipticity, [
], was calculated as,
|
(Eq. 1)
|
where
is the observed ellipticity, m
is the mean residue weight, c is the concentration in mg/ml,
and L is the path length in centimeters. The concentration
of Phd was determined by measuring tyrosine absorbance both at pH 7 and
in 0.1 M KOH using a Hewlett-Packard 8452A diode array
spectrophotometer. Protein concentrations were calculated by using
extinction coefficients of 1394 M
1
cm
1 (274 nm at pH 7) and 2377 M
1 cm
1 (294 nm in 0.1 M KOH) for the single tyrosine of Phd. For thermal denaturation experiments, samples in a buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 0.1 mM EDTA were equilibrated at each temperature for 1 min,
and the CD ellipticity at 222 nm was averaged for 2 min. Experiments
were performed at protein concentrations ranging from 0.5 to 15 µM. Melting curves were fit to a two-state transition
between native and denatured protein by nonlinear least squares fitting
using the program NONLIN for Macintosh (15).
DNA Binding Assays--
Equilibrium binding of Phd to its
operator DNA site and to DNA subsites was monitored by a polyacrylamide
gel mobility shift assay as described (16-18). Single-stranded DNA
oligomers were 5' end-labeled by incubation with
[
-32P]dATP and T4 polynucleotide kinase (New England
Biolabs) for 45 min at 37 °C. Enzyme was removed from the reaction
by phenol:chloroform extraction, and unincorporated nucleotides were
removed by using a G-25 Sephadex Quick Spin column (Boehringer
Mannheim). The second DNA strand was than annealed by mixing equimolar
amounts of the complementary oligonucleotides, heating to 90 °C, and
cooling slowly. Labeled oligomers were diluted to approximately 5 × 103 counts/min (~10 pM). Synthetic DNA
oligomers that were used included a
32-bp1 oligomer that
contained the 23-bp operator DNA site of Phd (underlined)
or 26-bp oligomers that included the 10-bp left or right
operator subsites (underlined).
As a control, a 27-bp oligomer that contained the P22 Arc
repressor operator was used.
DNA binding assays were performed at room temperature in binding
buffer containing 50 mM NaCl, 100 mM Tris-HCl
(pH 7.4), 1 mM EDTA, 100 µg/ml bovine serum albumin, and
0.02% Nonidet P-40. Serial dilutions of the Phd protein were added to
the labeled DNA, and the mixtures were incubated for at least 2 h.
After this time, glycerol was added to each sample (to a final
concentration of 5%), and the samples were loaded onto an 8%
polyacrylamide gel and electrophoresed at 250 V in TBE buffer (90 mM Tris borate, 2 mM EDTA). The gel-mobility
assays were quantified using a Molecular Dynamics PhosphorImager and
IMAGEQUANT software.
Stoichiometry Assays--
To determine the molar ratio of Phd
bound to its palindromic DNA subsites, increasing amounts of Phd were
added to 1 µM left subsite DNA in binding buffer and
incubated for 2 h. The concentration of DNA and protein in this
experiment was much higher than the Phd-DNA dissociation constant, to
ensure a nearly complete binding of the protein to the DNA. A small
amount (~1 nM) of radiolabeled DNA was also added to
quantify the ratio of bound to unbound DNA, following electrophoresis
on a 8% polyacrylamide gel at 250 V in TBE buffer.
 |
RESULTS |
Secondary Structure and Thermal Stability of Phd--
Phd was
purified to homogeneity as described under "Materials and Methods."
Fig. 1A shows the CD spectra
of Phd at 4 °C and 37 °C, and also at 37 °C in buffer plus
trimethylamine N-oxide, an osmolyte that stabilizes native
structure (19). The amount of secondary structure was estimated from
the CD spectrum (20, 21). At 37 °C in buffer alone, Phd seems to be
in a largely unfolded, random-coil conformation. However, at 4 °C or
at 37 °C in the presence of trimethylamine N-oxide, Phd
has a CD spectra indicative of a folded protein containing
approximately 45%
-helix (Fig. 1A).

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Fig. 1.
A, circular dichroism spectra. CD
spectra of Phd were taken in a 0.1-cm cuvette in buffer containing 100 mM NaCl, 50 mM Tris-HCl (pH 7.4), and 0.1 mM EDTA at 4, at 37, and at 37 °C in the presence of 2 M trimethylamine N-oxide (TMAO).
B, thermal denaturation. Thermal stability was determined by
monitoring CD ellipticity as a function of temperature in 100 mM NaCl, 50 mM Tris-HCl (pH 7.4), and 0.1 mM EDTA at protein concentrations of 1 or 15 µM.
|
|
Denaturation experiments monitored by CD were performed to determine
the thermal stability of Phd (Fig. 1B). The thermal
denaturation of Phd is cooperative, with a Tm of
about 25 °C. As expected, Phd is predominantly unfolded at 37 °C.
Superimposible denaturation curves were observed at Phd concentrations
of 1 and 15 µM, suggesting that the native form of Phd is
stable as a monomer (Fig. 1B). The thermal denaturation of
Phd was fully reversible in a reverse melt, indicating that the protein
can readily alternate between its folded and unfolded states.
Stabilization of Phd by DNA Binding--
To study the effect of
DNA binding on Phd structure, increasing amounts of a 32-bp oligomer
containing the phd operator site were added to a solution
containing Phd, and CD spectra were taken (Fig.
2A). DNA binding induces a
dose-dependent increase in Phd structure, suggesting that
the folding and site-specific DNA binding of Phd are coupled. The
ellipticity at 228 nm (a wavelength at which B-DNA does not contribute
to the CD signal, see Fig. 2B) is plotted as a function of
DNA concentration in Fig. 2C. The slope of the initial part
of the titration curve shows that the amount of operator DNA needed for
induction of fully folded Phd is roughly one-quarter of the protein
concentration, suggesting that Phd binds to its operator DNA as a
tetramer. When nonspecific DNA (a 27-bp DNA oligomer containing the
arc operator (22)) was added as a control, no increase in
the CD signal of Phd at 228 nm was observed over the range of DNA
concentrations tested (Fig. 2C).

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Fig. 2.
Induction of Phd structure by DNA
binding. A, CD spectra of 700 nM Phd in the
presence of 83, 180, or 330 nM phd operator DNA.
B, spectral contribution of phd operator DNA
alone. C, Phd ellipticity at 228 nm (where DNA does not
contribute to the CD signal) as a function of DNA concentration.
Squares, phd operator; triangles,
arc operator. The line is best fit of the first
six points for the Phd operator (R2 = 0.99).
|
|
Thermal denaturation of Phd was performed in the presence of increasing
amounts of the phd operator (Fig.
3). The Tm of Phd
increases as a function of the concentration of operator DNA (Fig.
3B), demonstrating stabilization of the native Phd structure by DNA binding. No significant change in the Tm of
Phd was observed when nonspecific DNA was added at concentrations up to
500 nM.

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Fig. 3.
Stabilization of Phd by operator DNA
binding. A, thermal denaturation of 700 nM
Phd by itself and with 160 nM phd operator DNA.
B, Tm of Phd as a function of the
concentration of phd operator DNA or nonspecific DNA
(arc operator).
|
|
Stoichiometry of Phd Binding to Operator Subsites--
To
determine the oligomeric form of Phd that binds to an operator subsite,
increasing amounts of Phd were added to a 1 µM solution
of left subsite DNA, and binding was assayed by a gel-mobility-shift assay (Fig. 4A). A single
bound complex was observed. At the DNA concentration used for this
experiment, almost all of the added Phd becomes DNA-bound (see below),
and the stoichiometry of binding can be calculated from the slope of
the rising portion of the titration curve (Fig. 4B). In the
experiment shown, this slope is 0.52, indicating that Phd binds to an
operator subsite as a dimer. This value is consistent with the finding
that four molecules of Phd are stabilized by binding to the intact
operator, which contains two subsites. Because Phd in solution is an
equilibrium mixture of folded and unfolded monomers, the subsite DNA
binding reaction involves both folding and dimerization of the
protein.

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Fig. 4.
Stoichiometry of Phd binding to subsite
DNA. A, increasing amounts of Phd (0.5, 1, 1.5, 2, 2.5, and 3 µM) were added to 1 µM unlabeled left
operator subsite DNA with 0.01 µM radiolabeled tracer,
the complexes were run on a native gel, and bound complex was
quantified using a PhosphorImager. B, plot of fraction
subsite DNA bound in A as a function of total Phd
concentration. Linear regression of the first five points gives a slope
of 0.502 (R2 = 0.99), indicating that Phd binds
as a dimer.
|
|
Equilibrium Binding of Phd to Operator DNA--
Fig.
5, A and B, show a
gel-mobility-shift assay of Phd binding to DNA fragments containing the
left and the right operator subsites, respectively. The assays were
performed using a DNA concentration low enough (~10 pM)
to allow the reaction mechanism to be deduced from the dependence of
binding on Phd concentration and an equilibrium dissociation constant
to be calculated. Two observations suggest that two Phd monomers bind
cooperatively to subsite DNA. First, only free DNA and the dimer-bound
species are observed as populated species; no intermediate band
corresponding to a 1:1 complex is evident in the gel-mobility-shift
assay (Fig. 5, A and B). Second, a Scatchard plot
of the binding data is concave downward as expected for a reaction
displaying positive cooperativity (Fig. 5C). Fits of this
binding data reveal that subsite DNA binding shows an approximate
second-order dependence on Phd concentration as expected for the
model,
where P represents a mixture of folded and unfolded Phd monomers
and S represents subsite DNA. At half-maximal binding,
Kd = [P]2 or about
6·10
16 M2 for the left subsite
and 1·10
14 M2 for the right
subsite binding experiments shown in Fig. 5.

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Fig. 5.
Equilibrium binding of Phd to subsites
DNA. Gel-mobility-shift assays of Phd binding to DNA fragments
containing the left (L) operator subsite (A) or
the right (R) operator subsite (B). C,
Scatchard plot (bound DNA in arbitrary Phosphor Imager units) of Phd
binding to the left and right operator subsites (~10 pM)
at room temperature in 50 mM NaCl, 100 mM
Tris-HCl (pH 7.4), 1 mM EDTA, 100 µg/ml bovine serum
albumin, and 0.02% Nonidet P-40. Designation: circles, left
subsite; squares, right subsite.
|
|
Gel-mobility-shift assays were also used to assay Phd binding to the
intact operator site (Fig. 6). In these
experiments, both Phd dimer-bound and tetramer-bound DNA species are
observed in the gel. The dimer-bound species are observed at lower Phd concentrations than the tetramer-bound species but continue to persist
as populated intermediates as the Phd concentration is raised.
Moreover, binding to the full-site DNA is observed over the same
general range of Phd concentrations as binding to the left subsite DNA
(compare Figs. 5A and 6A). Both of these
observations suggest that there is little, if any, mutual stabilization
between Phd dimers bound at adjacent operator subsites. Indeed, the
full-site binding curve can be fit by independent binding of Phd to the isolated left and right operator subsites (Fig. 6B).

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Fig. 6.
Equilibrium binding of Phd to intact operator
DNA. Gel-mobility-shift assay (A) and binding curve
(B) of Phd binding to the intact phd operator
(~10 pM). In B, the binding curve of Phd to
left (L) and right (R) operator subsites DNA is
also shown. Assay conditions were the same as described in the legend
to Fig. 5. The curves were fitted to a second-order binding isotherm:
fraction bound = max (P2/(P2 + Kd)), where max is the maximal binding value, P is
the free concentration of Phd, and Kd is the
equilibrium dissociation constant. Designation: circles,
left subsite; squares, right subsite; triangles,
full site.
|
|
Using the gel-mobility-shift assay, we attempted to measure the
dissociation kinetics of complexes of Phd with the left operator subsite and with intact operator DNA. In both cases, however, dissociation was complete by the first time point. These experiments allow us to place an upper limit of approximately 10 s on the half-live of these Phd-DNA complexes.
 |
DISCUSSION |
In the studies reported here, the Phd protein of the plasmid
addiction module of phage P1 was purified to homogeneity and assays
probing structural stability and DNA binding were performed. Phd has
the CD spectrum of a predominantly denatured protein at 37 °C, and
only folds at this temperature when bound to operator DNA, or when
stabilized by high concentrations of osmolytes. In its native state,
Phd has a CD spectrum of a protein that is roughly 45%
-helical.
Because denatured proteins are generally subject to proteolytic
destruction in E. coli (23), it seems likely that the
thermal instability of free Phd contributes to its degradation in the
cell (5). In a cell containing the phage P1 genome, free Phd should be
unfolded and sensitive to degradation, whereas operator-bound Phd
should be folded and less susceptible to degradation. Moreover, the
continual synthesis of new Phd maintains sufficient steady-state levels
of Phd to inhibit the toxicity of Doc. In a cell that has lost the P1
genome, there is no operator to bind, new Phd synthesis cannot occur,
and the degradation of free Phd eventually leads to Doc-mediated cell
death (5).
The phd operator overlaps the promoter for Phd and Doc
transcription, and Phd represses this operon and its own synthesis (12). This ensures that when Phd levels in a P1 lysogen fall too low,
transcription is derepressed and new synthesis of Phd can occur. Our
studies show that two Phd monomers bind cooperatively to each left and
right operator subsite. As a consequence, operator occupancy changes as
a function of the second power of the Phd concentration, and the system
is quite responsive to moderate changes in steady-state Phd levels. For
example, a 5-fold drop in Phd concentration would cause a 25-fold
increase in the ratio of free to bound operator. Although binding of
Phd to operator occurs in the 10 nM range, the half-life of
the protein-DNA complex appears to be less than 10 s. Such a rapid
dissociation rate might ensure a dynamic system capable of responding
rapidly to sudden decreases in Phd levels. If the half-life of the
Phd-operator complex were too long, a precipitous drop in Phd levels
might result in Doc toxicity before transcription could be derepressed to permit new synthesis.
Phd shares some sequence similarities with the phage P22 Arc repressor
and additional members of the ribbon-helix-helix family of
transcription factors and also has a DNA recognition site whose overall
size and organization is similar to the operators of many of these
ribbon-helix-helix proteins (12). Some of the findings here extend
these similarities. For example, Phd binds to each operator subsite as
a dimer and to the intact operator as a tetramer in a fashion analogous
to Arc repressor (24, 14). However, our studies show that Phd differs,
in one very important way, from Arc and its relatives. The intertwined
nature of the native Arc dimer permits folding only as a dimer and
dissociation of Arc dimers leads to denaturation (25). Phd, by
contrast, is capable of folding as a monomer. This difference between
Phd and Arc indicates that the native folds of these two proteins must be significantly different.
Another difference between Arc and Phd involves the presence or absence
of dimer-dimer cooperativity in binding to their respective intact
operators. Phd dimers bound to adjacent operator subsites do not appear
to be mutually stabilizing and still dissociate rapidly, whereas Arc
dimers bind in a highly cooperative fashion and stabilize a complex
with a half-life in excess of 1 h (26). As discussed above,
however, the short half-life of the Phd-operator complex may be
functionally important in allowing rapid transcriptional responses to
decreases in Phd concentration.
The P1 plasmid addiction system raises a number of intriguing
physiological issues about mechanism and bacterial cell death and also
provides a model system to study the interplay of protein folding,
protein degradation, protein-protein interactions, and protein-DNA
interactions. The studies reported here should provide a basis for
future biochemical, biophysical, and structural studies of many of
these issues.
 |
ACKNOWLEDGEMENTS |
We thank Roy Magnuson and Michael Yarmolinsky
for a gift of the pHAL20 plasmid and Carl Pabo and Tania Baker for use
of equipment.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants AI-15706 and AI-16892.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by postdoctoral fellowships from the European Molecular
Biology Organization and the Human Frontiers Science Foundation Program.
§
To whom correspondence should be addressed: MIT 68-571, Cambridge, MA 02139. E-mail: bobsauer{at}mit.edu.
The abbreviation used is:
bp, base pair(s).
 |
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