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INTRODUCTION |
It has been known for many years that induction of photosynthesis
gene expression in the anoxygenic phototroph, Rhodobacter capsulatus, is controlled by the RegB/RegA two-component
regulatory system (1-4). This circuit functions as a signal
transduction pathway that promotes synthesis of the photosynthetic
apparatus in an anaerobic environment. RegB is believed to be an
integral membrane histidine kinase that monitors the external
environment and signals for a switch to photosynthetic growth when the
oxygen concentration falls below a threshold level (~1%) (1-3).
This involves phosphorylation of the response regulator, RegA, which then stimulates the transcription of several key photosynthesis genes
(2-6). More recently, the RegB/RegA system has been implicated in the
regulation of other anaerobic processes including CO2 and N2 assimilation (7, 8). In fact, RegB/RegA homologs have now been isolated from a variety of purple bacteria including some
phototrophs, which do not alter photosynthesis gene expression in
response to oxygen, and some species that are nonphotosynthetic (9,
10). Collectively, these observations indicate that the RegB/RegA
circuit constitutes a highly conserved signal transduction system for
the global control of diverse metabolic processes.
Recently, we reported the isolation of a mutant strain of R. capsulatus (SD97*) that exhibits constitutively high
photosynthesis gene expression in the absence of a functional
regB gene (11). The mutant strain was found to contain a
RegA variant (RegA*) responsible for activation of operons (puf,
puc, and puh) that encode structural proteins of the
photosystem. DNase I footprint experiments with purified RegA*
demonstrated that it is a DNA-binding protein that interacts with
promoters for the puf and puc operons. This study
also indicated that RegA* may recognize localized features in DNA
structure rather than a specific nucleotide sequence to bind target
promoters (11). In most cases, phosphorylation of a response regulator
affects its ability to regulate gene expression. However, until
experiments are carried out in parallel with wild-type RegA, it is not
possible to determine whether RegA* activates gene expression because
it is phosphorylated in vivo by an alternative kinase(s) or
because it is a constitutively active DNA-binding protein.
To further probe the transcription activating function of RegA, we have
characterized various properties of RegA* and wild-type RegA using an
in vitro kinase assay. To facilitate this study, we
constructed a vector that overproduces a soluble form of RegB (RegB")
that lacks all of its putative transmembrane domains. We demonstrate
that purified RegB" will autophosphorylate in vitro when
incubated with ATP and that it can donate a phosphoryl group to
purified RegA or RegA*. By comparing the levels of RegA and RegA*
phosphorylation and examining how phosphorylation affected their
binding to the puc promoter, we have concluded that RegA* has an altered structural conformation that facilitates DNA binding, perhaps because it mimics the phosphorylated state of the wild-type protein. These results are consistent with the hypothesis that RegA* is
a constitutively active transcriptional regulator that does not require
phosphorylation to activate target genes.
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MATERIALS AND METHODS |
Construction of RegB" and RegA Overexpression Vectors--
We
constructed a vector that could be used to overexpress a
His6-tagged, truncated form of RegB (RegB"), which lacks
all of the putative transmembrane domains of the NH2
terminus. A DNA fragment encoding the cytoplasmic portion of
regB was amplified from the chromosome of R. capsulatus by polymerase chain reaction using the upstream primer
5'-CCATATGTCGGATGCGCTTTTCGC and the downstream primer
5'-CCTCGAGAACGATTGTGATCATCAGGC. The upstream and downstream
primers were designed to contain NdeI and XhoI restriction enzyme sites, respectively (underlined bases),
to facilitate cloning. The polymerase chain reaction product was initially cloned into the plasmid, pCRTM11 (Invitrogene),
and then subcloned into NdeI and XhoI restriction sites of the expression vector pET28a(+) (Novagen). The resulting plasmid pET28a(+):RegB" was transformed into the Escherichia
coli strain BL21(DE3) (12) to overproduce RegB" as described below.
A clone that overexpresses wild-type RegA was also constructed using
the same procedure as described previously for RegA* (11). DNA
sequences of the RegA and RegB" coding regions in both expression
constructs were confirmed using the ABI automatic sequencing system
(Perkin-Elmer).
Overexpression and Purification of RegB" and RegA--
A total
of 5 liters of Terrific Broth was innoculated with the E. coli strain BL21(DE3)/pET28a(+):RegB". The cultures were grown
with vigorous shaking at 37 °C until they reached an
A600 of 0.6, at which time
isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 1.0 mM. The cultures were grown for an
additional 3 h before the cells were harvested by centrifugation at 7600 × g at 4 °C for 10 min. The cell pellet was
washed once and then resuspended in 50 ml of 1× start buffer (2 mM HPO4 (pH 7.2), 1 M NaCl). Cells
were lysed by three passages through a chilled French pressure cell at
18,000 psi, and the resulting lysate was centrifuged at 18,000 × g for 1 h. The supernatant was filtered through a 0.45 µM Acrodisc (Gelman Sciences) and then loaded onto a 5 ml
HiTrap chelating column (Amersham Pharmacia Biotech) that had been
charged with NiSO4 and equilibrated with 1× start buffer.
The column was washed with 25 ml of 1× start buffer before RegB"
protein was eluted with 1× start buffer supplemented with 400 mM imidazole (pH 8.0). Elution fractions that contained significant amounts of RegB" protein, as determined by the Bradford assay (Bio-Rad), were pooled and dialyzed first against 20 mM HEPES (pH 8.0), 400 mM KCl, 5 mM
MgCl2, and 20% glycerol and then against 20 mM
HEPES (pH 8.0), 400 mM KCl, 5 mM
MgCl2, 2 mM dithiothreitol, and 50% glycerol.
The final RegB" concentration was determined by the
A205/A280 method described by Scopes (13), and
the protein preparation was stored at
80 °C. The procedure used to
overexpress and purify RegA and RegA* protein was the same as
previously reported (11).
Protein Phosphorylation Assays--
Protein phosphorylation
assays were conducted by incubating purified RegB", ATP/[
-32P]ATP (a typical kinase reaction contained 1.0 mM ATP and 200-400 µCi of [
-32P]ATP
(7000 Ci/mmol, ICN)), and either wild-type RegA or RegA* in 1×
footprint buffer (25 mM HEPES (pH 8.0), 150 mM
KCl, 5 mM MgCl2, 3 mM
CaCl2, 10 mM dithiothreitol, 25 µg/ml bovine
serum albumin, and 15% glycerol). The reactions were incubated at room temperature, and at various times, samples were removed, mixed with an
equal volume of 2× SDS-loading buffer, (100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol, 2% SDS, 0.1% bromphenol
blue, 10% glycerol), and placed on ice. The samples were later size fractionated by SDS-PAGE1
using a 12% gel. The 32P-labeled protein bands were
localized by autoradiography and excised from the gel, and the amount
of Cerenkov radioactivity they contained was measured by scintillation counting.
To compare the percentage of RegA/RegA* protein preparations that could
be phosphorylated, 640 pmol of RegB" in a 40-µl reaction was
incubated for 2 h at room temperature in the presence of 50 mM ATP/[
-32P]ATP in 1× footprint buffer.
The reaction was then diluted with 120 µl of 1× footprint buffer.
Samples consisting of 5 µl of the diluted kinase reaction were mixed
with an equal volume of 1× kinase buffer that contained various
amounts of either RegA or RegA*. These phosphotransfer reactions were
incubated at room temperature for 20 s before being terminated
with the addition of 10 µl of 2× SDS-loading buffer. The reactions
were then fractionated by SDS-PAGE, and the levels of RegA/RegA*
phosphorylation were quantitated as described above.
To compare the relative stabilities of RegA~P and RegA*~P,
approximately 175 pmol of RegB" was mixed with ATP/[
-32P]ATP in 1× kinase buffer in a total volume of 175 µl. The mixture was incubated at room temperature for approximately
2 h to allow RegB" to autophosphorylate. Then 50-µl samples were
removed from the reaction and mixed with an equal volume of 1×
footprint buffer that contained no protein, RegA, or RegA* (the final
concentrations of RegB", RegA, or RegA* in these mixtures was 5 µM). At various times, 10-µl samples were removed from
each of the three reactions and mixed with an equal volume of 2× SDS
loading buffer and then placed on ice. These samples were later
fractionated by SDS-PAGE, and the level of RegB", RegA, or RegA*
phosphorylation was quantified as described above.
DNase I Footprint Experiments--
A 180-base pair DNA fragment
that encompassed the puc promoter region was amplified by
polymerase chain reaction and purified as described previously (14).
The polymerase chain reaction product was stored in 25 mM
HEPES (pH 8.0), 50 mM NaCOOH, and 0.1 mM EDTA.
To prepare RegA~P/RegA*~P for use in footprint reactions, RegB" was
incubated at room temperature for approximately 2 h in the
presence of 1.0 mM ATP. RegA/RegA* protein was then added to the kinase reaction and incubated for 5 min before serial dilutions were made with 1× footprint buffer to obtain various
RegA~P/RegA*~P concentrations. 10-µl samples were removed from
each and mixed with approximately 5-10 pmol of a
32P-labeled DNA fragment in 1× footprint buffer at a total
volume of 20 µl. These DNA-binding reactions were incubated for 20 min at room temperature before a limited digestion was initiated with the addition of 5 µl of DNase I (400 ng/ml). After 5 min, the digestions were quenched with 180 µl of stop buffer (0.33 M NH4COOH, 55 mM EDTA, and 14 µg/ml yeast tRNA). The reactions were phenol-extracted once, ethanol
precipitated, and resuspended in 3 µl of formamide-loading buffer
comprised of 80% deionized formamide, 1× Tris-borate-EDTA buffer,
0.1% bromphenol blue, and 0.1% xylene cyanol FF. The recovery of
32P-labeled DNA for each reaction was measured by
scintillation counting. Samples that represented equal amounts of probe
were heated to 90 °C for 3 min and resolved by electrophoresis
through 8% urea-polyacrylamide gels made with Long
RangerTM acrylamide solution (FMC Bioproducts). The dried
gels were exposed to x-ray film for 7-10 days. The effective
concentration for 50% response (EC50) for RegA and RegA*
was calculated by comparing the extent of DNase I protection at various
protein concentrations using a PhosphorImager (Molecular Dynamics
Co.).
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RESULTS |
Characterization of RegB" Autophosphorylation--
In previous
studies, difficulties were encountered in the purification of RegB
because overproduced protein formed insoluble inclusion bodies (6). To
avoid this problem, we constructed a new vector, which expresses only
the cytoplasmic portion of RegB (RegB") fused to a "His tag" at its
NH2 terminus. SDS-PAGE analysis of cell lysates made from
cultures that expressed His-RegB" (hereafter called RegB") revealed
that a significant proportion (>50%) of the polypeptide was soluble.
The protein was subsequently purified to homogeneity by nickel column
chromatography (see "Materials and Methods").
Autophosphorylation of RegB" was assayed by incubating the purified
protein with [
-32P]ATP at room temperature. At various
times, samples were removed from the reaction, mixed with a denaturing
dye solution, and then chilled on ice. After fractionation by SDS-PAGE,
the level of RegB" phosphorylation was measured and the data plotted as
a function of time (Fig. 1). Typically,
we observed that the proportion of phosphorylated RegB" increased for
approximately 2 h before the reaction appeared to equilibrate. We
consistently observed that autophosphorylation reactions plateaued when
30-40% of the protein had become phosphorylated, indicating that a
significant fraction of the RegB" preparation was biologically active.
A 10-fold increase in ATP concentration did not significantly affect
the kinetics of autophosphorylation or the final level at equilibration
(data not shown). Therefore, the rate-limiting step in RegB"
autophosphorylation does not appear to be binding of ATP. Rather, the
rate may be limited by the ability of RegB" to catalyze the transfer of
phosphate from bound ATP to its histidine phosphoacceptor site or
possibly by the formation of RegB" dimers required for
autophosphorylation to commence.

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Fig. 1.
Autophosphorylation of RegB". An
autophosphorylation reaction was initiated by mixing purified RegB" and
32P-labeled ATP. At various time points indicated above the
blots in A (min), the samples were removed and later
fractionated by SDS-PAGE. The 32P-labeled proteins were
detected by autoradiography (A). The level of RegB"
phosphorylation in these protein bands was quantified (see "Materials
and Methods") and plotted as a function of time (B).
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RegA* Is Phosphorylated to a Greater Extent than Wild-type
RegA--
We examined the ability of RegB"~P to function as a
substrate for the transfer of phosphate to RegA or RegA* by conducting kinase assays that contained mixtures of these proteins. Samples were
removed and stopped at various times after the reactions were initiated
by the addition of [
-32P]ATP. The levels of RegA/RegA*
phosphorylation in the samples were measured following SDS-PAGE (Fig.
2). In repeated experiments, we observed
that phosphorylation of wild-type RegA increased for approximately
2 h after the addition of ATP. After this time, RegA
phosphorylation usually plateaued at a level of 5-10%. In contrast to
wild-type RegA, reactions with RegA* did not plateau, even when
incubated as long as 4 h (Fig. 2). Instead, RegA*~P continued to
accumulate so that 20-30% of the protein had become phosphorylated by
the end of the time course.

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Fig. 2.
Phosphorylation of RegA and RegA*.
Kinase reactions were initiated by mixing RegB",
32P-labeled ATP, and either RegA or RegA* with samples
removed at various time intervals (min), which were subsequently
fractionated by SDS-PAGE. A and B show
autoradiographic detection of phosphorylation of RegA and RegA*,
respectively. C, the levels of phosphorylation of RegA ( )
and RegA* ( ) as plotted as a function of time.
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Despite differences in the extent of phosphorylation, we consistently
observed that initial rates of phosphorylation were very similar for
both RegA and RegA* suggesting that autophosphorylation of RegB" might
be a rate-limiting step in the process. To test this possibility, we
attempted to measure the rate of phosphotransfer from RegB"~P to RegA
or RegA*. This experiment involved incubating RegB" with
[
-32P]ATP for 2 h to allow the formation of
RegB"~P and then adding RegA or RegA* to the reaction. At various
times samples were removed, and the extent of phosphotransfer was
measured as before. In this analysis, we observed that phosphotransfer
from RegB"~P to RegA or RegA* was complete before the first sample
was removed (10 s time point) from the reaction (data not shown).
Although the rate of phosphotransfer was too fast to measure using this
method, the experiment confirmed that the rate-limiting step for
RegA/RegA* phosphorylation occurs during RegB" autophosphorylation.
The Phosphoryl Group in RegA*~P Is Significantly More Stable than
That of RegA~P--
We interpreted the observation that a greater
proportion of RegA* became phosphorylated than wild-type RegA as
indicating one of two possibilities. The active fraction of the
wild-type protein preparation might be much lower than for RegA*.
Alternatively, the phosphoaspartate bond might be more stable in
RegA*~P than RegA~P. If so, it could have the effect of increasing
the equilibrium level of RegA*~P over that of the wild-type protein.
We attempted to measure the percent active fraction of RegA and RegA*
protein preparations by assaying DNA binding activity. However, these
experiments did not yield consistent results, probably because
unphosphorylated RegA has an extremely low affinity for its DNA
substrate. To overcome this problem, we measured the saturating ability
of RegA and RegA* to accept phosphate from a constant amount of
preformed RegB"~P. Several reports have concluded that the response
regulator, rather than the histidine kinase, is responsible for
catalyzing phosphotransfer (15-17). Consequently, a measure of
phosphotransfer activity provided a method of comparing the relative
proportions of active protein in RegA and RegA* preparations. The
experiment determined the amount of RegA or RegA* needed for maximal
phosphotransfer by mixing a constant amount of RegB"~P with various
amounts of either RegA or RegA*. Phosphotransfer reactions were
terminated 2 min after mixing the proteins, and levels of RegA~P or
RegA*~P were again measured after fractionation by SDS-PAGE. As
indicated in Fig. 3, maximal transfer of
phosphoryl groups from RegB"~P occurred at similar inputs of RegA and
RegA* (~50 pM). These data indicate that the percent
active fractions for RegA and RegA* preparations are reasonably similar
and, thus, cannot account for large differences in the extent of RegA
or RegA* phosphorylation observed for the time course experiments described earlier.

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Fig. 3.
Titration of a constant amount of RegB"~P
with various amounts of RegA or RegA*. A constant amount of RegB"
(20 pmol) that was taken from an equilibrated autophosphorylation
reaction was mixed with various amounts of either RegA or RegA*. After
20 s, these phosphotransfer reactions were quenched; the reactions
were fractionated by SDS-PAGE, and the amount of RegA/RegA*
phosphorylation for each reaction was quantitated. The results are
plotted as a function of the amount of RegA ( ) or RegA* ( ) added
to each phosphotransfer reaction.
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Next we examined the relative stability of the phosphate group on
RegA~P and RegA*~P by initiating a single round of phosphotransfer and then measuring the decay to RegA-OH or RegA*-OH. The assays were
initiated by mixing 32P-labeled RegB" with a large excess
of unlabeled ATP and either RegA or RegA* to allow rapid transfer of
labeled phosphate to the response regulator while masking subsequent
autophosphorylation or phosphotransfers. At various times, samples were
removed and quenched, and the level of RegA~P or RegA*~P that
remained was measured. (Previous experiments had determined that when
denatured and kept on ice, RegA~P/RegA*~P was stable for several
hours, indicating that little additional loss of phosphate occurred in the interval between quenching reaction samples and separation by
SDS-PAGE (data not shown). Fig. 4 shows
results obtained from one experiment that demonstrates an obvious
difference in RegA~P and RegA*~P stability. Whereas the amount of
RegA*~P was unchanged over a period of 4 h, the percentage of
RegA~P that remained after the same time period was 6-fold lower than
the original level.

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Fig. 4.
Relative stabilities of RegA~P and
RegA*~P. Samples from an equilibrated RegB" autophosphorylation
reaction were mixed with an equal volume of either RegA or RegA* in
buffer that contained a large excess of unlabeled ATP. Samples were
removed from the phosphotransfer reactions at various times and were
later fractionated by SDS-PAGE. The concentrations of phosphorylated
RegA ( ) or RegA*~P ( ) were measured for each sample and the
results are plotted as a function of time.
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RegB Functions as a Phosphatase for RegA~P--
Instability of
the phosphate in wild-type RegA~P could be a consequence of inherent
phosphatase activity that is present in the wild-type but not the
mutant RegA. Alternatively, RegB"-OH that is present in the kinase
reactions may be able to remove phosphate from RegA~P but not from
RegA*~P. To test these two possibilities we set up rapid RegB"~P to
RegA phosphotransfer reactions as described above with the exception
that various amounts of RegB"-OH were added to the reactions at the
initial round of phosphotransfer. If stability of the phosphate was
dependent on an inherent phosphatase activity of RegA, then the
stability would not be affected by the level of RegB"-OH. However, if
RegB"-OH was also functioning as a phosphatase, then the half-life of
phosphate on wild-type RegA would be dependent on the level of
RegB"-OH. The results of this experiment indicate that the half-life of phosphorylated RegA~P is 120 min at a 4:1 ratio of RegA to RegB, 60 min at a 1:1 ratio, and 13 min at a 1:4 ratio. This indicates that
increased stability of phosphate with RegA* is a consequence of reduced
dephosphorylation by RegB.
DNA Binding Affinities of RegA and RegA*--
Recently, we
reported that purified RegA* bound to promoter regions of the
puf and puc operons (11). Because phosphorylation has been reported to increase the DNA binding affinity of various response regulators, we decided to test whether phosphorylation affected this activity for wild-type RegA and RegA*. For this analysis
we performed DNase I protection assays on the puc promoter region using various amounts of RegA, RegA~P, RegA*, or RegA*~P. To
obtain roughly equivalent levels of RegA or RegA* phosphorylation, samples from equilibrated RegB" autophosphorylation reactions were
mixed with either RegA or RegA*. The mixtures were incubated for a few
minutes to facilitate phosphotransfer before serial dilutions were
prepared and added to the footprint assays. Typically we observed that
15-25% of the RegA/RegA* protein were phosphorylated in kinase
reactions that were conducted in this way. To test the effect of
unphosphorylated protein, serial dilutions of RegA or RegA* were added
directly to footprint assays.
As indicated by Fig. 5, RegA and RegA*
protected identical regions of the puc promoter from DNase I
digestion. This site is the same as we previously reported for RegA*
binding to the puc promoter (11). The only apparent
differences in these experiments was the amount of each protein
required to obtain a similar extent of DNase I protection (note in Fig.
5 that different ranges of protein concentration were used in testing
the various forms of RegA and RegA*).

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Fig. 5.
DNase I protection assay for the
puc promoter. Samples containing various pmol
amounts of RegA, RegA*, RegA~P, or RegA*~P (see "Materials and
Methods") were incubated with a DNA fragment containing the
puc promoter, in which the top strand had been
32P-labeled prior to a limited DNase I digestion. The
region of protection and the formation of two hypersensitive sites by
unphosphorylated RegA is indicated by lines and two
asterisks, respectively, on the left. Protection and
formation of a single hypersensitive site by RegA*, RegA~P, and
RegA*~P are indicated by lines and an asterisk,
respectively, on the right. For comparative analysis of DNA
binding affinities, the figure is from a single autoradiography of a
single gel containing footprint reactions that utilized the same DNA
probe.
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In the case of unphosphorylated wild-type RegA, a protein concentration
of 16 µM was required to obtain half-maximal protection. Even at the highest RegA concentrations, binding to the puc
promoter was very weak and is observed by DNase I hypersensitivity at
positions
56 and
57 rather than nuclease protection.
Phosphorylation of RegA to a level of 20% substantially improved its
affinity for DNA as demonstrated by significant nuclease protection
observable between 0.5 and 2 µM of phosphorylated RegA.
Analysis of half-maximal protection by PhosphorImager analysis
indicates that phosphorylation increased RegA binding by approximately
16-fold.
In the case of RegA*, the concentration of unphosphorylated protein
required for half-maximal DNase I protection was slightly less
(2.5-fold) than that observed with phosphorylated RegA. Phosphorylation of RegA* provided a further 6-fold increase in binding resulting in the
highest binding affinity as measured by half-maximal protection (0.8 µM). A comparison of the apparent binding affinities of
these proteins thus indicates that there is a 15-20-fold difference in
DNA binding affinity between unphosphorylated RegA and RegA*~P with
the overall order of affinity being RegA
RegA* < RegA~P < RegA*~P.
In addition to differences in binding efficiency, there is an apparent
qualitative difference among the type of DNA-protein complexes produced
by unphosphorylated wild-type RegA and other forms of the protein. This
difference is indicated by a change in the DNase I hypersensitivity of
protection patterns observed for RegA~P, RegA*, and RegA*~P. These
patterns do not exhibit hypersensitivity at position
56 as seen in
the RegA footprint, suggesting that the structure of a wild-type
RegA-DNA complex may be slightly altered when the protein is not phosphorylated.
Collectively, the DNase I footprint data indicate that phosphorylation
significantly increases DNA binding activity for wild-type RegA. The
similarity in the apparent binding affinities of RegA~P and RegA*
suggests that the mutant protein probably does not require phosphorylation to activate photosynthesis gene expression in vivo.
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DISCUSSION |
The RegB" polypeptide lacks the first 175 amino acid residues of
the wild-type protein. Despite this, it was evident in our experiments
that the truncated protein retains the ability to undergo
autophosphorylation and phosphotransfer. Therefore, the putative
transmembrane domains located in the NH2 terminus of RegB
are not essential for it to function as a kinase. Insertion of RegB
into the cytoplasmic membrane may orient the protein to enhance
dimerization, which is required for autophosphorylation and/or may be
important for the regulation of kinase activity (15). It is apparent
that the protein does not require an anaerobic environment to be
activated because we did not take steps to ensure that RegB" was kept
anaerobic during purification or in kinase assays. This suggests that
RegB activity may be inhibited by some mechanism in vivo to
prevent phosphorylation of RegA and induction of target genes under
aerobic conditions. Alternatively, the kinase and phosphatase
activities of RegB may be reciprocally regulated as observed in other
systems (15). It is possible that regulating RegB activity may involve
an interaction of the NH2 terminus of RegB with some other
protein. If so, the absence of the amino-terminal domain in RegB" might
render a constitutive kinase activity in vivo. We plan to
test this hypothesis by expressing the truncated protein in R. capsulatus.
Strain SD97* was isolated by selecting for elevated photosynthesis gene
expression in a regB null mutant grown under aerobic conditions (11). Consequently, RegA* must either be activated by a
heterologous kinase or have the ability to bind efficiently to target
promoters while unphosphorylated. Our DNase I footprint assays
demonstrate that unphosphorylated RegA* was able to bind the
puc promoter as well as RegA~P or RegA*~P. This supports
the view that RegA* is a constitutively active transcription factor that is directly responsible for abnormally high photosynthesis gene
expression. Presumably, this is because RegA* adopts a conformation that resembles the activated structure of RegA~P.
Most response regulators are composed of a receiver domain at its
NH2 terminus and an effector domain at its COOH terminus. They are believed to exist in dynamic equilibrium between at least two
different structural conformations that constitute active and inactive
states. It has been proposed that phosphorylation increases the
proportion of protein in the active conformation by disrupting
intramolecular interactions between the receiver and effector domains
(15, 18-21, 23). For some response regulators this permits the protein
to dimerize. For others, phosphorylation exposes the DNA-binding domain
(18). In either case, the ultimate effect is to increase the DNA
binding activity. The 10-fold increase in binding affinity that we
observed for the wild-type protein probably underestimates the effect
of phosphorylation in vivo where it is likely that the
proportion of RegA~P during anaerobic growth is greater than the
15-25% obtained in kinase reactions.
The effector domain of RegA is relatively short but contains an amino
acid sequence motif that is characteristic of a helix-turn-helix type
DNA-binding domain. This region is separated from the receiver domain
by a short "linker" comprised of four consecutive proline residues.
Based on the structural model for CheY and NarL, the point mutation in
RegA* (a serine for alanine substitution at position 95) is located in
the
4 region of the protein (24, 25). In fact,
constitutively active variants of other response regulators, including
Spo0A, NarL, and OmpR, also contain mutations in this region (26).
Because the
4 helix is proposed to lie in close
proximity to the linker region of RegA, we suspect that the SD97*
mutation may result in a realignment of domains that is mediated by the
stretch of prolines. This could produce a conformational change that
resembles the effect of RegA phosphorylation and result in constitutive
activation of genes within the RegA regulon.
Differences in the relative stabilities for RegA~P and RegA*~P
indicate that the two proteins have different structures. Inherent rates of phosphatase activity for different response regulators have
been found to vary widely, suggesting that they may be influenced by
protein conformation (27, 28, 30). Often these rates are enhanced
in vivo by phosphatase activity exhibited by the histidine
kinase. The observation that dephosphorylation of RegA~P is enhanced
by increasing amounts of RegB"-OH indicates that RegB" must indeed have
an inherent phosphatase activity for RegA~P. Presumably the mutant
protein assumes an altered conformation that inhibits the ability of
RegB" to bind and/or catalyze the dephosphorylation of RegA*~P.
Nothing is known about the mechanism used by RegA to activate
transcription at target promoters. Various DNA-binding response regulators have been shown to activate transcription by recruiting RNA
polymerase to the promoter or by catalyzing the formation of an open
complex between the polymerase and promoter. However, in most cases
that have been studied, phosphorylation of the response regulator does
not directly affect these activities. Instead, phosphorylation simply
influences the ability of the response regulator to bind target
promoters (18, 19, 22, 29, 32, 34-37). In the case of RegA, there is
an apparent correlation between the location of its binding sites at
the puf and puc promoters and those of additional
transcriptional regulators. For instance, puc promoter
activity is known to be under the control of the aerobic repressor,
CrtJ, which binds a palindrome that overlaps the RegA binding site for
this promoter (31). In addition, the RegA binding site for the
puf promoter is superimposed over a region of dyad symmetry
proposed to be the binding site for a transcriptional repressor that
functions under aerobic conditions (33). Thus, it appears that
aerobic/anaerobic control of the puf and puc
operons results from antagonistic interactions between RegA and various
transcriptional repressors through competition for overlapping binding
sites. If so, phosphorylation of RegA would have a direct affect on its
ability to contend with or perhaps displace DNA-bound repressors.
Consequently, the regulation of photosynthesis gene expression may then
depend on the relative affinities of multiple regulators that may each
be controlled in a redox-dependent manner. It will be
interesting to examine whether this is a common feature for additional
genes that fall within the RegB/RegA regulon.