(Received for publication, September 23, 1994; and in revised form, November 8, 1994)
From the
The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed
with cAMP (CRPcAMP). In addition, glucose stimulates pts operon expression in vivo. The pts promoter
region has a fructose repressor (FruR)-binding site (the FruR box) that
partially overlaps with one of the CRP
cAMP-binding sites. The
effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in
vivo glucose effect on pts operon expression is mediated
by FruR. In vitro, FruR can repress P1b transcription, which is activated by CRP
cAMP, and restore P1a transcription, which is repressed by CRP
cAMP. FruR
can displace CRP
cAMP from its binding site in the presence of RNA
polymerase even though FruR and CRP
cAMP can bind simultaneously
to their partially overlapping binding sites in the absence of RNA
polymerase. FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by
glucose. Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the
presence of fructose or in fruR
mutant cells
as in cells grown in the presence of glucose. These results suggest
that FruR alone does not mediate the in vivo glucose effect on pts operon expression.
The phosphoenolpyruvate:carbohydrate phosphotransferase system
(PTS) ()catalyzes the phosphorylation and transport of its
sugar substrates and acts as a major signal transduction system in
bacterial cells(1, 2, 3) . The PTS also
regulates the transport and metabolism of a variety of non-PTS sugars
as well as the activity of adenylate
cyclase(2, 4, 5) . In Escherichia
coli, the first two steps of the PTS phosphoryl transfer chain are
catalyzed by two general cytoplasmic proteins, Enzyme I, encoded by the ptsI gene, and HPr, encoded by the ptsH gene. In
addition, PTS sugar-specific membrane complexes are needed for the
transport of individual PTS sugars. Enzyme IIA
, a protein
needed for the transport of glucose, is encoded by the crr gene. The ptsH, ptsI, and crr genes
constitute the pts operon, located at 52 min on the E.
coli chromosome(6, 7, 8, 9) .
The pts operon is regulated in a complex fashion by at
least five promoters, P1a, P1b, P0a, P0b, and Px, which cluster into two groups, P1 and P0 (see Fig. 1)(10) . P1a, P1b, P0a, and P0b are
-RNA polymerase-dependent. P1a and P1b partially overlap and initiate transcription in vitro from positions +1 and +8, respectively. P1a and P1b are regulated inversely by CRP
cAMP: the
CRP
cAMP complex stimulates P1b and inhibits P1a (see Fig. 1B). P1a is active only when
the template is supercoiled. P0a initiates transcription at
position -100 and also needs supercoiled DNA as template. P0a is marginally stimulated by CRP
cAMP. When the template is
linear, transcriptional initiation switches from P0a to P0b, 3 base pairs farther upstream from P0a, and
becomes more CRP
cAMP-dependent. Px is recognized by
-RNA polymerase and initiates transcription from
position -94(10) . The regulatory circuits of pts promoters are summarized in Fig. 1B.
Figure 1:
A, nucleotide sequence of the
promoter region of the pts operon. The last codon of the cysK gene is boxed with an asterisk, and the
first codon of the ptsH gene is boxed. The -10
and -35 regions of the P0a and P1a promoters
are underlined. The -10 and -35 regions of the P0b and P1b promoters are overlined. The
transcription start site of each promoter is boxed with an arrow. The CRP0 and CRP1 sites are boxed and labeled.
The FruR-binding site is labeled as FruR box. B,
schematic diagram showing the regulatory circuit of promoters of the pts operon by CRPcAMP and FruR. Drawing is not to scale.
All numbering is relative to the transcription initiation site of P1b. +, activation; -,
repression.
pts operon expression is regulated both by CRPcAMP and glucose in vivo(7, 11, 12) . Both
CRP
cAMP and glucose have stimulatory effects on pts expression in vivo. Transcription increases in the
presence of CRP
cAMP as well as glucose(10, 12) .
The glucose-mediated and the cAMP-mediated activation occur
independently of each other(7, 10, 12) , even
though glucose lowers the level of cAMP in the
cell(4, 5) . There is no P0 expression in the
absence of CRP or cAMP in the cell, unless glucose is present. P1b expression needs both CRP
cAMP and glucose in the
cell(10) . These observations suggest the presence of a
repressor that is sensitive to glucose.
The fructose repressor,
FruR, plays a pleiotropic role in the transcriptional regulation of
many genes encoding enzymes of carbon and energy
metabolism(13, 14, 15, 16) . For
example, the expression of many enzymes including phosphoenolpyruvate
synthase, phosphoenolpyruvate carboxykinase,
fructose-1,6-diphosphatase, isocitrate lyase, and malate synthase is
activated by FruR. In addition, FruR represses the expression of the
fructose (fru) operon, which encodes the diphosphoryl transfer
protein, fructose-1-phosphate kinase, and Enzyme II. The
synthesis of some glycolytic enzymes is also subject to repression.
FruR consists of 334 amino acid residues and contains an amino-terminal helix-turn-helix DNA-binding motif, similar to other members of the GalR-LacI family of proteins(15, 16, 17, 18, 19, 20) . The fruR genes of E. coli and Salmonella typhimurium show 99% sequence identity, with no sequence differences in the proposed helix-turn-helix DNA-binding motif(15, 19) . Ramseier et al.(16) have identified many FruR-binding sites in E. coli and S. typhimurium, one of which is in the pts operon. In the pts operon, the center of the FruR-binding site is between the two promoter clusters P1 and P0, centered at 55.5 bp upstream from the P1b start site. The FruR-binding site partially overlaps the CRP1-binding site, which is centered at 42.5 bp upstream from P1b (see Fig. 1).
In this work, we studied the effect of FruR on pts operon expression in order to determine if FruR mediates the positive glucose effect on pts operon expression observed in vivo. We investigated the effect of FruR on transcription of the pts operon both in vitro and in vivo. We found that FruR repressed P1b transcription. However, FruR had almost no effect on the activity of the P0 promoter, which is regulated by glucose in vivo(10, 12, 21) . The significance of our findings is discussed.
Figure 5:
DNase I protection of pts DNA by
FruR (80 nM), CRP (40 nM), and RNA polymerase (20
nM). The supercoiled DNA was treated with DNase I in the
presence of various combinations of proteins as indicated at the top.
Each strand of treated DNA was probed with two different P-end-labeled primers. Numbering is relative to the P1a transcription initiation site. The results are summarized
on C. R (
), DNA region protected by RNA
polymerase; F (&cjs2113;), DNA region protected by FruR; C (&cjs2112;), DNA region protected by CRP
cAMP; CR (&cjs2108;), region protected by CRP
cAMP and RNA polymerase; FC (
), DNA region protected by FruR and CRP
cAMP; FCR (&cjs2110;), DNA region protected by FruR, CRP
cAMP,
and RNA polymerase;
, hypersensitive
site.
Figure 2:
Effects of FruR on the activity of the pts promoter in vitro. The plasmid pHX, which has
both P0 and P1 promoters, was used. A 85-base
transcript from P1a and a 78-base transcript from P1b are indicated as P1a and P1b, respectively. The
185- and 183-base transcripts from P0a and P0b were
not separated from each other well on this 8% sequencing gel and are
marked as P0. The transcripts from plasmid origin of
replication (106/107 bases) are marked as rep. Lane1, no CRPcAMP; lanes 2-7, 40 nM CRP
cAMP with the following concentrations of FruR,
respectively: 0, 5, 10, 20, 40, and 80 nM. A,
supercoiled DNA templates used for in vitro transcription
assay; B, linear DNA used for in vitro transcription
assay; C, scanning results of A; D, scanning
results of B. C and D cannot be compared
directly because rep activity used as an internal control
varies with the superhelical density of template
DNA.
Fructose 1-phosphate inhibits FruR binding to the fruB regulatory region in vitro and thus has been identified as the inducer of the fructose operon(16) . Induction of transcription by fructose 1-phosphate by neutralization of the FruR-mediated repression of the P1b promoter was tested by comparing the effects of fructose 1-phosphate and fructose 6-phosphate on FruR in a transcription assay (Fig. 3). Fructose 1-phosphate at 1 mM inhibited FruR repression of P1b almost completely, while fructose 6-phosphate had no effect under these conditions. The slight inhibitory effect of fructose 6-phosphate at 100 mM may be due to contaminating fructose 1-phosphate in the preparation of fructose 6-phosphate.
Figure 3:
Effects
of fructose 1-phosphate and fructose 6-phosphate on FruR function in in vitro transcription assay. 40 nM FruR was used as
explained under ``Experimental Procedures,'' and the results
were quantitated with an AMBIS
-scanner.
The FruR-binding site resides at position -55.5
from the transcription start site of P1b and partially
overlaps the CRP-binding site of P1 (Fig. 1)(16) . FruR could repress P1b transcription by displacing CRPcAMP from its binding site,
thus restoring P1a activity from CRP
cAMP inhibition.
This possibility was tested by studying the binding of FruR and
CRP
cAMP to the pts promoter region by DNA band mobility
shift and DNase I protection assays.
Figure 4:
Gel shift assay using the 640-bp DNA
fragment containing the pts promoter region. D, free
DNA; CD, DNA complexed with one molecule of CRPcAMP at
the CRP0 site; C
D, DNA complexed with two
molecules of CRP
cAMP at the CRP0 and CRP1 sites; FD, DNA
complexed with FruR; FCD, DNA complexed with FruR and one
molecule of CRP
cAMP; FC
D, DNA complexed with
FruR and two molecules of CRP
cAMP. The slowest migrating species
seen when CRP concentration is high is CRP bound
nonspecifically.
Fig. 4shows that the complex containing one FruR molecule and
one CRPcAMP molecule (FCD; Fig. 4, lane8) formed at low concentrations of CRP
cAMP in the
presence of 5 nM FruR. It seems that one more CRP
cAMP
molecule could bind to the FCD complex without displacing FruR, as
shown by the formation of a FC
D band (Fig. 4, lane10). This FC
D band began to form at
lower CRP
cAMP concentrations than C
D (Fig. 4,
compare lanes3 and 9) and was sharper than
C
D. This suggests that FruR may assist CRP
cAMP
binding to its weak CRP1 site. In other words, there may be a
cooperative interaction between CRP
cAMP and FruR at this site.
FruR and CRP
cAMP seem to bind to their partially overlapping
DNA-binding sites perhaps on opposite faces without displacing each
other. Such bindings were further tested by DNase I footprinting
experiments employing supercoiled DNA because FruR, as suggested above,
may bind more tightly to supercoiled DNA, and P1a is not
active when the template DNA is linear (Fig. 2).
There were slight differences in the DNase I
digestion pattern in the P0 region around positions -115
and -135 caused by FruR when CRPcAMP and RNA polymerase
were present (Fig. 5A, compare lanes2, 4, and 6 with lane8). These differences may be responsible for the slight
repression of P0 by high concentrations of FruR as seen in Fig. 2. It is likely that a change in DNA topology caused in
part by DNA bending due to CRP
cAMP binding to the CRP0 site and
FruR binding to the FruR box has an effect on DNase I activity. Similar
changes may repress P0 transcription.
Figure 6:
Transcription from pts promoters
in wild-type (SA2600) and fruR (SR500)
strains. Total RNA was isolated from cells grown in Tryptone broth with
glucose or fructose as indicated at the top. Primer extension analysis
of 30 µg of total RNA/reaction using primers complementary to
sequence 297-326 in Fig. 1was done as described under
``Experimental Procedures.'' Lanes 1-3,
wild-type strain (SA2600); lanes4-6, strain fruR
(SR500).
FruR may
be involved in the regulation of pts expression in an
alternative way. Cells do not need to increase pts operon
expression in the presence of fructose because fructose has its own
specific HPr-like protein(13, 14) . Consistently, the P1a activity was repressed in the presence of fructose in the
wild-type strain (Fig. 6, lane3). P1a activity was, nevertheless, constitutive in a fruR strain under the same conditions (Fig. 6, lane6).
The pts operon of E. coli is regulated in a
complex fashion by at least five promoters, P1a, P1b, P0a, P0b, and Px, that cluster into two
groups, P1 and P0. Transcription from each promoter
is modulated by several factors such as CRPcAMP, superhelical
density of the DNA, and
-RNA polymerase (10) . In addition, glucose positively controls pts operon expression in vivo by an unknown
mechanism(10, 12, 21) . Our previous study on
the transcriptional regulation of pts promoters indicated the
presence of a glucose-inducible repressor(10) . Knowledge of
the mechanism of pts operon regulation by glucose is essential
for a better understanding of this complex regulatory system.
FruR is a DNA-binding protein with 334 amino acid residues and contains a typical amino-terminal helix-turn-helix DNA-binding motif. FruR plays a pleiotropic role in the transcriptional regulation of many genes encoding enzymes of carbon and energy metabolism(14, 16) . We have tested (i) whether glucose induction is mediated by FruR, whose binding site is between the two pts promoters P0 and P1(16) , and (ii) whether glucose or one of its metabolic products acts as an allosteric modifier of FruR action. We used the S. typhimurium FruR protein for in vitro studies of pts promoters of E. coli because the fruR genes of E. coli and S. typhimurium are almost identical, with 99% sequence identity(17, 19, 20) . Furthermore, there is no difference in the helix-turn-helix DNA-binding motif of the FruR proteins (20) and in the FruR-binding sites of ptsH genes from E. coli and S. typhimurium(29) . As expected, the protein from S. typhimurium has been shown to bind to the FruR-binding site of the E. coli pts promoter region(16) .
In vitro transcription results showed that FruR repressed P1b transcription, which is activated in the presence of CRPcAMP (Fig. 2). FruR had no effect on P1a transcription in
the absence of CRP
cAMP, but it could restore P1a activity, which is repressed by CRP
cAMP. FruR slightly
repressed P0 transcription at high concentrations. The
mechanism of FruR repression of P1b in vitro was studied by
DNA band mobility shift assay and DNase I footprinting experiments.
FruR may repress P1b either by displacing CRP
cAMP from
its overlapping binding site or by another unknown mechanism while
maintaining CRP
cAMP at its binding site. The weak affinity of
CRP
cAMP for the CRP1 site and the restoration of P1a transcription, which is repressed by CRP
cAMP in the presence
of FruR, support the first possibility (Fig. 2).
The DNA band
mobility shift assay, however, revealed that CRPcAMP and FruR
could bind to their overlapping binding sites simultaneously in the
absence of RNA polymerase even though CRP
cAMP bound poorly to the
CRP1 site (Fig. 4). The simultaneous binding to overlapping
regions is possible when two proteins bind to opposite faces of the
DNA. It is interesting to note that DNA band mobility shift assays
showed that CRP
cAMP binds better to the CRP1 site in the presence
of FruR. FruR binding may change the DNA conformation to one to which
CRP
cAMP binds better. Alternatively, FruR may increase the local
CRP
cAMP concentration near the binding site by directly
interacting with CRP
cAMP. An example of a protein-protein
interaction between two proteins has been reported in the deoCp2 promoter, where CytR interacts with CRP
cAMP, forming a
bridge between the two DNA-bound CRP
cAMP complexes(30) .
DNase I footprinting using supercoiled DNA was employed for further
binding experiments in which RNA polymerase was included. The DNA band
mobility shift assay in which linear DNA was used could not be used for
the binding experiment in the presence of RNA polymerase because the
affinity of FruR and the activity of pts promoters are
influenced by the superhelical density of the DNA(10) . The
DNase I digestion pattern unique to CRPcAMP binding was
maintained in the presence of FruR, in agreement with DNA band mobility
shift assay results, but was absent in the presence of both FruR and
RNA polymerase (Fig. 5). The footprinting results suggest that
FruR and CRP
cAMP bind simultaneously to their overlapping binding
sites, but FruR displaces CRP
cAMP from its binding site in the
presence of RNA polymerase. In other words, FruR represses P1b by displacing CRP
cAMP from its binding site. This model also
explains the mechanism of restoration of P1a by FruR in the
presence of CRP
cAMP.
It is not known why P1b is
poorly expressed in vivo, even in a fruR strain. The predominant P1b is activated easily by
CRP
cAMP in vitro in the absence of FruR. Perhaps growth
conditions for optimal expression of P1b have not been
achieved. The large difference in affinity between CRP
cAMP and
FruR for their overlapping binding sites may be important for the
modulation of P1a and P1b transcription in
vivo. FruR binds to its overlapping binding site much better than
CRP
cAMP to its site (Fig. 4). Cells grown in the presence
of both glucose and exogenous cAMP express P1b most, but even
under these conditions, P1b is not very active(10) . P1a is the major promoter in vivo. The main reason
for the incomplete pts promoter switch from P1a to P1b in vivo is not competition between CRP
cAMP and FruR
binding to their overlapping binding sites because the effects are
similar in a fruR
strain (Fig. 6).
We confirmed that fructose 1-phosphate is the inducer of FruR by in vitro transcription assay (Fig. 3). Fructose
1-phosphate is produced from fructose via the fructose-specific
PTS(31) . There is no known mechanism for producing fructose
1-phosphate when glucose is metabolized in the cell. It is not clear
whether fructose 1,6-bisphosphate can be converted into fructose
1-phosphate or how efficient it is as an inducer. Glucose may provide
partial inactivation of FruR, or cells may not have enough
CRPcAMP for binding to the CRP1 site even when exogenous cAMP is
provided. Cells may need a high concentration of CRP
cAMP and
inactivation of FruR for CRP
cAMP to bind to the very weak CRP1
site in order to express P1b in vivo. This is unrealistic for
normal growth conditions because the conditions for inactivation of
FruR (for example, growth in the presence of glucose or fructose)
normally depress cAMP production in the cell. Cells may not have enough
CRP for P1b expression because we could not get full P1b expression even with fruR
cells (SR500)
grown in the presence of glucose and exogenous cAMP (data not shown).
The level of CRP may be low under these conditions. crp gene
expression is autoregulated and is reduced by glucose(32) .
Note that the incomplete promoter switch in vivo was also
noted in the E. coli gal operon(33, 34) ,
which has two promoters inversely regulated by CRP
cAMP as in pts P1a and P1b. The CRP-binding site of the gal promoter is also relatively weak.
Cells may maintain a certain
level of pts operon expression by steady expression of P1a and may modulate different levels of pts expression
mainly by changing P0 activity. In support of this
speculation, we have found that P1a mRNA is more stable than P0 mRNA. ()The importance of the modulation of P1a and P1b activity by CRP
cAMP in vivo in the regulation of pts expression remains to be
studied.
De Reuse et al.(12) have found that there
is very little CRPcAMP modulation of P1 promoter
activity in vivo and that P0 is important for pts regulation by glucose in vivo, in agreement with our
present results. We found that FruR did not significantly repress P0 transcription. There was only slight repression of P0 activity at high concentrations of FruR in vitro (Fig. 2). This may be due to partial termination of
transcription through blockage of elongation by FruR bound at its
binding site
55 bp downstream from P0a(35) . It
is also possible that the change in DNA topology caused by
CRP
cAMP binding to the CRP0 site and FruR binding to the FruR box
as shown by DNase I footprinting (near positions -115 and
-135; Fig. 5A, lane8) may have
an effect on P0 transcription at different levels. In any
case, that effect was not sufficient to account for the glucose effect
on pts expression (
2.5-fold increase in pts expression) seen in vivo(12) .
Fig. 6shows that glucose primarily increased P0 expression, whereas the fruR mutation could not increase P0 transcription in vivo. Furthermore, glucose could
activate P0 transcription in a fruR strain. These results suggest that FruR does not play an
important role in the induction of pts expression by glucose.
However, fructose reduced overall pts transcription in
wild-type cells (Fig. 6, lane 3) in a FruR-dependent
fashion since that effect disappeared in a fruR
background. Interestingly, it has been shown that the activities
of HPr and Enzyme I were induced to a maximal extent of 3-fold by
either glucose or fructose(36) . These observations suggest
that FruR and fructose may have effects on pts expression by
still unknown mechanisms.
It is possible that the glucose-induced changes (37) in the superhelical density of chromosomal DNA near the pts promoters have an important function in the modulation of P0 activity. It has been shown that the activity of P0 is sensitive to the superhelical density of the DNA template in vitro(10) .