From the Department of Food Science and Technology,
School of Agricultural Biotechnology and Center for Agricultural
Biomaterials, Seoul National University, Suwon 441-744 and
Plant
Pathology Division, National Institute of Agricultural Science and
Technology, Suwon 441-707, Korea
Received for publication, December 30, 2002, and in revised form, February 13, 2003
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ABSTRACT |
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Transcription of ptsG encoding
glucose-specific permease, enzyme IICBGlc, in
Escherichia coli is initiated from two promoters, P1 and P2. ptsG transcription is repressed by Mlc, a
glucose-inducible regulator of carbohydrate metabolism. The regulation
of ptsG P1 transcription is also under positive control by
cyclic AMP receptor protein and cyclic AMP complex (CRP·cAMP) as
observed in other Mlc regulon. We report here that Fis, one of the
nucleoid-associated proteins, plays a key role in glucose induction of
Mlc regulon. ptsG transcription was induced when wild-type
cells were grown in the presence of glucose. However, in a
fis mutant, the basal level of ptsG
transcription was higher but decreased when cells were grown in the
presence of glucose, which implies the possibility of regulatory
interactions among Fis, Mlc, and CRP·cAMP. Footprinting experiments
with various probes and transcription assays revealed that Fis assists
both Mlc repression and CRP·cAMP activation of ptsG P1
through the formation of Fis·CRP·Mlc or Fis·CRP
nucleoprotein complexes at ptsG P1 promoter depending on
the availability of glucose in the growth medium. ptsG P2
transcription was inhibited by Fis and Mlc. Tighter Mlc
repression and enhanced CRP·cAMP activation of ptsG P1 by
Fis enable cells to regulate Mlc regulon efficiently by selectively
controlling the concentration of enzyme IICBGlc that
modulates Mlc activity.
P-enolpyruvate:carbohydrate phosphotransferase
system of bacteria catalyzes concomitant uptake and
phosphorylation of sugars (1). In Escherichia coli,
glucose is translocated and phosphorylated via the membrane-bound
glucose permease, enzyme IICBGlc
(EIICBGlc),1
which is a component of glucose-specific P-enolpyruvate:carbohydrate phosphotransferase system (1). EIICBGlc is encoded by
ptsG located at 25 min of the chromosomal locus. Kimata
et al. (2) showed that functional CRP·cAMP complex is essential for the expression of ptsG, which is induced by
glucose (3-5), and that EIICBGlc is a key mediator of
diauxic growth in glucose-lactose medium and of inducer exclusion.
ptsG transcription is regulated through two global systems,
positively by CRP·cAMP and negatively by Mlc (6, 7), as observed in
all Mlc-regulated genes (8-12). Our group and two others (13-15) have
demonstrated that unphosphorylated EIICBGlc formed upon
glucose uptake can relieve the repression of Mlc regulon by
sequestering Mlc through direct protein-protein interaction (13-15).
Our group found that glucose-induced expression of ptsG is
enhanced significantly under heat shock and thus proposed that this
enhancement may counteract the highly increased mlc
expression, whereby normal glucose metabolism is maintained even at
high growth temperature (16). In addition to the regulations at the
transcriptional level, expression of ptsG is regulated
post-transcriptionally via modulation of ptsG mRNA
stability in response to glycolytic flux in the cells (17). These
observations suggest that the expression of ptsG is
regulated in a highly complex and dynamic manner in response to various
growth environments, as well as to the availability of glucose.
Several types of small DNA-binding proteins, known as
nucleoid-associated or histone-like proteins, exist in E. coli (18), among which Fis is the most abundant in exponentially
growing cells (19). Fis is known to be involved in site-specific DNA recombination and participates in the regulation of growth-related genes, including those for rRNA, tRNA, and DNA replications (20). It
also regulates genes related to the catabolism of sugars and nucleic
acids (21). The expression of fis is dependent on the growth
phase of cells; the expressed fis mRNA reaches maximum level at the early exponential phase and then decreases rapidly (22).
Fis exists at maximum over 50,000 molecules per cell, bringing about
the assumption that Fis binds to every 200 to 300 bp of E. coli chromosomal DNA (19, 22).
We found several Fis-like sites, which are homologous to the known
degenerate-consensus binding sequence of Fis upstream of ptsG gene. ptsG P1 transcription is induced by
glucose in the wild-type (wt) strain (6). However, the opposite effect
of glucose on the ptsG P1 activity in fis mutant
compared with wt raises the possibility that either Fis is involved in
Mlc-mediated repression or CRP·cAMP-dependent activation
of the promoter. We report here that E. coli can regulate
the expression of ptsG prior to the modulation of other Mlc
regulon because of the selective enhancement of
Mlc-dependent repression, as well as
CRP-dependent activation of ptsG in the presence
of Fis.
Materials--
Cyclic AMP was obtained from Sigma. RNA
polymerase saturated with Bacterial Strains and Growth Condition--
All E. coli strains used in this study are MC4100 (araD139
Plasmid Construction--
Basic cloning protocols described by
Sambrook and Russell (26) were used. PCR cloning of the ptsG
promoter was carried out using primers that have unique restriction
sites in their sequences. The clone was verified by DNA sequencing. The
supercoiled plasmid pGX12, containing the entire ptsG
promoter region, and pGX10, carrying only ptsG P1 promoter,
were made by inserting DNA fragments from base pairs Primer Extension Analysis--
Cells were grown aerobically at
37 °C overnight in LB. Overnight cultures were diluted 1:100 into
fresh LB with or without 1% glucose. Total RNA was isolated from
E. coli cells grown to mid-exponential phase
(A600 = 0.5-0.6) using TRIzol reagent
(Invitrogen). To study ptsG transcription, 50,000 cpm
of 32P-labeled primer PG1
(5'-AATTGAGAGTGCTCCTGAGTATGGGTGC-3', complementary to +74 to +102) was
coprecipitated with 30 µg of total cell RNA. Primer extension
reactions were performed as described by Ryu and Garges (27).
In Vitro Transcription Assay--
Single-round in
vitro transcription reactions were performed in a 20-µl total
volume containing 20 mM Tris acetate, pH 8.0, 3 mM magnesium acetate, 200 mM potassium
glutamate, 1 mM dithiothreitol, 1 mM ATP, 0.2 mM GTP, 0.2 mM CTP, 0.02 mM UTP, 10 µCi of [ Gel Shift Assay--
A DNA fragment covering ptsG P1
promoter region was amplified by PCR using a pair of primers, PG1 and
PGR3 (complementary to DNase I Footprinting Analysis--
DNA fragment carrying
ptsG promoter region was amplified by PCR using either
5'-end-labeled PG1 or PGR2 (5'-ATAACTTCGCCCGTCTGTTTCACATCG-3', Potassium Permanganate Reactivity Assay--
Supercoiled plasmid
DNA (2 nM), 20 nM RNA polymerase, and regulator
proteins were incubated for 20 min at 37 °C in 20 µl of transcription buffer containing 100 mM each of two
initiating nucleotides, ATP and UTP. After the addition of 2 µl of
100 mM KMnO4, the reaction mixture was
incubated for 30 s at room temperature. To terminate the reaction,
2 µl of 14 M Dimethyl Sulfate (DMS) Footprinting--
Two nM
supercoiled plasmid DNA and proteins were incubated in 40 µl of the
transcription buffer. DMS methylation was started by adding 2.5 µl of
DMS (150 mM). After incubation at 37 °C for 5 min, the
reaction was stopped by adding Fis Is Required for Both Full Repression and Activation of
ptsG--
Role of Fis on ptsG expression was examined by
analyzing the effects of fis mutation on ptsG
transcription with various genetic backgrounds using primer extension
assay. When wt was grown in the absence of glucose, only the basal
level of ptsG P1 expression was observed, whereas P1
promoter activity was highly induced in cells grown in the presence of
glucose (Fig. 1A). However, the effect of glucose on ptsG P1 expression was reversed in
the fis
It is possible that Fis also assists CRP-dependent
activation of ptsG P1, because P1 promoter activity
decreased markedly in the fis
In the case of ptsG P2 expression, the P2 promoter activity
was also induced by glucose (Fig. 1A) (7) and increased in the fis mutant (Fig. 1A, lanes 2 and
4). These results suggest that ptsG P2
transcription is inhibited by Fis, as well as Mlc, in vivo.
Fis, CRP, and Mlc Can Bind Simultaneously to ptsG P1, Forming
Nucleoprotein Complexes--
To examine whether Fis influences
ptsG expression directly, we performed DNase I footprinting
assay using ptsG promoter DNA region from
Two CRP sites have been identified at the ptsG promoter. We
observed two hypersensitive bands upon CRP·cAMP binding at CRP site I
centered at
Fis sites I and II partially overlapped Mlc site I located at RNA Polymerase Binding at ptsG P1 Promoter--
We also examined
the effect of Fis and CRP·cAMP on RNA polymerase binding to
ptsG P1 using DMS footprinting experiment. When Fis was
incubated with supercoiled pGX12 carrying both P1 and P2 promoters of
ptsG, Fis binding to site I protected guanine residues at
When Fis, CRP·cAMP, and RNA polymerase were added at various
combinations, complete protection of ptsG P1 Transcription Is Better Repressed by Mlc in the
Presence of Fis--
Influence of Fis on the Mlc repression of
ptsG P1 was studied through in vitro
transcription assay. Addition of Mlc reduced the CRP-activated P1
promoter activity to 50% of its full activity (Fig.
5A). However, the P1 activity
was repressed further to 32% when both Mlc and Fis were added to the
reaction. The effect was specific to ptsG P1, because the
repression of ptsH P0 promoter activity by Mlc was not
affected by Fis (data not shown). These findings, together with the
DNase I footprinting results, strongly suggest that more effective
repression of the ptsG P1 activity could be accomplished
through the formation of nucleoprotein complex containing Fis at
ptsG P1.
Fis Increases CRP-dependent ptsG P1 Activity--
We
also investigated the effect of Fis on CRP-dependent
transcription activation of ptsG using in vitro
transcription assay. ptsG P1 is known as a typical class II
CRP-dependent promoter (6, 7). RNA polymerase alone could
not initiate transcription from the P1 promoter, and the addition of
Fis showed no effect on the P1 transcription (Fig. 5B).
However, P1 transcription was activated in the presence of CRP·cAMP,
and Fis further increased CRP-activated transcription of P1 (Fig.
5B). However, Fis did not affect the activation of
ptsH P0 by CRP·cAMP (data not shown).
In the case of P2 promoter, 308-nucleotide transcripts were made from
the P2 promoter regardless of the CRP·cAMP presence in the reactions
(Fig. 5B). As expected from DNase I footprinting experiment,
addition of Fis repressed transcription initiation at P2 promoter,
which suggests that Fis binding at site V represses the P2 promoter activity.
Fis Enhances CRP-dependent Open Complex Formation at
ptsG P1 Promoter--
To analyze the effect of Fis on the
ptsG transcription, open complex formation at the promoter
was probed with potassium permanganate. When RNA polymerase was
incubated with supercoiled pGX12 carrying the entire ptsG
promoter region, open complex was not formed at ptsG P1
regardless of the presence of Fis in the reaction. Addition of
CRP·cAMP increased the KMnO4 reactivity at positions +2,
Fis sites I and II were sufficient to show the Fis effect on the open
complex formation at P1 promoter. Fis could enhance CRP-dependent open complex formation at ptsG P1
promoter, even when DNA containing only Fis site I and site II (pGX10)
was used as a template (Fig. 6A, compare lanes 7,
8, and 9). Similar results were obtained through
gel shift assay using DNA fragment ranging from In this report, we demonstrate that two different types of
nucleoprotein complexes containing Fis are formed at ptsG P1
promoter in response to glucose in the growth medium for optimal
regulation of ptsG transcription. Several lines of evidences
suggest that Fis assists both repression by Mlc and activation by
CRP·cAMP of the ptsG P1.
Mlc site I is located between Fis sites I and II and partially overlaps
both sites. Fis and Mlc could bind simultaneously to their binding
sites of ptsG promoter (Fig. 3), and Mlc showed better
repression of ptsG P1 in the presence of Fis (Fig. 5). It is
possible that Mlc and Fis repress ptsG P1 independently; however, their effects are additive, because we could not detect cooperative binding between Fis and Mlc to their respective binding sites (data not shown). Corepression of ptsG P1 by Fis and
Mlc is distinct from the mechanism described by Nasser et
al. (32) that one of the two divergent promoters is repressed by
either Fis or CRP.
CRP·cAMP binding to site I centered at What is the biological significance of these findings in
ptsG expression? As E. coli cells are grown in
the presence of glucose, ptsG expression is highly induced,
because Mlc action is relieved via direct interaction with
unphosphorylated EIICBGlc during the glucose transport
(13-15). However, transport of glucose into the cells results in the
reduction of both CRP and cAMP (36). Thus, the level of ptsG
expression is a sum of the two effects, i.e. derepression of
Mlc and catabolite repression upon glucose uptake. Plumbridge (7)
reported that ptsG expression in wt grown with glucose was
42% compared with that in mlc
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70, nucleotide triphosphates,
[
-32P]ATP, and [
-32P]UTP were
purchased from Amersham Biosciences. The cycle sequencing kit
was from Epicentre Technologies (Madison, WI).
argF-lacU169 rpsL150 thiA relA1 flb5301 deoC1 ptsF25 rbsR)
derivatives. To construct a fis mutant strain, SR507,
fis::Km region of SA37 strain (23) (a gift from S. Altuvia) was transferred into MC4100 by P1 transduction. SR504 (MC4100
ptsG::Cm) was made by P1 transduction of the
CmR region of ZSC112L
G strain (24) (a gift
from B. Erni). fis::Km was transferred into SR505
(MC4100 mlc::Tc) (16) and SR504, through which
SR575 (MC4100 fis
mlc
) and SR574 (MC4100
fis
ptsG
),
respectively, were constructed. All strains were grown in Luria-Bertani (LB) medium (25) aerobically at 37 °C.
305 to +132 and
75 to +132 between EcoRI and PstI sites in
front of the rpoC terminator in plasmid pSA600, respectively
(27).
-32P]UTP (800 Ci/mmol), 2 nM
supercoiled DNA template, 5-20 nM RNA polymerase, 100 µg/ml bovine serum albumin, and 5% glycerol. Regulator proteins such
as CRP, Mlc, or Fis were added to the reaction as described under
"Results." All components except nucleotides were incubated at
37 °C for 10 min. Transcription was started by the addition of
nucleotides containing 200 µg/ml of heparin and terminated after 10 min by adding 20 µl of formamide loading buffer. RNA was resolved by
electrophoresis on 6% polyacrylamide gel containing 8 M
urea. The amounts of transcripts were measured using a phosphorimage analyzer (BAS2500; Fuji Photo Film Co.).
54 to
78). PCR products were purified from
6% polyacrylamide gel as described by Sambrook and Russel (26) and
labeled with [
-32P]ATP. Purified protein(s), DNA
probe, and 50 µg/ml of poly(dI-dC)·poly(dI-dC) were mixed in the
same buffer used for in vitro transcription at a 15-µl
total volume. The binding mixture was incubated for 20 min at 37 °C.
In the reaction containing RNA polymerase, heparin (200 µg/ml) was
added and further incubated for 5 min. The reaction was analyzed by
electrophoresis on 5% polyacrylamide gel. In the reaction containing
CRP·cAMP complex, 200 µM of cAMP was also added into
the gel and the running buffer in the upper reservoir.
248 to
222). The PCR product was purified using polyacrylamide gel. Purified
protein(s) and DNA fragment were incubated in 40 µl of in
vitro transcription assay buffer. Five microliters of DNase I
solution (10 ng of DNase I per reaction) was added to the binding
mixture, which was then placed at room temperature for 1 min. DNase I
reaction was terminated by the addition of 200 µl of stop solution
containing 0.4 M sodium acetate, 10 mM EDTA,
and 100 µg/ml yeast tRNA. After phenol extraction and ethanol precipitation, the pellet was dissolved in a sequencing dye and resolved on 8% polyacrylamide gel containing 8 M urea.
-mercaptoethanol was added. The reaction
was precipitated, and the pellet was washed with 70% ethanol and
resuspended in water, which was used as a template for five cycles of
PCR with end-labeled PG1 primer (28). The PCR products were resolved on
6% sequencing gel.
-mercaptoethanol. The reaction was
precipitated with ethanol in the presence of sodium acetate and salmon
sperm DNA. The pellet was resuspended in water, which was used as a
template for five cycles of PCR as described for the potassium
permanganate assay. The PCR products were analyzed on 6% sequencing gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain. ptsG P1 expression
in fis
strain grown in the absence of glucose
was markedly increased compared with wt (Fig. 1A,
lanes 1 and 2) and was reduced in the presence of
glucose. These results suggest that Fis is required for both the
repression of ptsG P1 by Mlc and the activation of ptsG P1 under limited concentration of CRP·cAMP (6, 7). However, the interaction between Mlc and EIICBGlc was not
affected by Fis, because the P1 expression also increased in the
absence of Fis, even in ptsG mutant that cannot produce EIICBGlc (Fig. 1B, lane 2). These
observations imply that Fis is involved directly in the Mlc binding
and/or repression of ptsG P1 promoter.
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Fig. 1.
Fis effects on ptsG
expression in E. coli analyzed by primer
extension analysis. In vivo effects of Fis on
ptsG expression were studied using primer extension assay.
Thirty micrograms of total E. coli RNA, extracted at
mid-exponential growth phase, was coprecipitated and annealed with
end-labeled PG1 primer. Reactions were performed as described under
"Experimental Procedures." The products were resolved on 6%
sequencing gel. A, expression of ptsG in E. coli MC4100 strain (lanes 1 and 3) and
isogenic fis mutant strain (lanes 2 and
4) was examined. The strains in lanes 1 and
2 and lanes 3 and 4 were grown in LB
without and with 1% glucose, respectively. B,
ptsG P1 expression was monitored at various mutation
backgrounds. ptsG P1 mRNA from
ptsG and fis
ptsG
strains grown with glucose were analyzed
as shown in lanes 1 and 2, respectively.
mlc mutant (lanes 3 and 6) and
fis mlc double mutant strains (lanes 4,
5, 7, and 8) were grown in the
presence (lanes 3-5) or absence (lanes 6-8) of
1% glucose. cAMP (2 mM) was added into the culture media
of lanes 5 and 8.
strain when
cells were grown in the presence of glucose (Fig. 1A,
lanes 3 and 4). The effect was not dependent on
Mlc, because the ptsG P1 promoter activity high in
mlc mutant also decreased in mlc and
fis double mutant regardless of the presence of glucose (Fig. 1B, lanes 3-8). We also determined the
effect of exogenous cAMP on ptsG P1 expression in the
fis
mlc
strain based
on the report of González-Gil et al. (29) that crp expression is increased in fis
strain. However, cAMP addition to the medium did not restore the P1
promoter activity (Fig. 1B, lanes 5 and
8), and the level of mlc expression was not
changed in the fis
strain compared with wt
(data not shown). These results imply that reduction of the
CRP-dependent P1 expression in the fis mutant cannot be explained by changes in the level of either Mlc or
CRP·cAMP. Furthermore, the reduction of ptsG P1
transcription in the fis
mlc
strain grown without glucose was lower
than that in the fis
strain grown in the
presence of glucose (Fig. 1). Thus, Fis effect is more prominent in the
CRP-dependent ptsG P1 transcription activation under limited intracellular concentration of CRP·cAMP.
248 to +103 (all
numberings in this study are based on the transcription start site of
the ptsG P1 promoter as shown in Fig.
2). At least five putative Fis-binding
sites were found on the ptsG promoter DNA (see Figs. 2 and
3), each of which showed high homology
with the Fis-binding sequence (20, 30). Fis sites I and II, centered at
positions +3 and
19, respectively, were detected between +18 and
32
of the ptsG promoter region. Fis site II was positioned
between the CRP and Mlc sites of the P1 promoter, and Fis site I
partially overlapped the Mlc site, as well as the P1 transcription
start site (Fig. 2B). Fis occupied from positions
65 to
90 (site III), which overlapped CRP site II centered at position
95. Fis also protected DNA from positions
105 to
130 (site IV),
and Fis site V centered at
145 overlapped the P2 promoter (see Figs.
2 and 3).
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Fig. 2.
Regulatory region of ptsG
gene in E. coli. A, schematic
diagram of ptsG promoter region. The two transcription
initiation sites of ptsG (7) are shown with
arrows. All numberings of this figure are based on the
transcription start point of the P1 promoter. The binding sites of two
major regulators of ptsG expression, CRP and Mlc, are
indicated in boxes. B, nucleotide sequences of
ptsG promoter region. The binding sites of CRP and Mlc are
indicated with gray boxes. The five protection regions
against DNase I digestion by Fis binding are shown with dashed
lines under the sequence. The best matched sequence with the
proposed 15 bp of highly degenerate Fis binding site,
Gnn(c/t)(A/g)(a/t)(a/t)(T/A)(t/a)(t/a)(T/c)(g/a)nnC (20), is indicated
with a dot over the sequence.
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Fig. 3.
DNase I footprinting analysis. Effect of
Fis on the binding of Mlc and CRP at the ptsG promoter is
shown. The ptsG promoter DNA fragment, end-labeled on the
lower strand, was incubated with individual regulator proteins or in
combinations as shown at the bottom of the figure. The
products were analyzed on 8% acrylamide gel containing 8 M
urea. Final concentrations of the proteins were 20, 60, and 10 nM CRP, Fis, and Mlc, respectively. The protection regions
by the binding of proteins are indicated with vertical lines
on the right side of the figure. The positions of the two
hypersensitive cleavages by CRP binding are shown with
arrowheads.
40.5, which is a typical pattern of CRP binding (Fig. 3,
lane 4) (31). It is likely that both CRP·cAMP and Fis could bind to the ptsG P1, because the two bands, which
appeared upon CRP·cAMP binding, remained, and DNA protection by Fis
at sites I and II was maintained in the presence of both CRP·cAMP and
Fis (Fig. 3). CRP·cAMP bound at CRP site II protected DNA from
85
to
105. In the presence of both CRP·cAMP and Fis, the hypersensitive cleavage site around
90 generated by Fis binding at
site III disappeared, suggesting the possibility that these two
proteins compete for the binding sites (Fig. 3, lane 3).
However, the relevance of CRP binding at site II in ptsG
expression has not yet been fully elucidated (6).
8.
Presence of Mlc protected DNA between +5 and
17 from DNase I attack
(Fig. 3, lane 6). Fis and Mlc could bind simultaneously to
the P1 promoter, even though their binding sites overlapped. The weak
bands remaining around the Mlc binding site in the presence of Fis were
further weakened by Mlc addition (Fig. 3B, compare lanes 2 and 5). At the same time, Fis binding at
sites I and II was observed clearly in the presence of Mlc as shown by
the protection of two bands around positions
32 and +18 (Fig. 3).
When all three regulators, Fis, CRP, and Mlc, were present a similar
DNase I cleavage pattern observed between Fis and CRP and between Fis and Mlc was reproduced (Fig. 3, lane 8). These findings
suggest that all these proteins could bind to ptsG P1
promoter simultaneously, forming a highly ordered protein·DNA complex.
5 and +11 but increased DMS reactivity of
3 G. Fis binding
at site II also generated characteristic changes in DMS reactivity at
ptsG P1 promoter, increasing reactivity at
25 G and near
20 G (Fig. 4). Binding of CRP·cAMP to
site I completely protected G residues at
47,
45, and
38 (Fig. 4,
lane 7). Consistent with our DNase I footprinting
experiment, DMS reactivity patterns at the P1 promoter shown in the
presence of either Fis or CRP·cAMP were retained in the presence of
both Fis and CRP·cAMP (Fig. 4, lane 10). Addition of RNA
polymerase alone did not change DMS reactivity at the ptsG
P1 promoter, and the presence of Fis showed no effect on RNA polymerase
binding to the P1 promoter (Fig. 4, lanes 2, 3,
5, and 6). However, strong protection of G
residues at
5 and
3 by RNA polymerase binding was detected in the
presence CRP·cAMP (Fig. 4, lanes 8 and 9).
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Fig. 4.
Effects of Fis and CRP·cAMP on RNA
polymerase binding to ptsG P1 promoter. DMS
footprinting was performed to investigate RNA polymerase interactions
at ptsG P1 promoter. Two nM supercoiled pGX12
was incubated with proteins as shown at the bottom of the
figure and treated with DMS. Changes in DMS reactivity at guanine
residues on the top strand of ptsG promoter are indicated
with arrows. Concentrations of RNA polymerase, CRP, and Fis
were 20 or 40, 40, and 80 nM, respectively.
5 G and significant reduction of
DMS reactivity at
3 G was observed, whereas DMS reactivity at +11 G
was restored (Fig. 4). However, the altered DMS reactivity by Fis
binding to site II and CRP binding to site I was retained under the
same condition. These results strongly suggest that ptsG P1
promoter can be occupied simultaneously by CRP·cAMP (at site I), Fis
(at site II), and RNA polymerase, but Fis bound to site I is excluded
by RNA polymerase in the presence of CRP·cAMP.
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Fig. 5.
Effects of Fis on transcription of
ptsG P1 analyzed by in vitro
transcription assay. A, to examine the effect of
Fis on Mlc repression of ptsG P1 promoter activity,
single-round in vitro transcription assay was carried out.
The supercoiled template was incubated with CRP (40 nM),
Fis (80 nM), and/or Mlc (10 nM). After
incubation with 20 nM RNA polymerase, the reaction
was started and stopped by adding NTP solution containing heparin and
loading dye, respectively, and analyzed on 6% sequencing gel. The
106/107-nucleotide rep transcripts were used as internal
controls for normalization of in vitro transcription
results. B, effects of Fis on transcription activation of
ptsG by CRP·cAMP in vitro. Two nM
supercoiled template was incubated with 40 nM CRP and/or 50 nM Fis. Subsequently, 5 nM RNA polymerase was
added, and the template was further incubated to form an open complex.
After the addition of heparin (200 µg/ml), the reactions were started
and stopped by adding NTP solution and loading dye, respectively, and
were resolved on 6% sequencing gel.
2, and
6, and the KMnO4 reactivity of these bases were
further increased in the presence of both CRP·cAMP and Fis (Fig.
6A). These results show that
Fis can enhance CRP-dependent open complex formation at
ptsG P1 promoter, resulting in the increased transcription initiation at the promoter.
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Fig. 6.
Fis increases CRP-dependent open
complex formation at ptsG P1. A, to
study Fis effect on promoter opening at ptsG P1,
KMnO4 footprinting assay was performed. KMnO4
reactivity patterns at top strand of pGX12 (lanes 1-6) and
pGX10 (lanes 7-9) are indicated with arrows.
Concentrations of RNA polymerase, CRP, and Fis were 20, 40, and 50 (lanes 3, 5, and 8) and 100 nM (lanes 6 and 9), respectively. The
reaction in lane 1 was treated with KMnO4 in the
absence of protein. B, effect of Fis on the open complex
formation at ptsG P1 was studied further using heparin
challenge experiment. Two nM end-labeled DNA fragment
carrying only P1 promoter region (+102 to 78) was incubated with
proteins as shown at the bottom of the figure. Heparin was
added to lanes 5-8 before gel loading. Each protein·DNA
complex is indicated with arrow. Concentrations of proteins
were 20, 40, and 60 nM of RNA polymerase, CRP, and Fis,
respectively.
78 to +102 of
ptsG promoter. Incubation of either Fis or CRP·cAMP with
the DNA probe generated a single retarded band (Fig. 6B).
When both Fis and CRP·cAMP were present in the reaction, Fis·DNA
complex disappeared, whereas Fis·CRP·DNA complex was observed, an
indication that Fis and CRP could bind simultaneously to the promoter
region (Fig. 6B, lane 4). Incubation of the DNA
probe with RNA polymerase only and subsequent heparin challenge did not
generate any RNA polymerase·DNA complex as expected from
KMnO4 reactivity assay (Fig. 6B, lane
5). When the same reaction was done in the presence of CRP·cAMP,
a heparin-resistant complex was detected, which was further increased
by the addition of Fis (Fig. 6B, compare lanes 7 and 8). However, addition of Fis only showed no effect on
the stable RNA polymerase·DNA complex formation. These results
suggest that CRP binding to site I is sufficient for the effective open
complex formation at ptsG P1 promoter, and Fis binding to
sites I and II can coactivate ptsG P1 transcription with CRP·cAMP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40.5 is essential for the
activation of ptsG P1 promoter, a typical class II
CRP-dependent promoter (33, 34). Fis further increased
CRP-dependent transcription of ptsG P1 promoter
(Fig. 5). However, Fis alone showed no effect on the transcription
initiation at P1 promoter. Our KMnO4 reactivity assay
demonstrated that the increased ptsG P1 transcription
initiation by Fis results from enhanced open complex formation at the
promoter in the presence of both CRP·cAMP and Fis (Fig. 6). Moreover,
nucleoprotein complex formations on CRP site I and Fis site II are
responsible for the increased open complex formation at ptsG
P1 promoter, because Fis could increase the open complex formation,
even when the shortened ptsG promoter without the Fis sites
III, IV, and V was used as a DNA template (Fig. 6), and Fis binding to
site I was excluded upon RNA polymerase binding to the P1 promoter in
the presence of CRP·cAMP (Fig. 4). Thus, to our knowledge, this is
the first case known in class II CRP-dependent promoter that Fis assists CRP-dependent transcription. This is
different from the coactivation of proP P2 promoter by Fis
and CRP·cAMP, where the CRP binding occurs at
121 (35). We are
presently attempting to elucidate the mechanisms of how Fis assists
CRP-dependent activation, because no evidence of
cooperative binding could be found between Fis and CRP.
strain grown
with glycerol, which indicates the degree of catabolite repression in
this gene. The level of ptsG expression during E. coli growth on glucose appears to be sufficient to derepress Mlc action completely, considering that the level of ptsG
expression was almost equal in both mlc+ and
mlc
cells grown in the presence of glucose
(7). Regulation of manXYZ, one of the Mlc regulon, is
mediated also by CRP·cAMP and Mlc (10). CRP binding to
40.5 of
manX promoter, which has been categorized as a class II
CRP-dependent promoter, is essential for its expression
(10). Other than ptsG, manX regulation is subject
to more severe catabolite repression upon glucose uptake; the promoter
activity in mlc+ cells grown with glucose was no
less than 20% of mlc
cells grown with
glycerol (10). Thus, we propose that Fis can alleviate catabolite
repression on ptsG P1 through the formation of coactivating
complex with CRP in cells grown with glucose. On the other hand, upon
glucose depletion in the medium, the phosphorylated form of
EIICBGlc begins to appear, after which Mlc is free from
sequestration by EIICBGlc (14). Under this circumstance,
Mlc may bind more preferentially to ptsG P1 promoter than to
other competing promoters through the interaction with Fis·CRP
nucleoprotein complex already formed at the promoter region.
Consequently, this selective shut-off of ptsG expression
will lead to the efficient repression in other Mlc-regulated genes.
Speculating that ptsG expression triggers cascades of gene
expressions for glucose uptake, Fis facilitates rapid adaptation of
E. coli to different nutritional environment through the
formation of nucleoprotein complex with either CRP·cAMP in the
presence of glucose or Mlc in the absence of glucose.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. B. Erni and S. Altuvia for gifts of bacterial strains and to Dr. Y. H. Lee for providing purified Fis protein.
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FOOTNOTES |
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* This work was supported in part by Korea Research Foundation Grant KRF-2001-015-DP0503.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.
§ Recipient of a grant for doctoral candidates provided by Korea Research Foundation (KRF-2001-908-GN0016).
¶ Recipient of the graduate fellowship provided by the Ministry of Education through the Brain Korea 21 Project.
** To whom correspondence should be addressed. Tel.: 82-31-290-2584; Fax: 82-31-293-4789; E-mail: sangryu@snu.ac.kr.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M213248200
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ABBREVIATIONS |
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The abbreviations used are: EIICBGlc, enzyme IICBGlc; CRP, cyclic AMP receptor protein; wt, wild-type; DMS, dimethyl sulfate.
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