From the Research Center for New Bio-Materials in Agriculture, Department of Food Science and Technology and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea and the ¶ School of Biological Sciences, Seoul National University, Seoul 151-742, Korea
Received for publication, February 26, 2001, and in revised form, April 17, 2001
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ABSTRACT |
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Mlc is a global regulator of carbohydrate
metabolism. Recent studies have revealed that Mlc is depressed by
protein-protein interaction with enzyme IICBGlc, a
glucose-specific permease, which is encoded by ptsG. The
mlc gene has been previously known to be transcribed by two
promoters, P1(+1) and P2(+13), and have a binding site of its own gene
product at +16. However, the mechanism of transcriptional regulation of the gene has not yet been established. In vitro
transcription assays of the mlc gene showed that P2
promoter could be recognized by RNA polymerase containing the heat
shock sigma factor When Mlc is overproduced on a multicopy plasmid in
Escherichia coli grown in the presence of glucose, it causes
reduction of acetate accumulation and E. coli makes large
colonies (1). Mlc has been proposed to be a new global regulator of
carbohydrate metabolism (2-5). It has been reported that Mlc regulates
manXYZ encoding enzyme II of the mannose PTS (4),
malT encoding the activator of maltose operon, and
mlc itself negatively (2). Moreover, ptsG
encoding enzyme IICB of the glucose PTS (EIICBGlc) and the
pts operon encoding general PTS proteins also proved to be
repressed by Mlc (3, 5-8). The Mlc regulon is also under the positive
control of the CRP1·cAMP
complex. It has been discovered that repression of the Mlc regulon is
relieved in cells grown in the media containing glucose or other PTS
sugars (7). It has been shown that the unphosphorylated EIICBGlc can sequester Mlc from its binding sites by direct
protein-protein interaction to induce expression of the Mlc regulon in
response to glucose (9-11).
The mlc gene encoding a 44-kDa Mlc protein is located around
35 min of chromosomal locus (1). This gene was also identified as the
same allele of the dgsA gene (4, 12, 13). Decker et
al. (2) have shown that mlc transcription starts from
two promoters called upstream "+1" and downstream "+13" and
there exists one Mlc-binding site centered at +16. In addition, a
highly conserved CRP-binding site is present within the mlc
promoter. However, the detailed mechanisms of transcriptional
regulation of mlc have not yet been reported.
The majority of the E. coli promoters are recognized by the
RNA polymerase containing the housekeeping sigma factor,
Materials--
Cyclic AMP was obtained from Sigma, RNA
polymerase saturated with Bacterial Strains--
MC4100 (araD139
Plasmid Construction and Preparation--
Basic cloning
protocols used were described in Sambrook and Russell (27). Polymerase
chain reaction cloning of the mlc promoter was
carried out using primers that have unique restriction sites in their
sequences. The clone was verified by DNA sequencing. The supercoiled
plasmid pMX, which contains the mlc promoter region, was
made by inserting the DNA segment from base pair Primer Extension--
Cells were grown aerobically at 30 °C
in tryptone broth (1% Bacto-tryptone, 0.8% NaCl) either in the
presence or absence of 0.2% glucose. At A600 = 0.5, growth temperature was shifted to 42 °C and incubation was
continued for the designated time and total E. coli RNA was
purified using Trizol reagent (Life Technologies, Inc.) to study the
heat shock effects on mlc and ptsG transcription. Purified RNA was resuspended in sterile distilled water. To study mlc transcription, 32P-labeled primer MLC7
(5'-ATTTTAGTGATACTGGCAGGAGCCAGTTGC-3'), which is complementary to +162
to +192, was co-precipitated with 40 µg of total cell RNA. The primer
PG1 (5'-AATTGAGAGTGCTCCTGAGTATGGGTGC-3', complementary to +74 to +102)
and the primer P11 (6) were co-precipitated with 30 µg of total RNA
to study ptsG and pts transcription,
respectively. The pellet was resuspended in 20 µl of 250 mM KCl, 2 mM Tris-HCl, pH 7.9, and 0.2 mM EDTA. Primer extension reactions were done as described
by Ryu and Garges (29).
Purification of RNA Polymerase Holoenzyme Containing
In Vitro Transcription Assay--
Reactions were done as
described by Ryu and Garges (29) in a 25-µl volume containing the
following: 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
[ The mlc Promoter Is Recognized by Both E
The purified Mlc could inhibit transcription from both promoters but
the degree of repression was dependent on the kind of Transcription of mlc Was Modulated Less Than 2-fold by
Glucose--
We studied further the regulation of mlc
transcription in vivo by primer extension assay. The wild
type strain MC4100 and its isogenic mutant strains were grown at
30 °C in the presence or absence of glucose then total RNA was
extracted as described under "Experimental Procedures."
Transcription from both P1 and P2 promoter was detected and the level
of expression from two promoters was similar when the wild type cells
were grown in the absence of glucose (Fig.
4A, lane 2). However, when
cells were grown in the presence of glucose, transcription from P1 was
increased slightly while that from P2 was not detectable (Fig. 4A,
lane 1). These results establish that P1 and P2 promoter
activity was dependent on CRP·cAMP in vivo in accordance
with the in vitro transcription assay results as described
above and that the overall expression level of mlc was
changed less than 2-fold by glucose.
We analyzed the mlc expression in SR505 (MC4100,
mlc::Tn10) to test the autoregulatory effect of
Mlc in vivo. The level of transcription could be analyzed in
SR505 because Tn10 was inserted in the C-terminal region of
the mlc gene. Transcription from both promoters was
increased clearly when the mlc:: strain was grown in the
absence of glucose (Fig. 4A, compare lanes 2 and
4), suggesting that Mlc acts as a repressor of the
mlc promoter. A strong stimulatory effect of CRP·cAMP on
mlc P2 promoter activity could also be seen when we compared
the P2 promoter activities in SR505 grown in the presence and absence
of glucose (Fig. 4A, lanes 3 and
4).
The P2 Promoter of mlc Is a Bona Fide Heat Shock
Promoter--
In vitro transcription assay showed that the
P2 promoter of mlc was recognized by E
We also examined changes in the mlc expression upon heat
shock by primer extension analysis. Growth temperature of a wild type
strain, MC4100, was shifted from 30 to 42 °C, and cells were further
incubated for the designated time as described under "Experimental Procedures." As shown on Fig. 4B, temperature upshift
caused dramatic changes in the mlc expression. The
mlc expression reached the maximum level 5 min after heat
shock (Fig. 4B, lanes 3 and 4), when
the intracellular concentration of E Transcription of ptsG Is Increased by Heat Shock and Glucose
Induction of ptsG Requires E
As shown on Fig. 5A, induction of ptsG P1
transcription by glucose was reduced significantly in the
It has been suggested that the mlc gene has two
promoters, P1 and P2, which are separated by 12 bases and autoregulated
by its product (2). Here, we report that the transcription of the
mlc gene is regulated in a highly sophisticated manner and that heat shock It is known that the expression level of several genes encoding
transcriptional repressors such as galS (33),
nagC (34), purR (35), and trpR (36) of
E. coli is low and that their expression level is not
modulated much in various growth conditions. It seems likely that both
CRP·cAMP and Mlc work together in E. coli to maintain the
level of Mlc optimum in response to availability of glucose. All genes
known to be regulated negatively by Mlc, such as manXYZ,
malT, ptsG, and pts, are also
regulated positively by CRP·cAMP (2-8). In vitro
transcription assay with E Repression of the P2 transcription when cells were grown in the
presence of glucose implies that the action of CRP·cAMP is dominant
over the self-repression by Mlc in the regulation of the mlc
P2 promoter. The low binding affinity of Mlc to its own promoter that
is 10 times weaker than that to ptsG or pts P0
promoters (9) seems to be a major reason for the low influence of Mlc on regulation of its own gene. In vitro transcription assay
revealed that each promoter of mlc has a different
sensitivity to Mlc (Fig. 3). When cells were grown in the absence of
glucose, a similar level of expression from both P1 and P2 was observed
even though the P2 transcription is more sensitive to Mlc repression
probably because the intracellular concentration of Mlc is limiting in E. coli (3). The condition seems to be similar to the
in vitro transcription condition where both CRP·cAMP and a
small amount of Mlc were present as shown in lane 6 of Fig.
3A. However, P1 was as active as P2 when the mlc
strain was grown in the absence of glucose. These results imply that
the concentration of intracellular CRP·cAMP is lower than that of
CRP·cAMP used for in vitro transcription reactions (40 nM) (39). When Mlc was induced and the concentration of
CRP·cAMP was lowered by the addition of glucose in the growth medium,
the P1 promoter was activated slightly while the P2 promoter was
repressed because the P2 promoter is active only in the presence of
CRP·cAMP. This situation is similar to the in vitro
transcription condition where neither CRP·cAMP nor Mlc were present
(lane 1 of Fig. 3A). Therefore, the addition of
glucose in the growth medium resulted in the reduction of
mlc expression by about half. These results also agree with
the previous report by Decker et al. (2) that expression of
mlc is reduced by half when cells were grown in the presence
of glucose by measuring the E To investigate whether the increased level of mlc expression
resulting from heat shock can influence Mlc-dependent gene
expression, we analyzed changes in ptsG expression by heat
shock. The P1 expression of ptsG was increased significantly
by heat shock only when cells were grown in the presence of glucose
regardless of the presence of Mlc (Fig. 5A). These results
suggest that activation of ptsG P1 by heat shock was not
mediated by Mlc or CRP·cAMP even though Mlc repression might be
dominant over activation of ptsG P1 by heat shock. We have
reported that the unphosphorylated form of EIICBGlc
sequesters Mlc from its target promoters upon glucose uptake by direct
protein-protein interaction (9). Therefore, glucose is required to
maximize the level of dephosphorylated EIICBGlc necessary
to sequester Mlc that is increased by heat shock. However, contrary to
the case of mlc P2 transcription in which E32 (E
32) as well as
E
70, while P1 promoter is only recognized by
E
70. The cyclic AMP receptor protein and cyclic AMP
complex (CRP·cAMP) increased expression from P2 but showed negative
effect on transcription from P1 by E
70, although it had
little effect on transcription from P2 by E
32 in
vitro. Purified Mlc repressed transcription from both promoters, but with different degrees of inhibition. In vivo
transcription assays using wild type and mlc strains
indicated that the level of mlc expression was modulated
less than 2-fold by glucose in the medium with concerted action of
CRP·cAMP and Mlc. A dramatic increase in mlc expression
was observed upon heat shock or in cells overexpressing
32, confirming that E
32 is involved in
the expression of mlc. Induction of ptsG P1 and pts P0 transcription by glucose was also dependent on
E
32. These results indicate that
E
32 plays an important role in balancing the relative
concentration of Mlc and EIICBGlc in response to
availability of glucose in order to maintain inducibility of the
Mlc regulon at high growth temperature.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 (14, 15). Several genes that are necessary to respond
to various environmental or nutritional changes require specific recognition by RNA polymerase associated with the alternative sigma
factors,
32 (16),
E (17),
54 (18), or
S (19). The heat shock
response in E. coli is mediated by E
32 (20)
and it is known that expression of at least 26 genes is induced by heat
shock in E. coli (21). Many essential genes in E. coli have multiple promoters including one recognized by E
32 in order to respond to various environmental
conditions (22-24). It has been shown that the pts P0
promoter is recognized by E
32 as well as
E
70 (25) as is expected for a system as central to
carbohydrate metabolism as the PTS. In this work, we studied the
transcriptional regulation of the mlc gene in
vitro as well as in vivo and the role of
E
32 in maintaining glucose-dependent
induction of the Mlc regulon at high growth temperature.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70, nucleotide triphosphates,
[
-32P]ATP, and [
-32P]UTP were
purchased from Amersham Pharmacia Biotech. The cycle sequencing kit was
from Epicentre Technologies (Madison, WI).
argF-lacU169rpsL150 thiA relA1 flb5301 deoC1 ptsF25 rbsR)
was used as a wild type strain in this study. The mlc strain
SR505 is the same as KD413 (MC4100,
mlc::Tn10Tet). To construct the
rpoH-deleted mutant, SR701 (SR702,
rpoH),
rpoH allele of
KY1603 (26) was transferred to SR702 (MC4100, suhX1 (6))
using P1 transduction. To study the effect of
32 on
mlc expression, a plasmid pKV10 in which the rpoH
gene is under control of tac promoter was introduced into MC4100.
272 to +105 (all of
these numberings are based on the transcription start site of the P1
promoter of mlc in Fig. 1) between the EcoRI and the PstI sites in front of the rpoC terminator in
plasmid pSA600 (28). Supercoiled DNA was prepared by Concert kit (Life
Technologies, Inc.) in RNase-free condition for in vitro
transcription assay.
32--
E. coli MC4100 cells harboring the
pKV10 plasmid were grown on 2 × LB (1% Bacto-tryptone, 0.5%
yeast extract, 2% NaCl) in a 5-liter fermenter at 30 °C in the
presence of 1 mM
isopropyl-1-thio-
-D-galactopyranoside. The cells were
harvested 10 min after a temperature shift from 30 to 42 °C at
A600 = 2.5 in late exponential growth phase. RNA polymerase was purified by fast protein liquid chromatography according
to Harger et al. (30) with some modifications as described by Sukhodolets et al. (31). Crude RNA polymerase was
obtained from 10 g of wet cell paste by Polymin P precipitation
and the drained pellet was solubilized with TGED (10 mM
Tris, pH 7.9, 5% (v/v) glycerol, 0.1 mM EDTA, 0.1 mM dithiothreitol) containing 0.2 M NaCl
(TGEDN). This enzyme solution was loaded on a
single-stranded DNA-agarose column (Amersham Pharmacia Biotech)
pre-equilibrated with TGEDN. After the column was washed
with 3 column volumes of TGEDN, RNA polymerase was eluted
with a linear salt gradient (from TGEDN to TGED containing
1.0 M NaCl) at a flow rate of 2 ml/min. The elution of RNA
polymerase was monitored by the presence of
' on a sodium dodecyl
sulfate-polyacrylamide gel. The fractions containing RNA polymerase
(from 0.3 to 0.5 M NaCl gradient) were loaded onto a 5/5
Mono-Q column (Amersham Pharmacia Biotech), and RNA polymerase
containing
32 was eluted with a linear gradient of NaCl
(0.3 M to 0.5 M in TGED) at 1 ml/min. The yield
for E
32 was 1.0~1.5 mg.
-32P]UTP (800 Ci/mmol), 2 nM supercoiled
DNA template, 20 nM RNA polymerase saturated with
70 or
32, 100 µg/ml bovine serum
albumin, and 5% glycerol. Additional regulators such as 40 nM CRP, 100 µM cAMP, or 0-1.5
µM Mlc were added to the reaction as needed. All
components except nucleotides were incubated at 37 °C for 10 min.
Transcriptions were started by the addition of nucleotides and
terminated after 10 min by the addition of 25 µl of formamide loading
buffer (80% formamide, 89 mM Tris base, 89 mM
boric acid, 2 mM EDTA, 0.05% bromphenol blue, 0.05%
xylene cyanol). RNA was resolved by electrophoresis on an 8 M urea, 6% polyacrylamide gel. The amounts of transcripts were measured using a phosphoimage analyzer, BAS2500 (Fuji Photo Film
Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 and
E
32 in Vitro and Its Activity Is Modulated by CRP·cAMP
and Mlc--
As shown in Fig. 1, there
is a CRP-binding site centered at
58.5 and one Mlc-binding site
centered at +16 within the mlc promoter region (2). The
sequence of the P2 promoter region has a partial homology with a known
consensus sequence (CTTGAAA, 11~16 base pairs, CCCATNT) of the
promoters recognized by E
32 (20). To investigate how the
two promoters of the mlc gene are regulated, we performed
in vitro transcription assays with supercoiled DNA template,
purified proteins, and either E
70 or E
32.
When E
70 was used, transcripts were made from both the
P1 and P2 promoters (Fig. 2, lanes
1 and 2). However, E
32 could initiate
transcription only from P2, as expected from the sequence of the
promoter region of mlc shown in Fig. 1. No transcription from P1 by E
32 was detected (Fig. 2, lanes 3 and 4). The specificity of E
32 used for the
in vitro transcription assay was confirmed by the lack of
"rep" transcript that is transcribed only by
E
70. Interestingly, CRP·cAMP showed a negative effect
on P1 transcription by E
70, while it exerted a positive
effect on P2 transcription directed by E
70. However,
CRP·cAMP had little effect on E
32-directed
transcription from P2.
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Fig. 1.
Nucleotide sequence of the promoter region of
mlc. The transcription start points of P1(+1) and
P2(+13) are shown with arrows. The 10 and
35 region of
the P1 promoter is underlined and that of P2 is indicated
with a line over the sequence. The known consensus
10 and
35 sequence recognized by E
32 is shown in
lowercase (20). The binding site of CRP and Mlc is indicated
as box. The start codon of mlc, GTG, is also
indicated. All of the numbering of this sequence is based on the
transcription start point of the P1 promoter.
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Fig. 2.
The effect of CRP·cAMP on mlc
transcription in vitro. The supercoiled DNA
template, pMX, was used for the in vitro transcription by
E 70 (lanes 1 and 2) and
E
32 (lanes 3 and 4) in the absence
(lanes 1 and 3) and presence (lanes 2 and 4) of the CRP·cAMP complex. The transcripts from the
plasmid origin of replication (106/107 nucleotides) are marked as
rep. The 132-nucleotide transcript from P1 and the
120-nucleotide transcript from P2 are indicated.
factor and
the promoter (Fig. 3). Transcription from
the P2 promoter by E
70 was most sensitive to the
repression by Mlc, and half-repression of E
70-directed
P2 expression by Mlc was obtained at the level of 11.5 and 6.5 nM of the protein in the presence and absence of
CRP·cAMP, respectively (Fig. 3A). However, about 100-fold
more Mlc (0.7 and 0.6 µM of Mlc in the presence and
absence of CRP·cAMP, respectively) was required in order to repress
the P1 transcription (Fig. 3B). For half-repression of the
E
32-directed P2 promoter activity, 20 and 12.5 nM Mlc were required in the presence and absence of
CRP·cAMP, respectively (Fig. 3C). Repression of P1
transcription and induction of E
70-directed P2
transcription by CRP·cAMP shown in Fig. 2 were also observed in the
presence of Mlc (compare lanes 1-4 with
lanes 5-8 in Fig. 3, A and
B).
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Fig. 3.
The autoregulatory effect of Mlc on its own
gene in vitro. In vitro transcription
was carried out using pMX as a DNA template. Forty nM of
CRP·cAMP was added to the reactions as marked in the top
of the figures. A, E 70-directed transcription
at low level of Mlc. The amount of Mlc used in each lane is following:
lanes 1-4 and lanes 5-8 contains 0, 6.3, 12.5, and 25 nM Mlc in the reaction, respectively. B,
E
70-directed transcription at high level of Mlc. 0.25, 0.5, 1.0, and 1.5 µM Mlc was added to the lanes
1-4 and 5-8, respectively. C,
E
32-directed transcription in the presence of Mlc.
Lanes 1-5 and 6-10 contain 0, 12.5, 25, 50, and
100 nM Mlc, respectively.
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Fig. 4.
In vivo transcription analysis and
heat shock response of mlc. Primer extension
analysis using 40 µg of total RNA per reaction was carried out as
described under "Experimental Procedures." The start site of each
promoter was marked with the name of each promoter. Sequence ladder was
generated using the same end-labeled primer used for primer extension
assay. (A) Wild type strain, MC4100 (lanes 1 and
2), MC4100 mlc::Tn10
(lanes 3 and 4), rpoH
strain (SR701, lanes 5 and 6), and MC4100
harboring pKV10 in which rpoH gene is under control of
tac promoter (lanes 7 and 8) were
grown at 30 °C in the presence or absence of glucose. In the case of
MC4100 carrying pKV10,
isopropyl-1-thio-
-D-galactopyranoside was added to
overexpress
32. B, changes in mlc
transcription in response to heat shock were monitored using primer
extension analysis. Duration of heat shock was indicated on
top.
32 as
well as E
70. We analyzed expression of mlc in
the
rpoH strain using primer extension assay.
SR701 strain (MC4100, suhX1,
rpoH)
can grow at 30 °C because it expresses a high level of GroELS due to
an IS1 element inserted upstream of the groE gene
(6, 26). Overexpression of GroELS did not affect mlc
expression (data not shown). As shown on Fig. 4A
(lanes 5 and 6), the expression level of
mlc was decreased but the overall expression pattern was not
changed in the
rpoH cell compared with wild
type. However, P2 transcription was increased and its activity was not
much affected by glucose in cells overexpressing
32
(Fig. 4A, lanes 7 and 8). It is also
interesting to note that even the activity of P1 that is not recognized
by E
32 in vitro was increased significantly
when cells overexpressing E
32 was grown in the presence
of glucose (Fig. 4A, lane 7).
32 is known to be at
the highest level (20, 32). When cells were grown in the presence of
glucose (Fig. 4B, odd lanes), expression of both
P1 and P2 was increased transiently after heat shock as in the cells
overexpressing
32 (Fig. 4A). The increased
level of mlc expression from both P1 and P2 upon heat shock
was decreased sharply after 15 min at 42 °C in the presence of
glucose. Activation of mlc by heat shock was not dependent
on the presence of glucose. The cells grown in the absence of glucose
showed relatively little change in the P1 transcription but a high
level of activation in the P2 transcription in response to heat shock
(Fig. 4B, even lanes). However, heat shock effect
was not observed in the
rpoH strain (data not
shown), confirming that activation of mlc transcription by
heat shock was dependent on E
32. These results show that
E
32 can recognize the P2 promoter in vivo and
E
32-directed transcription from P2 becomes more
prominent when cells were grown at a high temperature or overexpressing
32.
32--
How does the highly
increased level of mlc expression in heat-shocked cells
affect glucose induction of Mlc regulon? To answer this question, we
analyzed changes in the ptsG transcription upon heat shock.
It has been known that transcription of the ptsG gene in
E. coli encoding the major membrane-bound glucose
transporter, EIICBGlc, is initiated from a major promoter,
P1, and a minor promoter, P2, and that both P1 and P2 transcription is
regulated negatively by Mlc (3, 5). There were no changes in P2
transcription of ptsG by heat shock (data not shown). But P1
transcription of ptsG was increased 5 min after heat shock
when wild type cells were grown in the presence of glucose (Fig.
5A, compare lanes 1 and 3) despite that mlc expression was also
increased at this condition as shown in Fig. 4B. However,
ptsG P1 transcription was not changed by heat shock when
cells were grown in the absence of glucose. Activation of
ptsG P1 transcription by heat shock was also seen in the
mlc strain (Fig. 5A, lane
7), suggesting the possibility that Mlc was not involved in the
activation of ptsG P1 by heat shock. In that expression of
both EIICBGlc and its negative regulator Mlc was increased
by heat shock, it seems that there is another mechanism(s) to maintain
glucose induction of the Mlc regulon under the heat shock
condition.
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Fig. 5.
Effects of heat shock RNA polymerase and Mlc
on ptsG P1 and pts P0 transcription
analyzed by primer extension analysis. A, heat shock
effects on ptsG P1 transcription. Total RNA was extracted
from cells grown at 30 °C to A600 = 0.5 in
the presence or absence of glucose (lanes 1, 2, 5, 6, 9, and 10) or cells held at 42 °C for 5 min before
RNA extraction (lanes 3, 4, 7, 8, 11, and
12). B, effects of heat shock RNA polymerase on
glucose induction of pts P0 transcription. The
pts P0 transcription was not induced by glucose in
rpoH strain.
rpoH strain. From these results, together with
our previous data (9), it could be assumed that the Mlc regulon could
not be induced by glucose because not enough EIICBGlc was
available to sequester Mlc in the
rpoH strain.
As expected, pts P0, one of several genes known to be under
Mlc control (6-8), was not induced in the
rpoH strain (Fig. 5B). These
results support the view that the expression of EIICBGlc
and Mlc is balanced in response to glucose uptake even when the cells
were heat-shocked and that E
32 plays an important role
in this regulation. However, it should be noted that heat shock still
increased P1 transcription of ptsG in the
rpoH strain albeit to a lower degree when
cells were grown in the presence of glucose (Fig. 5A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor,
32, is involved in its transcription.
70 and CRP·cAMP showed two
opposite effects on each promoter of mlc, that is the
positive effect on P2 and the negative effect on P1 (Fig. 2). This can
be explained based on the fact that the CRP-binding site of P2
centered at
71.5 to the transcription start site is more compatible
for a functional CRP site (37) compared with that of P1 centered at
58.5 to the transcription start site. However,
E
32-directed P2 transcription was insensitive to
CRP·cAMP. The P2 promoter of mlc should be a good model
system to assess the effect of
factor on transcription activation
by CRP·cAMP because it has been known that CRP·cAMP activates
transcription by direct protein-protein interaction with the
-subunit of RNA polymerase (38).
-galactosidase activity of the
mlc-lacZ fusion. Level of Mlc expression can vary precisely
in response to the available sugars but the variation range is less
than 2-fold in that the availability of unphosphorylated EIICBGlc may be more critical than the intracellular level
of Mlc for induction of the Mlc regulon by glucose as shown in our
previous report (9).
32 is involved in the transcription of the
mlc gene. In vitro transcription assay with
E
32 showed that E
32 could recognize the
P2 promoter of the mlc gene. Transcription of P2 was
increased when
32 was overexpressed (Fig.
4A). Moreover, mlc expression was increased upon
heat shock. It is known that the intracellular concentration of
32 in E. coli increases from 15-20-fold
within 5 min then declines to a new steady-state level severalfold
higher than the preshift level in response to temperature shift from 30 to 42 °C (20, 32). Transcription from P2 recognized by
E
32 was induced transiently to an extraordinary level
upon heat shock when cells were grown in the absence of glucose (Fig.
4B, lane 4). The level of mlc
transcription was changed parallel to the changes in intracellular
concentration of
32. In addition, P2 transcription was
activated upon heat shock even when cells were grown in the presence of
glucose. These results imply that the major RNA polymerase which
activated the P2 transcription upon heat shock was E
32
because E
32 was less sensitive to Mlc than
E
70 and the P2 transcription by E
32 was
not dependent on CRP·cAMP as revealed by the in vitro
transcription assay (Fig. 3). It is not clear why the P1 transcription
was reduced in the
rpoH strain and activated
by heat shock or when cells overexpressing
32 were grown
in the presence of glucose even though P1 promoter was not recognized
by E
32 in vitro. Because heat shock should
exert pleiotropic effects by regulating transcription of various genes
(21), further study on the mechanism of heat shock is needed for a
better understanding of these phenomena.
32
plays a major role in its regulation, it is likely that additional factors independent of E
32 are involved in regulation of
the ptsG expression because ptsG P1 expression
was increased partially upon heat shock even in the
rpoH strain grown in the presence of glucose.
It means that two separate mechanisms involving
E
32-dependent and
E
32-indendent activation of ptsG P1 may work
additively for full activation of ptsG P1 by glucose when
cells were heat-shocked. The importance of E
32 in
glucose induction of the Mlc regulon was manifested by the fact that
glucose induction of ptsG could not be observed in the
rpoH strain. The inability of glucose to
activate ptsG expression resulted in an insensitivity of the
pts P0 promoter to glucose in
rpoH
strain. We are trying to elucidate the mechanism of activation of
ptsG transcription by heat shock in the absence of
E
32 in order to understand the general role of
E
32 in regulation of genes involving carbohydrate metabolism.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. C. Park for providing KD413 and Dr. T. Yura for providing bacterial strains and a pKV10.
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FOOTNOTES |
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* This work was supported in part by Grant 2000-2-20200-006-3 from the Basic Research Program of the Korea Science & Engineering Foundation.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 the Brain Korea21 Project
§ Supported by the Brain Korea21 Project.
To whom correspondence should be addressed: Dept. of Food
Science and Technology and School of Agricultural Biotechnology, Seoul
National University, Suwon, 441-744 Korea. Tel.: 82-31-290-2584; Fax:
82-31-293-4789; E-mail: sangryu@snu.ac.kr.
Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M101757200
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ABBREVIATIONS |
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The abbreviations used are: CRP, cyclic AMP receptor protein; PTS, phosphoenolpyruvate: carbohydrate phosphotransferase system.
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