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Previous Article | Next Article 
Journal of Bacteriology, July 2001, p. 3910-3918, Vol. 183, No. 13
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.13.3910-3918.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Activation from a Distance: Roles of Lrp and
Integration Host Factor in Transcriptional Activation of
gltBDF
Ligi
Paul,1
Robert M.
Blumenthal,2 and
Rowena G.
Matthews1,3,*
Biophysics Research
Division1 and Department of Biological
Chemistry,3 University of Michigan, Ann
Arbor, Michigan 48109, and Department of Microbiology and
Immunology, Medical College of Ohio, Toledo, Ohio
436142
Received 23 January 2001/Accepted 13 April 2001
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ABSTRACT |
The leucine-responsive regulatory protein (Lrp) binds to three
sites centered 252, 216, and 152 bp upstream of the transcription start
site of the Escherichia coli glutamate synthase operon
(gltBDF) and activates transcription. Activators of
70-dependent promoters usually bind closer to the 
35
hexamer of the core promoter sequence. To study the mechanism by which
Lrp-dependent activation occurs over this relatively large distance,
the gltBDF upstream region was sequentially replaced with
corresponding portions from the well-characterized
70-dependent promoter lacZYAp. The
glt-lac promoter hybrids were placed upstream of
lacZ, allowing transcriptional activity to be monitored via
-galactosidase assays. Even replacing all gltBDF sequences downstream of and including the 35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the 35 hexamer and the proximal Lrp binding site (48
to 128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a
consensus binding sequence, this region seemed likely to be a binding
site for integration host factor (IHF). Experiments to study the
effects of a himD mutant on expression of a
gltB::lacZ transcriptional fusion,
gel mobility shift analyses, and DNA footprinting assays were used to
confirm the direct participation of IHF in gltBDF promoter
regulation. Based on these results, we suggest that IHF plays a crucial
architectural role, bringing the distant Lrp complex in close proximity
to the promoter-bound RNA polymerase.
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INTRODUCTION |
Escherichia coli
synthesizes glutamate under ammonia-limiting conditions using the
enzymes glutamate synthase, specified by the gltBDF operon,
and glutamine synthetase, specified by glnA. The genes
gltB and gltD specify the large and small
subunits of glutamate synthase, respectively (7), and
gltF specifies a protein of unknown function (8,
20). The gltBDF operon is positively regulated by the
leucine-responsive regulatory protein (Lrp) (12, 13) and
is transcribed at very low levels in the absence of Lrp
(12). The coregulator for Lrp is leucine, and exogenous
leucine reduces the expression of gltBDF about twofold, although this level is still substantially above baseline transcription in the absence of Lrp. Thus, relative to more responsive operons, gltBDF is a leucine-independent member of the Lrp regulon
(12).
There are three Lrp binding sites centered 246, 215, and 152 bp
upstream of the gltBDF transcription start site
(35); the closest of these sites is ~110 bp from the
35 hexamer. The presence of such distal regulatory sites is unusual
in 70-regulated promoters, although it has precedence in
promoters that are recognized by 54 (6, 9,
28). In 54-dependent promoters, upstream elements
or activators binding far upstream of the core promoter sequence are
brought closer to the RNA polymerase by a looping mechanism that is
sometimes assisted by DNA-bending proteins (9), raising
the question of whether additional regulatory proteins are required for
activation of gltBDF transcription. Upon binding the
glt DNA, the Lrp dimer bends the DNA towards itself;
occupancy of all three sites results in both phased hypersensitivity of
the region from 126 to 264 (35) and compaction of the
DNA in this region (D. E. Wiese II, M. Young, R. G. Matthews,
and C. Bustamante, unpublished data), suggesting formation of a
nucleosome-like structure. It is not known whether Lrp activates
transcription by a mechanism involving direct contact with RNA polymerase.
In this study, we have examined the role of the core promoter and
downstream sequences in Lrp-dependent activation of the gltBDF operon. We show that Lrp-dependent activation
persists even when the entire region downstream of and including the
35 hexamer is replaced by the corresponding region from the
lac operon. We also demonstrate that integration host factor
(IHF) binds to the region between the proximal Lrp binding site and the
35 hexamer and positively regulates gltBDF transcription.
We propose that IHF-induced bending of DNA positions bound Lrp close to
RNA polymerase for transcriptional activation of the gltBDF operon.
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MATERIALS AND METHODS |
Strains and growth conditions.
The strains used in this
study are listed in Table 1. Cultures
were grown in LB medium (30) or glucose minimal MOPS
(morpholinopropanesulfonic acid) medium (25) at 37°C.
The antibiotics ampicillin (80 to 100 µg/ml), tetracycline (20 µg/ml), and chloramphenicol (25 µg/ml), were added to the medium as
indicated below.
Replacement of the gltBDF upstream region with the
lac operon sequences and construction of 
lysogens.
Fusions of the glt and lac operon regions (Fig.
1) were constructed from fragments of
glt and lac operons obtained by PCR amplification
using the primers listed in Table 1. The glt regions were
amplified from plasmid pBE10 (13), and the lac
regions were amplified from E. coli strain W3110 genomic DNA
purchased from Sigma Aldrich (St. Louis, Mo.).
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FIG. 1.
Maps and -galactosidase activities of
glt-lac chimeric operon fusions. The chimeric promoters were
introduced as a single copy into the attB site of E. coli strain PS2209 (W3110  lac). The
gltBDF sequences are shown with a black line, and the
lac sequences are shown by a gray hatched line. The base
pairs that were changed to introduce restriction sites in the chimeras
are indicated in lowercase letters in the sequences shown at the
bottom. The region from 48 to 128 in the gltBDF operon
that was replaced with a fragment from the cat gene in
strain LP1048 is shown as an open rectangular box. The box on the far
left of each construct indicates the 3' portion of the proximal Lrp
binding site that extends from 142 to 161. The 35 and 10
hexamers are also represented by boxes and are shown underlined in the
chimeric sequences at the bottom; arrows and the underlined A indicate
the start sites of transcription determined for the native
gltBDF (26) or lacZYA
(23) promoters. The -galactosidase activity from each
chimeric strain in the presence or absence of Lrp is presented on the
left side of the construct diagrams. The -galactosidase activities
from the chromosomal lacZ gene in E. coli strain
W3110 (lrp+) and isogenic strain BE1
(lrp::Tn10) are also shown for
comparison. NA, not applicable.
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The PCR mixtures contained 2 ng of template plasmid DNA or 200 ng of
E. coli genomic DNA, 1 U of Vent polymerase (New
England Biolabs, Beverly, Mass.), 200 mM concentrations of each primer, and 200 µM concentrations of each deoxynucleoside triphosphate (Life
Technologies, Rockville, Md.) in thermopol reaction buffer [10 mM KCl,
10 mM (NH4)2SO4, 20 mM Tris-HCl (pH
8.8), 2 mM MgSO4, and 0.1% Triton X-100] in a total
volume of 100 µl. The reaction was carried out for 20 cycles of
96°C for 30 s, 60°C minus 0.5°C/cycle for 1 min, and 72°C for
1.5 min, followed by 10 cycles of 96°C for 30 s, 50°C for 1 min, and 72°C for 1.5 min. The PCR products were purified using a
Qiaquick PCR purification kit (Qiagen Inc., Valencia, Calif.). A single
deoxyadenosine was added to the 3' end of the products by incubating
them at 72°C for 20 min with 5 U of Taq DNA polymerase
(Life Technologies) and 200 µM dATP in 20 mM Tris-HCl (pH 8.4)
containing 50 mM KCl and 2 mM MgCl2. The products were
resolved on a 1.0% agarose gel, purified using a Qiaquick gel
extraction kit (Qiagen), and cloned into E. coli strains
XL-1 Blue or DH5
using the pGEM-T vector (Promega, Madison, Wis.).
The sequences of the cloned fragments were verified using the ABI PRIZM
dye terminator cycle sequencing method (Applied Biosystems, Foster
City, Calif.) at the University of Michigan DNA Sequencing Core Facility.
Restriction enzyme sites were incorporated into the primers used for
PCR (Table 1), allowing ligation of the glt and
lac operon sequences (Fig. 1). All the glt
fragments had an EcoRI site at the 5' end introduced from
the primer glt1, and all the lac fragments had an
EcoRV site at the 3' end introduced from the primer lac1.
The ligated glt-lac fragments were introduced into plasmid
pRS528 (31) cut with EcoRI and
EcoRV.
The glt-lac chimera in strain LP1008 was generated using
fragments derived by amplification of glt DNA using primers
glt1 and glt2 (406 to +8) and amplification of lac DNA
using primers lac2 and lac1 (+1 to +1166); primers glt2 and lac2 each
contain a PstI site, allowing ligation of the fragments
together. The glt and lac fragments are numbered
with respect to their transcription start sites (23, 26).
The glt-lac chimera in strain LP1023 was obtained using the
primer pairs glt1 and glt3 (406 to 23) and lac3 and lac1 (24 to
+1166). Primers glt3 and lac3 each contain a PstI site,
which was used to ligate the two fragments together.
The chimera in strain LP1035 was generated using primers glt1 and glt4
(406 to 35) and lac4 and lac1 (36 to +1166). Primer glt4 has a
HindIII site that is present in the glt sequence,
and primer lac4 has a HindIII site upstream of the
lac sequence.
The chimera in strain LP1048 was constructed by replacing the
glt sequence between 48 and 128 in strain LP1008 with a
fragment of the chloramphenicol acetyltransferase gene
(cat). The cat fragment was amplified from the
plasmid vector pKK232-8 (Pharmacia) using the primers cat1 and cat2.
The flanking regions were amplified from pLP1008, which contains the
glt-lac chimera introduced into strain LP1008, with primers
glt1 and glt6 for the upstream flanking region and glt7 and lac1 for
the downstream flanking region. Primer glt6 contains an NruI
site, and primer cat1 contains complementary sequence, allowing
ligation of the upstream fragment with the cat insert.
Primer glt7 contains a PstI site, as does primer cat2, allowing ligation of the downstream fragment to the cat insert.
The glt region of strain LP1000 (Table 1) was generated
using primers glt1 and glt5 (406 to +246), resulting in a fragment with an EcoRI site at the 5' end and a BamHI site
at the 3' end which was ligated into plasmid pRS415 (31)
cut with EcoRI and BamHI.
The glt-lac fusions from the pRS vectors were transferred
into the chromosome of E. coli strain PS2209 (W3110 lac)
at the attB site using RZ5 (2) according to
the procedure described in the work of Weise et al. (35).
Single-copy lysogens were identified using the PCR method described by
Powell et al. (27).
Transferring lrp and himD mutations into
lysogens.
An lrp-201::Tn10
allele from the E. coli strain BE1 (13) and a
himD::cat allele from E. coli strain RJ1413 (from Robert Osuna, University at Albany,
Albany, N.Y.) were transferred into the lysogens carrying
glt-lacZ fusions by P1 vir transduction (24). The transductants were selected on LB
medium-ampicillin plates containing tetracycline or chloramphenicol.
-Galactosidase assays.
The lysogens were grown in
glucose minimal MOPS medium supplemented with 0.4 mM isoleucine and 0.6 mM valine (13). IPTG (isopropyl--D-thiogalactopyranoside; 0.5 mM) was added
to the cultures to avoid any effect of the lac repressor on
the expression of glt-lac fusion constructs. Samples for
-galactosidase assays were taken throughout the growth period, and
the assays were carried out as described by Ernsting et al.
(13). The absorbances of the cultures at 420 nm were
plotted versus the -galactosidase levels. The points were fitted by
linear regression, and the slope of the line indicates the
-galactosidase activity of the culture (35). Only
samples taken before the cultures reached an
A420 of 1.0 were used for the slope
determinations. The -galactosidase levels from the wild-type
chromosomal lacZ in Lrp+ (W3110) and
Lrp
(BE1) backgrounds were used as controls to account
for the effect of Lrp on lacZ transcription.
Gel mobility shift assays.
The DNA fragments used for gel
mobility shift assays were amplified by PCR from pBE10
(13). The DNA was labeled at the 5' end using T4
polynucleotide kinase (Life Technologies) and
[
-32P]ATP (7,000 Ci/mmol; ICN Biomedical Research
Products, Costa Mesa, Calif.) at 37°C for 45 min. The unincorporated
nulceotides were removed by passing the labeling reaction mixtures
through a Biospin 6 gel filtration column (Bio-Rad Laboratories,
Hercules, Calif.). Lrp was purified from an overexpressing E. coli strain (JWD3) using the procedure described earlier
(13). Purified IHF was a gift from Steven D. Goodman
(University of Southern California). The labeled DNA fragment (1.15 nM)
was incubated with various concentrations of Lrp and/or IHF in a buffer
containing 20 mM Tris-acetate (pH 8.0), 0.1 mM EDTA, 0.1 mM
dithiothreitol, 50 mM NaCl, 4 mM Mg acetate, 12.5% glycerol (vol/vol),
and 200 ng of poly(dI-dC) · poly(dI-dC) (Amersham Pharmacia
Biotech, Pistcataway, N.J.) in a total volume of 20 µl. The mixtures
were left at room temperature for 5 min before being incubated at
28°C for 15 min. The samples were loaded directly onto a 6%
polyacrylamide gel (8.3 cm wide by 7.3 cm long by 1.0 mm) in 0.5× TBE
(45 mM Tris-borate [pH 8.3], 0.1 mM EDTA) and electrophoresed at 12 mA and 4°C. The electrophoresis buffer contained 0.5× TBE and 5 mM
MgCl2. The gel was fixed in a solution of 10% acetic acid
and 10% methanol for 15 min and dried at 80°C. Biomax MS film
(Kodak, Rochester, N.Y.) was used for autoradiography. The gels were
scanned using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.),
and the intensities of the bands were determined using ImageQuant version 1.2 software. The data were fitted to the Michaelis-Menten equation using Kaleidagraph (Synergy Software, Reading, Pa.).
DNase I footprinting.
Footprinting assays were carried out
as described earlier (5). The gltBDF DNA
fragment used for footprinting of the template strand was amplified by
PCR from plasmid pBE10 (13) using primers glt1 and glt2
and digested with the restriction enzyme HincII, resulting
in a fragment from 176 to +8. For footprinting of the nontemplate
strand, DNA was amplified with primers gltFP and glt5 and digested with
Bsp1286I, resulting in a fragment from 203 to +161. The
DNA fragments were labeled at the glt2 and gltFP ends for the template
and nontemplate strands, respectively, using T4 polynucleotide kinase
and [-32P]ATP as described above for gel mobility
shift assays. Various concentrations of IHF were incubated with the
labeled DNA in a buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM
MgCl2, 1 mM CaCl2, 2 mM dithiothreitol, 50 µg
of bovine serum albumin per ml, and 2 µg of poly(dI-dC) · poly(dI-dC) per ml in a total volume of 20 µl. The mixtures were
incubated at 28°C for 30 min, after which 0.05 U of RNase-free DNase
I (amplification grade from Life Technologies) was added to each tube.
DNase I digestion was stopped after 2 min by adding 700 µl of a stop
solution (645 µl of 100% ethanol, 5 µl of saturated ammonium
acetate, and 5 µg of yeast tRNA) (5). The precipitated
DNA fragments were resolved on a 6% acrylamide gel containing 8 M urea
(30). A dideoxy sequencing reaction of the
gltBDF upstream region with the primer corresponding to the
labeled end, carried out using a T7 Sequenase version 2.07-deaza-dGTP kit (Amersham Pharmacia Biotech), was used to generate the ladder.
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RESULTS |
Effect of replacing the gltBDF promoter regions with
those of the lac promoter on Lrp-dependent activation.
To study the role of the DNA sequences downstream of the proximal Lrp
binding site centered at 152 in the regulation of the glutamate
synthase (gltBDF) operon of E. coli, these
downstream sequences were systematically replaced with the
corresponding regions of the lacZYA operon. Single copies of
the chimeric constructs were introduced into E. coli strain
PS2209 (W3110 lac) at the att site by
lambda integration. Transcription from these hybrid constructs was
monitored using lacZ as the reporter gene. Figure 1
illustrates the sequence replacements and the -galactosidase activities of each construct. As described below, replacing the gltBDF region downstream of and including the 35 hexamer
with the corresponding regions of the lac operon did not
abolish Lrp-dependent regulation. Thus, Lrp is clearly able to regulate
the well-characterized 70-dependent promoter
lacZYAp from its binding sites more than 110 bp upstream of
the 35 hexamer.
In strain LP1008, the region downstream of +8 of the
gltBDF operon was replaced by the lac
operon sequence. Bases at +4 and +7 were changed (C to T and T to A,
respectively) to introduce a PstI site, which was used for
ligating the glt and lac fragments. In strain
LP1008, lacZ was still under the control of Lrp and showed a
nearly 30-fold reduction in -galactosidase levels in an
Lrp background relative to the wild-type strain.
Strain LP1023 has a chimeric promoter, with the 35 hexamer being from
gltBDF and the 10 hexamer being from the lac
operon. The sequence in the spacer region derived from glt
was changed in two positions (T to G at 23 and T to C at 25) to
generate a PstI site for preparing the construct, and the
spacer sequence downstream of 23 was from the lac operon.
The length of the spacer sequence is 17 bp as in glt; the
wild-type lac spacer region is 18 bp. This construct showed
an extremely high level of -galactosidase activity: 200-fold higher
at both the basal and activated levels compared to levels in strain
LP1008. Nonetheless transcription from this chimeric promoter was
activated over 20-fold by Lrp.
In strain LP1035 the 35 hexamer and the entire region downstream of
it have been replaced with the corresponding sequences from the
lac operon. The distance between the proximal Lrp binding site and the 35 hexamer was 1 bp shorter than in the other constructs (111 versus 110 bp). The -galactosidase level from this construct was reduced sevenfold in an Lrp background, but the basal
level of transcription was higher than in strain LP1008.
In the gltBDF operon, the proximal Lrp binding site centered
at 152 and the RNA polymerase recognition sequence at 34 are farther apart (more than 110 bp) than is usually seen in other 70-dependent promoters. To see if the intervening region
is involved in transcriptional regulation of the glt operon,
the region from 48 to 128 was replaced with a portion of the
chloramphenicol acetyltransferase (cat) gene of the same
length (LP1048). This replacement had no effect on basal transcription
but completely abolished Lrp-mediated activation.
IHF is required for positive regulation of transcription from the
gltBDF promoter.
In preliminary experiments we used
surface-enhanced laser desorption and ionization (SELDI) ProteinChip
technology (Ciphergen Biosystems, Fremont, Calif.) to identify proteins
that might bind to the upstream region of the gltBDF operon.
In this approach, biotin-labeled DNA fragments of the glt
region (from 406 to +132) were attached to streptavidin-coated chips
and incubated with cell extracts. The masses of proteins that bound to
the DNA fragments were determined using mass spectroscopy. This binding
experiment was carried out under nonstringent conditions, and there
were multiple peaks corresponding to various proteins that bound the DNA nonspecifically. Even though the results of these experiments were
not conclusive, they suggested that two polypeptides with molecular
masses of 10.6 and 11.2 kDa bound to the gltBDF upstream region. The molecular masses of these polypeptides matched those of the
two subunits of IHF. These preliminary results led us to focus our
study on the possible role of IHF in gltBDF transcription.
Upstream of the gltBDF promoter, within the region replaced
by the cat fragment in strain LP10048, there are two regions
resembling the consensus IHF binding sequence WATCAANNNNTTR,
where W is A or T and R is A or G (10, 17). The
region from 95 to 83 (TTTCAGTCATTTA) has two mismatches
and the overlapping region from 91 to 79 (AGTCATTTAATAA)
has three mismatches to the consensus sequence. An insertion in
the himD gene encoding the subunit of IHF
(himD::cat) was transferred by P1
vir transduction into strain LP1000, which contains a
gltB::lacZ transcriptional fusion. The
IHF strain had a -galactosidase level over 30-fold
lower than that of the IHF+ strain (Fig.
2).
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FIG. 2.
Effect of IHF on gltB-lacZ expression. The
-galactosidase activity of the gltB-lacZ transcriptional
fusion, integrated as a single copy into the attB site of
isogenic IHF+ and IHF
(himD::cat) strains (LP1000 and LP2000,
respectively) constructed from E. coli strain PS2209 (W3110
lac), is shown. The cultures were grown in glucose
minimal MOPS medium, and samples were taken at intervals during the
growth. The optical density of the cultures was measured at 420 nm. The
-galactosidase activities for the cultures were calculated from the
slopes of the lines and were 1,180 for strain LP1000 and 39 for strain
LP2000.
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Purified IHF binds to the upstream region of the gltBDF
promoter.
To see if IHF affects gltBDF transcription
directly, gel mobility shift assays were carried out using purified IHF
and the region upstream of the gltB translational initiation
codon. Assays with both the region from 406 to +246 that included the
first 30 bp of the coding region of gltB and a shorter
fragment (406 to +8) showed similar band shift patterns. A gel
mobility shift assay with the shorter fragment is shown in Fig.
3A. Migration of the DNA fragment was
retarded in the presence of IHF. IHF binds to the gltBDF
upstream region with an apparent Kd of 5.7 ± 0.3 nM (Fig. 3B). A DNA fragment from 406 to +246 but with the
region from 48 to 128 replaced with an equivalent length of the
cat gene sequence did not bind IHF under identical binding
conditions (not shown).
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FIG. 3.
Gel mobility shift analysis of IHF binding to
glt DNA. (A) The assays used a DNA fragment containing the
region from 406 to +8 of the gltBDF promoter region and
various amounts of purified IHF. The reaction mixtures contained 1.15 nM DNA and 0 to 100 nM IHF. (B) The free DNA in the above gel mobility
shift assay was quantitated using a phosphorImager and used to
calculate the bound DNA. The data were fitted using the
Michaelis-Menten equation. The Kd of IHF for the
gltBDF upstream region was 5.7 ± 0.3 nM. (C) Lrp and
IHF binding to gltBDF DNA (324 to +246). The PCR fragment
from 406 to +246 was digested with the restriction enzyme
NsiI to obtain the above-described fragment. The reaction
mixtures contained 1.6 to 100 nM Lrp and 6.3 to 100 nM IHF as noted
above the lanes. In the presence of both Lrp and IHF in the reaction
mixture, we observed an additional band corresponding to the ternary
complex, which was not present when either Lrp or IHF alone was used in
the reactions; at saturating concentrations of Lrp and IHF, most of the
DNA migrated in this band.
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When both Lrp and IHF were present in the gel mobility shift assays, we
observed an additional band that was not present in assays with either
IHF or Lrp alone. The position of the shifted DNA fragment
corresponding to the ternary complex depended on the distance of the
IHF and Lrp binding sites from the ends of the DNA fragment used for
the assay (37). When the glt DNA fragment from
324 to +246 was used, the band corresponding to the ternary complex
was seen between those corresponding to IHF or Lrp alone (Fig. 3C). In
the presence of saturating amounts of Lrp and IHF, most of the DNA was
bound to both IHF and Lrp.
The binding site of IHF on the gltBDF upstream DNA was
determined using DNase I footprinting assays of both strands of the DNA
(Fig. 4). IHF protected a 41-bp region
from 115 to 75 on the nontemplate strand and a 38-bp region from
113 to 76 on the template strand (Fig.
5). The bases 78 on the nontemplate strand and 82 on the template strand were made hypersensitive to
DNase I digestion by IHF binding. Upstream of the IHF binding consensus
site there are two short tracts of adenine (106 to 109 and 113 to
115), both of which were protected in the DNase I footprinting
assays. High-affinity IHF binding sites are often preceded by a poly
(A) tract (21), whose narrow minor groove helps optimize
IHF-DNA contacts (29). In Fig.
6, the sequences upstream of the
gltBDF operon of E. coli are compared with those in other genera of Enterobacteriaceae: Salmonella
enterica serovar Typhimurium LT2, Klebsiella
pneumoniae, and Yersinia pestis. The approximate
distance between the proximal Lrp binding site and the IHF site is
conserved among the different genera, but the sequence of the
intervening region is not highly conserved except for the poly (A)
region. Though the sequence of the IHF binding site is not conserved
among the four genera, the cytosine that is present in all known
natural IHF binding sites (16) is conserved. An adenine
following the cytosine is also generally conserved (17),
but this residue is a T rather than an A in K. pneumoniae.
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FIG. 4.
DNase I footprinting analysis of IHF binding to the
glt DNA. DNA fragments extending from 176 to +8 of the
template strand (the strand complementary to the mRNA sequence) (A) or
from 203 to +161 of the nontemplate strand (B) of the
gltBDF operon were footprinted in the presence of 0 to 100 nM IHF. Dideoxy sequencing reactions of the gltBDF promoter
region with the primer glt2 (for the template strand) and gltFP (for
the nontemplate strand) were used to generate the ladders. IHF
protected the region (shown in brackets) from 113 to 76 on the
template strand and the region from 115 to 75 on the nontemplate
strand against DNase I digestion. The positions of the sequences are
given with respect to the transcription start site.
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FIG. 5.
Diagramatic representation of the region upstream of the
gltBDF promoter that was protected against DNase I digestion
by IHF binding. Open circles indicate protection against cleavage in
the presence of IHF, and the filled circles indicate unchanged
cleavage. Where no circles are shown, the DNA was not cleaved by DNase
I in the absence of IHF. The two gray circles indicate hypersensitivity
to DNase I digestion due to IHF binding. The underlined sequences
indicate the region protected from DNase I digestion.
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FIG. 6.
Comparison of sequences upstream of the
gltBDF operon from E. coli (4) with
corresponding sequences from related genera: S. enterica
serovar Typhimurium LT2 (WUGSC 99287/stmlt2-contig1496),
K. pneumoniae (WUGSC 573/kpneumo B KPN.contig272), and
Y. pestis (Sanger 632/Y pestis Contig850). The sequences of
the related genera were obtained from unfinished genomes using a BLAST
search (1), and the alignment was generated using CLUSTAL
W (33). Bases conserved in all four genera are shown in
bold, and the shaded regions indicate the proximal Lrp binding site,
the poly(A) region upstream of the IHF binding site, the consensus IHF
binding site (95 to 83), and the 35 and 10 hexamers. The
non-E. coli sequences are prepublication communications from
the Genome Sequencing Center at Washington University and The Sanger
Centre.
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DISCUSSION |
glt::lac hybrid promoters are
activated by Lrp.
To study the minimum DNA sequence required for
Lrp-dependent regulation, the gltBDF operon sequence
downstream of and including the 35 hexamer was sequentially replaced
with the corresponding regions of the lacZYA operon. The
effects of these substitutions on transcription were studied by
monitoring the activity of the reporter gene lacZ (Fig. 1).
The gltBDF sequences downstream of 23, including the 10
hexamer, are not required for Lrp-dependent activation of
transcription. Strain LP1023 has the 35 hexamer of gltBDF,
the 10 hexamer of the lac operon, and a hybrid spacer
region, yet Lrp-dependent activation (23.4-fold) was similar to that of
strain LP1008 (27.5-fold), in which the gltBDF core promoter
regions were intact. -Galactosidase expression in strain LP1023 was
>200-fold higher than in strain LP1008 and over 10-fold higher than
the IPTG-induced level from the native chromosomal lacZ gene
in W3110. Apparently, the combination of the 35 and the 10 hexamers
or the hybrid spacer region affected the strength of the promoter. A
study using random sequences in the spacer region of Lactococcus
lactis promoters in L. lactis and E. coli
backgrounds has shown that the sequence of the spacer region can affect
promoter strength up to 400-fold (22).
Lrp was able to activate transcription even when the gltBDF
core promoter sequence (the 35 hexamer and downstream sequence) was
completely replaced with that of the 70-dependent
lac promoter in strain LP1035. The lac promoter
normally requires catabolite activator protein for activation. Dove et al. (11) have shown that any DNA binding protein that
contacts RNA polymerase can activate transcription by strengthening the interaction of RNA polymerase with the promoter region. The extent of
Lrp-dependent activation was lower in LP1035 than in strain LP1023 with
the chimeric promoter or in strain LP1008 with the gltBDF
core promoter. It is possible that the longer spacer region between the
35 and 10 hexamers or the shorter region between the proximal Lrp
binding site and the 35 hexamer in strain LP1035 affected
transcriptional activation. Alternatively, there may be something about
the gltBDF 35 hexamer that makes it more amenable to
Lrp-dependent activation. In the E. coli fis promoter,
changes in the 35 hexamer, but not in the 10 hexamer, reduce the
response to stringent control (34).
The native gltBDF promoter is presumed to be recognized by
70, but evidence for this is not definitive. The
promoter is recognized in vitro by purified 70 and core
polymerase (B. R. Ernsting, unpublished data), and the sequence
and spacing of the 35 and 10 hexamers relative to the transcription
start site defined by primer extension are consistent with those of
moderately expressed 70-dependent promoters. The
experiments reported here provide definitive evidence that Lrp dimers,
bound more than 110 bp upstream of the 35 hexamer, can activate
transcription of a 70-dependent promoter, since the
lac operon is controlled by what is probably the
best-categorized such promoter.
IHF binding is required for Lrp-dependent activation of the
gltBDF operon.
The mechanism by which the
leucine-responsive regulatory protein (Lrp) regulates the genes under
its control is not completely understood. In some operons, e.g.,
gcv, Lrp exerts its effect in tandem with other regulatory
proteins (32); in others, such as ilvIH, Lrp is
sufficient for regulation (36). Lrp has been reported to
play an architectural role in the transcription of the gcv
operon (32). Although the gltBDF operon of
E. coli is positively regulated by Lrp (13), in
vitro transcription with purified Lrp produced minimal activation of
transcription from the gltBDF promoter (B. R. Ernsting,
unpublished results). This observation suggested that additional
factors might be involved in the transcriptional activation of the
gltBDF operon of E. coli.
The 110-bp distance between the proximal Lrp binding site (centered at
152) and the 35 hexamer of the gltBDF operon is unusual for a 70-dependent promoter. When a 91-bp sequence in
this region (128 to 48) was replaced with a fragment of the
cat gene (strain LP1048), transcription was reduced almost
40-fold. Results of SELDI ProteinChip experiments suggested that IHF
might bind to the gltBDF upstream sequence. The presence of
a consensus IHF binding sequence in this region further supported this
idea. In vivo assays using a himD mutant (Fig. 2) confirmed
that IHF is required for activation of transcription from the
gltBDF promoter.
While this work was in progress, a global transcript analysis of a
himA mutant was published (3). Mutation in
himA led to a 7.1-fold reduction in gltD
transcript levels in microarrays. This effect was not as drastic as the
approximately 30-fold effect seen in our assays with the
himD mutant or with a strain where the IHF binding region
was replaced by heterologous DNA (strain LP1048). It is possible that
functional himD homodimers were formed in the
himA mutant used for the microarray assay, resulting in activation of the gltBDF operon transcription to a certain
extent (3, 38).
We confirmed that IHF binds to the gltBDF upstream region
using gel mobility shift and footprinting assays (Fig. 3 to 5). The two
overlapping matches to the consensus IHF binding sequence in the
glt upstream region (95 to 83 and 91 to 79) are
protected in footprinting assays. Site-specific mutational studies have to be conducted to identify the site actually recognized by IHF. The
activation of the gltBDF operon by Lrp is totally dependent on the presence of IHF, since introduction of an Lrp mutation in strain
LP1048 did not lead to a further decrease in -galactosidase level.
IHF has been shown to be involved in transcriptional regulation of many
operons on its own or in concert with additional activator proteins
(14, 18). The crystal structure of IHF bound to DNA shows
that this protein bends DNA at an ~160° angle (29).
This facilitates looping of DNA, allowing an upstream element or
upstream-bound activator protein to come in close contact with RNA
polymerase. Transcriptional activation of the gltBDF operon
depends completely on the presence of both proteins, suggesting that a
DNA looping mechanism may be involved in gltBDF regulation.
The proposed IHF-induced bend in the gltBDF upstream region
would bring the proximal Lrp binding site close to the region just
upstream of the 35 hexamer, a position where activators of
70-dependent promoters usually bind (19).
So far, activation of transcription by direct contacts between Lrp and
RNA polymerase has not been documented for any promoter. The effect of
IHF on transcription from the gltBDF promoter is phase
dependent: transcription from the gltBDF promoter was
reduced fivefold when the IHF binding site was taken out of phase with
respect to the 35 hexamer and the Lrp binding sites by 5-bp
insertions at 40 and 120 (D. E. Weise II, unpublished results).
It is not known at present whether IHF acts as a direct regulator of
gltBDF transcription or whether it plays a structural role
in bringing the transcriptional machinery together. When IHF plays an
architectural role, it can be replaced by an intrinsically bent region
of DNA (15). Such an experiment remains to be done for the
gltBDF operon. Since the IHF binding site and the Lrp binding sites upstream of the glt promoter are conserved in
S. enterica, K. pneumoniae and Y. pestis, the
glutamate synthase operons of enteric bacteria appear to be regulated
by a common mechanism.
Our results suggest an exception to the general rule that
transcriptional activators of 70-dependent promoters
bind close to the 35 hexamer (19). When a confirmed
binding site for IHF is present, activators may bind farther upstream
and retain function. Since the proximal Lrp binding site in
gltBDF operon is positioned 110 bp upstream of the 35 hexamer, an intermediate IHF binding site appears to be essential to
Lrp-mediated activation. Future experiments will probe for evidence of
direct contact between Lrp and/or IHF and RNA polymerase.
| |
ACKNOWLEDGMENTS |
We thank Steven D. Goodman (University of Southern California)
for providing us with purified IHF, William S. Brusilow (Wayne State
University) for RZ5, Robert Osuna (University at Albany) for strain
RJ1413, and Ruth Van Bogelen (Pfizer, Ann Arbor, Mich.) and Lisa
Bradbury (Ciphergen Biosystems) for their assistance with the SELDI
ProteinChip experiment. We thank the Genome Sequencing Center,
Washington University, St. Louis, Mo., and The Sanger Centre for
communication of DNA sequence data prior to publication.
This work was supported by a grant (MCB 9807237) from the National
Science Foundation to R.G.M. and R.M.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biophysics
Research Division, 4028 Chemistry, 930 N. University Ave, University of Michigan, Ann Arbor, MI 48109-1055. Phone: (734) 764-5257. Fax: (734)
764-3323. E-mail rmatthew{at}umich.edu.
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
T. L. Madden,
A. A. Schäffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402[Abstract/Free Full Text].
|
| 2.
|
Angov, E., and W. S. A. Brusilow.
1988.
Use of lac fusions to measure in vivo regulation of expression of Escherichia coli proton-translocating ATPase (unc) genes.
J. Bacteriol.
170:459-462[Abstract/Free Full Text].
|
| 3.
|
Arfin, S. M.,
A. D. Long,
E. T. Ito,
L. Tolleri,
M. M. Riehle,
E. S. Paegle, and G. W. Hatfield.
2000.
Global gene expression profiling in Escherichia coli K12. The effects of integration host factor.
J. Biol. Chem.
275:29672-29684[Abstract/Free Full Text].
|
| 4.
|
Blattner, F. R.,
G. I. Plunkett III,
C. A. Bloch,
N. T. Perna,
V. Burland,
M. Riley,
J. Collado-Vides,
J. D. Glasner,
C. K. Rode,
G. F. Mayhew,
J. Gregor,
N. Davis,
W. H. A. Kirkpatrick,
M. A. Goeden,
D. J. Rose,
B. Mau, and Y. Shao.
1997.
The complete genome sequence of Escherichia coli K-12.
Science
277:1453-1474[Abstract/Free Full Text].
|
| 5.
|
Brenowitz, M.,
D. F. Senear, and R. E. Kingston.
1994.
DNA-protein interactions, p. 12.4.1-12.4.11.
In
V. B. Chanda (ed.), Current protocols in molecular biology, vol. 2. John Wiley & Sons, Inc., New York, N.Y.
|
| 6.
|
Buck, M.,
S. Miller,
M. Drummond, and R. Dixon.
1986.
Upstream activator sequences are present in the promoters of nitrogen fixation genes.
Nature
320:374-378[CrossRef].
|
| 7.
|
Castano, I.,
F. Bastarrachea, and A. A. Covarrubias.
1988.
gltBDF operon of Escherichia coli.
J. Bacteriol.
170:821-827[Abstract/Free Full Text].
|
| 8.
|
Castano, I.,
N. Flores,
F. Valle,
A. A. Covarrubias, and F. Bolivar.
1992.
gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression.
Mol. Microbiol.
6:2733-2741[CrossRef][Medline].
|
| 9.
|
Collado-Vides, J.,
B. Magasanik, and J. D. Gralla.
1991.
Control site location and transcriptional regulation in Escherichia coli.
Microbiol. Rev.
55:371-394[Abstract/Free Full Text].
|
| 10.
|
Craig, N. L., and H. A. Nash.
1984.
E. coli integration host factor binds to specific sites in DNA.
Cell
39:707-716[CrossRef][Medline].
|
| 11.
|
Dove, S. L.,
J. K. Joung, and A. Hochschild.
1997.
Activation of prokaryotic transcription through arbitrary protein-protein contacts.
Nature
386:627-630[CrossRef][Medline].
|
| 12.
|
Ernsting, B. R.,
M. R. Atkinson,
A. J. Ninfa, and R. G. Matthews.
1992.
Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli.
J. Bacteriol.
174:1109-1118[Abstract/Free Full Text].
|
| 13.
|
Ernsting, B. R.,
J. W. Denninger,
R. M. Blumenthal, and R. G. Matthews.
1993.
Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?
J. Bacteriol.
175:7160-7169[Abstract/Free Full Text].
|
| 14.
|
Friedman, D. I.
1988.
Integration host factor: a protein for all reasons.
Cell
55:545-554[CrossRef][Medline].
|
| 15.
|
Goodman, S. D.,
S. C. Nicholson, and H. A. Nash.
1992.
Deformation of DNA during site-specific recombination of bacteriophage : replacement of IHF protein by HU protein or sequence directed bends.
Proc. Natl. Acad. Sci. USA
89:11910-11914[Abstract/Free Full Text].
|
| 16.
|
Goodman, S. D.,
N. J. Velten,
Q. Gao,
S. Robinson, and A. M. Segall.
1999.
In vitro selection of integration host factor binding sites.
J. Bacteriol.
181:3246-3255[Abstract/Free Full Text].
|
| 17.
|
Goodrich, J. A.,
M. L. Schwartz, and W. R. McClure.
1990.
Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF).
Nucleic Acids Res.
18:4993-5000[Abstract/Free Full Text].
|
| 18.
|
Goosen, N., and P. van de Putte.
1995.
The regulation of transcription initiation by integration host factor.
Mol. Microbiol.
16:1-7[CrossRef][Medline].
|
| 19.
|
Gralla, J. D., and J. Collado-Vides.
1996.
Organization and function of transcription regulatory elements, p. 1232-1245.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 1. ASM Press, Washington, D.C.
|
| 20.
|
Grassl, G.,
B. Bufe,
B. Muller,
M. Rosel, and D. Kleiner.
1999.
Characterization of the gltF gene product of Escherichia coli.
FEMS Microbiol. Lett.
179:79-84[CrossRef][Medline].
|
| 21.
|
Hales, L. M.,
R. I. Gumport, and J. F. Gardner.
1996.
Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.
Nucleic Acids Res.
24:1780-1786[Abstract/Free Full Text].
|
| 22.
|
Jensen, P. R., and K. Hammer.
1998.
The sequence of the spacers between the consensus sequences modulates the strength of prokaryotic promoters.
Appl. Environ. Microbiol.
64:82-87[Abstract/Free Full Text].
|
| 23.
|
Maizels, N. M.
1973.
The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli.
Proc. Natl. Acad. Sci. USA
70:3585-3589[Abstract/Free Full Text].
|
| 24.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 25.
|
Neidhardt, F. C.,
P. L. Bloch, and D. F. Smith.
1974.
Culture medium for enterobacteria.
J. Bacteriol.
119:736-747[Abstract/Free Full Text].
|
| 26.
|
Oliver, G.,
G. Gosset,
R. Sanchez-Pescador,
E. Lozoya,
L. M. Ku,
N. Flores,
B. Becerril,
F. Valle, and F. Bolivar.
1987.
Determination of nucleotide sequence of glutamate synthase structural genes of Escherichia coli K-12.
Gene
60:1-11[CrossRef][Medline].
|
| 27.
|
Powell, B. S.,
D. L. Court,
Y. Nakamura,
M. P. Rivas, and C. L. Turnbough, Jr.
1994.
Rapid confirmation of single copy lambda prophage integration by PCR.
Nucleic Acids Res.
22:5765-5766[Free Full Text].
|
| 28.
|
Reitzer, L. J., and B. Magasanik.
1986.
Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter.
Cell
45:785-792[CrossRef][Medline].
|
| 29.
|
Rice, P. A.,
S. Yang,
K. Mizuuchi, and H. A. Nash.
1996.
Crystal structure of an IHF-DNA complex: a protein induced DNA U-turn.
Cell
87:1295-1306[CrossRef][Medline].
|
| 30.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis (ed.).
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 31.
|
Simons, R. W.,
F. Houman, and N. Kleckner.
1987.
Improved single and multicopy lac based cloning vectors for protein and operon fusions.
Gene
53:85-96[CrossRef][Medline].
|
| 32.
|
Stauffer, L. T., and G. V. Stauffer.
1999.
Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon.
Microbiology
145:569-576[Abstract].
|
| 33.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 34.
|
Walker, K. A.,
C. L. Atkins, and R. Osuna.
1999.
Functional determinants of the Escherichia coli fis promoter: roles of 35, 10, and transcription initiation regions in the response to stringent control and growth phase-dependent regulation.
J. Bacteriol.
181:1269-1280[Abstract/Free Full Text].
|
| 35.
|
Weise, D. E., II,
B. R. Ernsting,
R. M. Blumenthal, and R. G. Matthews.
1997.
A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli.
J. Mol. Biol.
270:152-168[CrossRef][Medline].
|
| 36.
|
Willins, D. A., and J. M. Calvo.
1992.
In vitro transcription from the Escherichia coli ilvIH promoter.
J. Bacteriol.
174:7648-7655[Abstract/Free Full Text].
|
| 37.
|
Wu, H.-M., and D. M. Crothers.
1984.
The locus of sequence-directed and protein-induced DNA bending.
Nature
308:509-513[CrossRef][Medline].
|
| 38.
|
Zulianello, L.,
E. de la Gorgue de Rosny,
P. van Ulsen,
P. van de Putte, and N. Goosen.
1994.
The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers.
EMBO J.
13:1534-1540[Medline].
|
Journal of Bacteriology, July 2001, p. 3910-3918, Vol. 183, No. 13
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.13.3910-3918.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
