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Previous Article | Next Article 
J Bacteriol, April 1998, p. 1803-1807, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Promoter Characterization and Constitutive
Expression of the Escherichia coli gcvR Gene
Angela C.
Ghrist and
George V.
Stauffer*
Department of Microbiology, The University of
Iowa, Iowa City, Iowa 52242
Received 9 October 1997/Accepted 20 January 1998
 |
ABSTRACT |
The Escherichia coli glycine cleavage repressor protein
(GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational
start site was determined by N-terminal amino acid sequence analysis of
a GcvR-LacZ fusion protein. Primer extension analysis of the
gcvR promoter region identified a primary transcription
start site 27 bp upstream of the UUG translation start site and a minor
transcription start site approximately 100 bp upstream of the
translation start codon. The -10 and -35 promoter regions upstream of
the primary transcription start site were defined by mutational
analysis. Expression of a gcvR-lacZ fusion was unaltered in
the presence of glycine or inosine, molecules known to induce or
repress expression of gcv, respectively. In addition, it
was shown that gcvR-lacZ expression is neither regulated by
the glycine cleavage activator protein (GcvA) nor autogenously
regulated by GcvR. From DNA sequence analysis, it was predicted that
the translation start codon of the downstream bcp gene
overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region
of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the
bcp promoter region demonstrated that bcp has
its own promoter within the gcvR coding sequence. These
results show that gcvR and bcp do not form an
operon. Furthermore, the deletion of bcp from the
chromosome had no effect on gcv-lacZ expression.
| |
INTRODUCTION |
The Escherichia coli
glycine cleavage enzyme system catalyzes the cleavage of glycine into
carbon dioxide, ammonia, and 5,10-methylenetetrahydrofolate (9). Glycine is required for both protein and purine
biosynthesis, while 5,10-methylenetetrahydrofolate serves as a
one-carbon donor in the biosynthesis of purines, methionine, thymine,
and numerous methylated products (15). Three components of
the glycine cleavage enzyme system, the GcvT, GcvH, and GcvP proteins,
are encoded by the gcv operon (17). Induced by
glycine (13, 17, 28) and repressed by purines (8,
27), it appears that expression of the gcv operon is
regulated in order to balance cellular requirements for glycine and
one-carbon units.
Currently, four proteins, the leucine-responsive regulatory protein
(Lrp), the purine repressor protein (PurR), the glycine cleavage
activator protein (GcvA), and the glycine cleavage repressor protein
(GcvR), have been shown to be involved in regulating expression of the
gcv operon. Lrp is a global regulatory protein involved in
the control of transcription of numerous genes involved in amino acid
metabolism (6) and is required for normal induction of
gcv (11, 21). Lrp binds to multiple sites
upstream of the gcv promoter, suggesting a direct role for
Lrp in gcv expression (21). Whether Lrp interacts
with RNA polymerase, one of the other regulatory proteins, or plays a
structural role by bending DNA is unknown.
PurR is a negative regulator of many genes involved in nucleotide
metabolism (30), including gcv (27).
PurR mediates a twofold decrease in gcv transcription in
response to purine supplementation and has been shown to bind to the
gcv control region, overlapping the gcv
transcription start site (20, 27).
GcvA and GcvR work in concert to further regulate gcv
expression. In glucose minimal medium, GcvR negatively regulates
gcv expression, resulting in a low, basal level of
gcv expression (8). In glycine-supplemented
cultures, GcvR repression is relieved, and GcvA activates
gcv expression (8, 28). In purine-supplemented cultures, both GcvA and GcvR are required to mediate a PurR-independent repression of gcv (8, 27). GcvA has been shown to
bind to three sites upstream of the gcv promoter, and
mutations have verified that all three sites are required for normal
GcvA-mediated activation and repression of gcv
(29).
It has been shown that gcv expression can be altered by
changing the ratio of GcvA to GcvR (8). Overexpression of
GcvA leads to constitutive activation of gcv, even in the
absence of glycine, while overexpression of GcvR causes superrepression
of gcv, even in the absence of purines. Therefore, it is
important to understand how the expression of gcvA and
gcvR is regulated. Expression of gcvA is
negatively autoregulated and is unaffected by glycine or purine
supplementation (26). It has also been shown that GcvR has
no effect on gcvA expression (8). In this study,
we characterized the promoter region of gcvR and
investigated transcriptional regulation of gcvR. In
addition, we show that bcp, a gene located immediately
downstream of gcvR, is not in an operon with gcvR
and plays no role in the regulation of gcv expression.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
The E. coli K-12
strains and plasmids used in this study are described in Table
1.
Media and growth conditions.
The complex medium used was
Luria-Bertani (LB) broth (14). The glucose minimal (GM)
medium used was minimal salts (25) supplemented with 0.4%
glucose, phenylalanine (50 µg/ml), and thiamine (1 µg/ml).
Additional supplements were added, where indicated, at the following
concentrations: inosine, 50 µg/ml; glycine, 300 µg/ml; ampicillin,
150 µg/ml; and kanamycin, 30 µg/ml. 
lysogens carry the
cI857 mutation resulting in a temperature-sensitive repressor and were grown at 30°C. All other strains were grown at
37°C.
Nucleotide sequencing.
Dideoxynucleotide DNA sequencing was
performed by using a Sequenase version 2.0 sequencing kit (United
States Biochemical, Cleveland, Ohio).
Construction of gcvR-lacZ and bcp-lacZ
translational fusions.
The gcvR-lacZ fusion plasmid
pGS343 was constructed by cloning the EcoRI-SspI
fragment carrying the control region and initial coding sequence of
gcvR from plasmid pGS334 (Fig.
1) into the EcoRI-SmaI sites of the lac fusion
plasmid pMC1403 (7), forming an in-frame fusion of
gcvR to the lacZYA genes. Plasmids bearing mutations in the -10 (-13A) and -35 (-36A) regions of the
gcvR-lacZ fusion promoter (pGS393 and pGS394, respectively)
were created by the PCR megaprimer mutagenesis procedure
(18), using primers GcvR7
(5'-GCATACATCAATCAGAACGG-3') and GcvR8
(5'-GCATGTTTTTTTTATGCATTCCTTAAG-3') (mutations
are underlined; see Fig. 2). Plasmid pGS343 was used as a template.
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FIG. 1.
Diagram of the EcoRI-HindIII
fragments of plasmids pGS334 and pGS434. The locations and directions
of transcription of gcvR and bcp are indicated by
arrows. The EcoRI sites of both fragments as well as the
HindIII site of the pGS334 fragment were PCR generated.
A, AflII; H, HindIII; RI, EcoRI;
RV, EcoRV; P, Ppu10I; Ss, SspI; St,
StuI.
|
|
The bcp-lacZ fusion plasmid pGS430 was constructed by
cloning the EcoRI-EcoRV fragment carrying
gcvR and the initial coding sequence of bcp from
pGS334 (Fig. 1) into the EcoRI-SmaI sites of
pMC1403. Plasmid pGS431, carrying a gcvR promoter mutation upstream of a bcp-lacZ fusion, was constructed in two steps.
First, the EcoRI-AflII fragment carrying the
wild-type gcvR promoter was deleted from plasmid pGS334
(Fig. 1) and replaced with the same fragment carrying the -13A mutation
from pGS393. This plasmid was designated pGS429. Then, a
bcp-lacZ fusion was constructed downstream of this
gcvR promoter mutation by cloning the
EcoRI-EcoRV fragment from pGS429 into the
EcoRI-SmaI sites of pMC1403.
All fusions were cloned into bacteriophage gt2 (16) as
previously described (23), and the resultant phage were used
to lysogenize the appropriate strains. Lysogens were tested for the presence of a single copy of the phage by infection with phage cI90c17 (19).
Purification and N-terminal amino acid sequencing of a GcvR-LacZ
fusion protein.
Wild-type strain GS162 carrying the
gcvR-lacZ fusion plasmid pGS343 was grown in LB-ampicillin,
and the GcvR-LacZ fusion protein was extracted and partially purified
by affinity chromatography (22). To further purify the
fusion protein, approximately 5 µg of fusion protein was
electrophoresed on a sodium dodecyl sulfate-10% polyacrylamide (PA)
gel and electroblotted onto a polyvinylidene difluoride membrane. The
sequence of the first 14 amino acids was determined by automated
N-terminal amino acid sequencing at the University of Iowa Protein
Facility.
Primer extension analysis.
To determine the location of the
gcvR promoter, primer extension mapping was performed with a
Promega (Madison, Wis.) primer extension kit. Wild-type strain GS162
carrying the gcvR+ plasmid pGS334 was grown in
LB-ampicillin, and total RNA was isolated as previously described
(3). Primer extension reactions were carried out by the
Promega protocol, using the 32P-labeled primer GcvR4
(5'-GCAACTACTGACATGACGGGTG-3'), which hybridizes 71 bp
downstream of the gcvR translation start site. Reaction products were electrophoresed on a 5% PA gel next to DNA sequencing reactions also generated with primer GcvR4.
To determine the location of the bcp promoter, primer
extension mapping was performed as described below, using SuperScript II RNase H
reverse transcriptase (RT) (Gibco BRL,
Gaithersburg, Md.). Total RNA was isolated from strain GS597 as
previously described (3). Approximately 5 µg of RNA and
either 100 fmol (reaction 1) or 1 pmol (reaction 2) of
32P-labeled primer Bcp2 (5'-CTCCGTCTTGATCCGGCAAGC-3')
were mixed in 1× RT buffer (50 mM Tris-HCl [pH 8.3], 75mM KCl,
3 mM MgCl2), heated to 60°C for 10 min, and then allowed
to anneal at 25°C for 10 min. Extension reactions were carried out in
1× RT buffer, 400 nM deoxynucleoside triphosphates 100 µg of bovine
serum albumin per ml, and 1 mM dithiothreitol in a total volume of 10 µl at 42°C for 30 min, using 10 (reaction 1) or 100 (reaction 2) U
of RT. Reactions were stopped by adding 8 µl of sequencing stop mix. The products of each reaction were electrophoresed on a 5% PA gel next
to DNA sequencing reactions also generated with primer Bcp2.
-Gal assays.
-Galactosidase (-Gal) enzyme assays
were performed as described by Miller (14). All results are
the average of two or more assays, with each sample being determined in
triplicate. All standard deviations are 
12% of the mean.
Construction of a gcvR-bcp chromosomal deletion.
An approximately 1.9-kb fragment carrying gcvR and
bcp was cloned by PCR amplification of Kohara phage 424 (10), using primers GcvR1
(5'-GAATTCGCAATTACCGGAATGCGCC-3') and Bcp1
(5'-GCGCATGGGCATTCACCGGACGGC-3'). This fragment was then
digested with EcoRI and HindIII, and the resulting fragment was cloned into the
EcoRI-HindIII sites of plasmid pGS433, which
is a pBR322 (5) derivative in which the SspI site
has been replaced with a SmaI site. This plasmid was designated pGS434 (Fig. 1). A deletion from codon 35 of gcvR
through codon 49 of bcp (Fig. 1) was made by digesting
pGS434 with SspI and StuI. The deleted fragment
was then replaced with a blunt-ended, SalI-HindIII fragment carrying the kanamycin
resistance gene (neo) from Tn5 (4).
This plasmid was designated pGS437.
Initial attempts to integrate this deletion into the chromosome by
linear transformation were unsuccessful, most likely due to a limiting
amount of homologous DNA flanking the neo gene. Therefore,
an additional 2 kb of flanking DNA was introduced upstream of the
neo gene by cloning an EcoRI-Ppu10I
fragment from Kohara phage 424 carrying approximately 2.3 kb of DNA
upstream of the gcvR coding sequence into the
EcoRI-Ppu10I sites of pGS437, creating plasmid
pGS451. This plasmid was digested with EcoRI and
HindIII and used to perform a linear transformation of
strain GS1066 (recD::Tn10). Transformants were selected on LB-kanamycin and checked for ampicillin sensitivity by spotting on LB-ampicillin. One kanamycin-resistant, ampicillin-sensitive transformant was designated GS1111.
Nucleotide sequence accession number.
The nucleotide
sequence data reported here are in the GenBank database under accession
no. AFO23337.
| |
RESULTS AND DISCUSSION |
Nucleotide sequence of gcvR.
Plasmid pGS334 carries the
E. coli gcvR gene and a portion of the downstream
bcp gene on an approximately 1-kb
EcoRI-HindIII fragment (Fig. 1). In this
study, the nucleotide sequence of most of this PCR-generated fragment
was determined (Fig. 2). This sequence differs from the original sequence of this region as determined by
S. C. Andrews et al. (GenBank accession no. M37689) but is
identical to the sequence of this region as determined by F. R. Blattner et al. and Y. Yamamoto et al. (GenBank accession no. ECAE000335 and D90877, respectively).
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FIG. 2.
Complete nucleotide and deduced amino acid sequences of
the E. coli gcvR gene and partial nucleotide and deduced
amino acid sequences of the E. coli bcp gene. The
gcvR and bcp transcription start sites (+1), -10 and -35 promoter regions, and ribosome binding sites (RBS) are
underlined and in boldface. A potential secondary gcvR
transcription start site and its promoter are underlined. Mutations in
the gcvR promoter constructed in vitro are shown below the
sequence. The gcvR amino acid sequence in boldface was
verified by N-terminal amino acid sequencing.
|
|
gcvR translation start site.
To determine where
translation of gcvR begins, a gcvR-lacZ fusion
plasmid was constructed, the GcvR-LacZ fusion protein was purified, and
the sequence of the first 14 N-terminal amino acids was determined
(Fig. 2; see Materials and Methods). This sequence indicates that
translation initiates at a UUG codon located 9 bp downstream of a
potential ribosome binding site (Fig. 2). The formylmethionine encoded
by the UUG translation initiation codon is apparently removed from the
nascent polypeptide. These data suggest that GcvR is a 189-amino-acid
protein with a molecular mass of 20.6 kDa. A similarity search
performed with the BLAST (1) program failed to identify any
proteins with significant similarity to the deduced amino acid sequence
of GcvR.
Only 1% of all sequenced E. coli genes use a UUG codon to
initiate translation (91% use AUG; 8% use GUG) (12), and
in such cases, translational efficiency is reduced three- to fivefold (24). Since overproduction of the GcvR protein results in
repression of the gcv operon (8), the use of this
codon as the gcvR translation start site may serve to reduce
translation of gcvR, preventing superrepression of
gcv.
gcvR promoter mapping.
To determine the location
of the gcvR promoter, primer extension mapping was performed
on RNA isolated from the wild-type strain GS162 bearing the multicopy
gcvR+ plasmid pGS334. As shown in Fig.
3A, two primer extension products were
synthesized. The major primer extension product (P1) migrated in
alignment with an A residue located 27 bp upstream of the
gcvR translation start site (Fig. 2). This A residue was
designated +1. The minor primer extension product (P2) indicates that a
second transcription start site may also exist approximately 100 bp
upstream of the gcvR translation start codon. A potential
70 promoter sequence is present upstream of both start
sites (Fig. 2).
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FIG. 3.
Primer extension analysis of gcvR and
bcp transcripts. Primer extension products were synthesized
as described in Materials and Methods and are marked by arrows. DNA
sequencing ladders were generated with the same primer as that used in
the primer extension reactions. The nucleotide sequence of the
complementary strand of each promoter region is indicated to the left.
The nucleotide designated +1 for each promoter is indicated with an
asterisk. (A) gcvR primer extension analysis. Lanes: A, C,
G, and T, DNA sequencing ladders; 1, primer extension products (P1,
primary transcription start site; P2, minor transcription start site).
(B) bcp primer extension analysis. Lanes 1, primer extension
reaction 1; A, C, G, and T, DNA sequencing ladders; 2, primer extension
reaction 2.
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|
To verify the -10 and -35 regions of the promoter sequence upstream of
the primary start site, a mutation was introduced in each region, and
the effect of each mutation on promoter function was determined.
Wild-type strain GS162 was lysogenized with phage carrying either
wild-type (gcvR-lacZ) or mutant
(gcvR-113A-lacZ and gcvR-36A-lacZ) fusions.
The resulting lysogens were grown in GM medium and GM medium
supplemented with either glycine or inosine (molecules known to induce
or repress gcvT-lacZ expression, respectively) and assayed
for -Gal activity (Table 2).
Expression of the wild-type gcvR-lacZ fusion is not affected
by the addition of glycine or inosine, suggesting that these molecules
do not indirectly affect gcv operon expression by altering
gcvR expression. The -13A mutation resulted in a 15-fold
decrease in gcvR-lacZ expression compared to the wild-type
level, and the -36A mutation resulted in a 9-fold decrease in
gcvR-lacZ expression compared to the wild-type level,
regardless of supplementation. Thus, it appears that the primary
transcription start site determined by primer extension mapping is
correct.
The importance of the minor promoter of gcvR is still
unclear. If this promoter functions in vivo to control expression of gcvR, it appears to be relatively weak compared to the
primary promoter, since the -13A and -36A mutations in the primary
promoter eliminate more than 90% of gcvR-lacZ expression
under all growth conditions (Table 2). These results are consistent
with the primer extension results which show that nearly all of the
gcvR mRNA transcript initiates from the primary promoter
(Fig. 3A).
gcvR expression is not regulated by GcvA or GcvR.
Since it is known that GcvR-mediated regulation of gcv
requires GcvA, it is possible that GcvA regulates gcvR
expression. To test this hypothesis, lysogens
GS162gcvR-lacZ and GS1029gcvR-lacZ (gcvA) were grown in GM medium and GM medium supplemented
with either glycine or inosine and assayed for -Gal activity. As
shown in Table 3, GcvA has no effect on
gcvR-lacZ expression. This was true for all growth
conditions.
Since many regulatory proteins are known to regulate their own
expression, we tested the ability of GcvR to regulate
gcvR-lacZ expression. Lysogens GS162gcvR-lacZ
and GS1053gcvR-lacZ (gcvR) were grown in
GM medium and GM medium supplemented with either glycine or inosine and
assayed for -Gal activity. As shown in Table 3, GcvR does not
autoregulate its own expression. This is true for all growth
conditions. Furthermore, when GS162gcvR-lacZ was grown in
rich (LB) medium, or when GM medium-grown cells were harvested from
stationary phase rather than exponential phase, there was no
significant change in -Gal activity (data not shown), suggesting
that gcvR expression does not respond to any other component
in LB medium or to growth phase. Thus, whatever the role is for GcvR in
the negative regulation of the gcv operon, the mechanism
appears to be independent of changes in GcvR levels in the cell.
The bcp gene is not in an operon with gcvR.
The bcp gene, encoding the bacterioferritin comigratory
protein, is located immediately downstream of gcvR. It is
predicted that this open reading frame, first reported by Andrews et
al. (2), encodes a 156-amino-acid protein of unknown
function. Our sequence of gcvR suggests that gcvR
and bcp may form an operon, as the UAA stop codon of
gcvR overlaps the predicted AUG start codon of
bcp (Fig. 2). To test this hypothesis, a translational bcp-lacZ fusion was constructed downstream of the wild-type
gcvR gene as well as downstream of a mutant gcvR
gene carrying the -13A promoter mutation described above. Both fusions
were cloned into phage gt2, and the phage was used to lysogenize the
wild-type strain GS162. The resulting lysogens were grown in GM medium
and assayed for -Gal activity. The lysogen carrying the wild-type gcvR promoter had 325 U of activity, and the lysogen
carrying the mutant gcvR promoter had 323 U of activity.
Thus, a mutation in the -10 region of the primary gcvR
promoter, which results in a 15-fold decrease in gcvR-lacZ
expression, has no effect on the expression of a downstream
bcp-lacZ fusion. These results suggest that gcvR
and bcp do not form an operon.
To further demonstrate that gcvR and bcp do not
form an operon, we tested whether bcp has its own promoter
within the gcvR coding sequence. Primer extension mapping
was performed on RNA isolated from strain GS597. As shown in Fig. 3B,
transcription of bcp begins at a G nucleotide, 8 bp
downstream of a potential 70 promoter and 41 bp upstream
of the putative bcp translation start site as predicted by
Andrews et al. (2) (Fig. 2). The presence of multiple
products in lane 2 compared to the presence of a single product in lane
1 is most likely due to nonspecific primer annealing and extension due
to the 10-fold increase in the amount of primer and RNA polymerase used
in this extension reaction.
The bcp gene has no effect on the regulation of
gcv expression.
Even though gcvR and
bcp are transcribed separately, their proximity still
suggested that bcp might play a role in the regulation of
gcv expression. To test whether bcp plays a role
in gcv expression, we constructed strain GS1111, which
carries a chromosomal deletion of both the gcvR and
bcp genes (see Materials and Methods). Lysogens GS162gcvT-lacZ, GS1053gcvT-lacZ
(gcvR), and GS1111gcvT-lacZ (
gcvR
bcp), each transformed with the single-copy vector pGS311, and
lysogen GS1111gcvT-lacZ, transformed with the single-copy gcvR+ plasmid pGS338, were grown in GM medium
and GM medium supplemented with either glycine or inosine and assayed
for -Gal activity (Table 4).
GS1053gcvT-lacZ[pGS311] and
GS1111gcvT-lacZ[pGS311] had similar levels of
gcvT-lacZ expression under all conditions. In addition,
transformation of GS1111gcvT-lacZ with pGS338 resulted in
wild-type levels of gcvT-lacZ expression under all
conditions, confirming that bcp is not involved in the
regulation of gcv expression.
Possible roles for GcvA and GcvR in the regulation of
gcv.
Expression of the gcv operon is induced in
the presence of glycine and repressed in the presence of purines
(8, 13, 17, 27, 28). Since overproduction of GcvA leads to
activation of gcv, while overproduction of GcvR results in
repression of gcv (8), it is possible that
glycine and inosine alter gcv expression indirectly by
altering the ratio of GcvA to GcvR. However, previous results (8,
26) as well as those presented here demonstrate that glycine and
inosine have no effect on the expression of gcvA and
gcvR. Both genes also appear to be transcribed
constitutively with respect to medium richness and growth phase.
Since both GcvA and GcvR are required for normal regulation of
gcv, it is also possible that they regulate gcv
indirectly by regulating the expression of one another. However, it has
been shown that no reciprocal regulation occurs. Only a twofold
autoregulation by GcvA occurs.
Several models to explain how GcvA and GcvR interact to regulate
gcv expression have been proposed. In one model, GcvA
homocomplexes function as activators, while GcvA-GcvR heterocomplexes
function as repressors. In this model, the coregulators determine the
type of complex formed; glycine leads to the formation of activation complexes, while inosine leads to the formation of repression complexes. Increasing the amount of either GcvA or GcvR would force the
formation of activation or repression complexes, respectively. In a
second model, GcvR may synthesize the corepressor required for the
repressor function of GcvA. In a gcvR mutant, insufficient corepressor would lead to constitutive gcv expression. If
GcvR is overproduced, too much corepressor causes superrepression of gcv. In another model, GcvR may negatively regulate
gcv by modifying the structure of GcvA, changing it from an
activator to a repressor in response to the coregulators. Purification
of GcvR protein and identification of the actual coregulators of
gcv expression should help us to determine which of these
models, if any, is correct.
| |
ACKNOWLEDGMENT |
This work was supported by Public Health Service grant GM26878
from the National Institute of General Medical Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, 3-315A Bowen Science Building, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7791. Fax: (319) 335-9006. E-mail: george-stauffer{at}uiowa.edu.
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Abstract
Introduction
