 |
INTRODUCTION |
The regulation of transcription of
the Bacillus subtilis pyr operon, which encodes the
enzymes of de novo UMP biosynthesis, has been extensively studied in
this laboratory (for a review, see reference 31).
Expression of this operon is governed by a transcriptional
attenuation mechanism. When uridine nucleotide levels are elevated in
the cells, the protein encoded by the first gene in the operon,
PyrR, acts to promote termination of transcription at three attenuation
sites located in the 5' end of the operon. PyrR brings about
termination by binding to pyr mRNA at specific sites and
altering its secondary structure such that formation of a downstream
transcription terminator is favored.
By contrast, very little is known about the regulation of
expression of pyrG, the gene encoding CTP synthetase,
in B. subtilis or other bacteria. The
pyrG gene is not part of the pyr operon in B. subtilis. The gene was identified by Trach et al.
(32), who called it ctrA, as a gene lying
between rpoE, which encodes the 
subunit of RNA
polymerase, and spo0F. Expression of B. subtilis pyrG is not coordinated with that of the pyr
operon. This conclusion comes from the experiments of Asahi et
al. (3), who showed that in wild-type cells both uridine
and cytidine repressed the pyr operon enzymes but
not CTP synthetase. In a cytidine deaminase-deficient mutant, which
cannot convert cytidine to uridine, cytidine was able to repress CTP
synthetase but was no longer able to repress genes of the
pyr operon. These results suggest that repression of
the pyr operon is brought about by uridine
nucleotides, in accord with our present understanding of the system
(31), but that pyrG is specifically
repressed by cytidine nucleotides. Nothing is known about the
mechanism of this repression.
The only other study of the regulation of bacterial pyrG of
which we are aware is that of West and O'Donovan (36).
These authors reported that repression of pyrG by cytidine
could be demonstrated only in a Salmonella enterica serovar
Typhimurium strain in which the cytidine deaminase gene
cdd was inactive and in which a leaky UMP kinase
(pyrH) gene brought about abnormally low levels of
pyrimidine nucleoside di- and triphosphates. Again, no information
about the mechanism of this repression is available.
In the present study we identified the site of transcriptional
initiation for B. subtilis pyrG. We showed that repression is responsive to cytidine nucleotide levels and is independent of both
uridine nucleotides and PyrR-dependent attenuation. To do this, it was
necessary to determine pyrG expression in mutant strains in
which nucleotide pools could be manipulated by blockage of the de novo
biosynthetic pathway (using pyrB and pyrDII
mutants) or by inactivation of genes involved in interconversion of
uridine and cytidine nucleotides (using cdd and
pyrG mutants) (Fig. 1). Primer
extension analysis and mutational analysis of the pyrG 5'
leader indicates that repression is brought about by a transcriptional antitermination mechanism that involves an attenuator lying between the
pyrG promoter and its coding region.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1.
Genes for pyrimidine nucleotide metabolism in
B. subtilis. Known pathways for de novo
biosynthesis and salvage are shown. Transport proteins implicated in
uptake of pyrimidines are indicated by shaded boxes. Genes that were
disrupted in strains used in this study to characterize the repression
of pyrG are indicated in boldface.
|
|
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, media, and growth conditions.
Bacterial strains used in this study are listed in Table
1. LB medium (22) was used
as the rich liquid medium and the solid medium (1.5% agar) for both
Escherichia coli and B. subtilis. Buffered
minimal medium (19) was used as the chemically defined growth medium. Histidine (50 µg/ml) was used for growth of B. subtilis strain DB104 and its derivatives. To starve B. subtilis strain HC11 for pyrimidines, 100 µg of orotate per ml
was used. To grow B. subtilis in the presence of excess
pyrimidines, either 50 µg of uracil per ml plus 50 µg of uridine
per ml or 200 µg of cytidine per ml was used. For selection of
antibiotic resistance, antibiotics were used at the following
concentrations: sodium ampicillin, 100 µg/ml; chloramphenicol, 6 µg/ml for B. subtilis and 20 µg/ml for E. coli; spectinomycin, 100 µg/ml; erythromycin 1 µg/ml.
Bacterial transformation, isolation, manipulation, and analysis
of DNA and RNA.
Transformation of E. coli was carried
out as described by Sambrook et al. (26). Transformation
of B. subtilis followed the procedure of Contente and
Dubnau (6).
Plasmid DNA from E. coli was isolated as described by
Sambrook et al. (26) and purified using the plasmid
Miniprep kit from Qiagen. B. subtilis chromosomal DNA
was isolated by the protocol of Wilson (37). PCR was
performed with VentR or Taq DNA
polymerase in a Perkin-Elmer Cetus DNA thermal cycler by the procedure
recommended by the manufacturer. DNA sequencing was done at the Genetic
Engineering facility in the University of Illinois Biotechnology Center.
Total RNA was extracted from B. subtilis according to
the method of Saxild et al. (29). Cells were harvested in
exponential phase at a cell density of approximately 90 Klett units
except for strain HC11 grown on orotate and DB104
pyrG::erm grown with limiting cytidine, which
were harvested at 60 to 65 Klett units. To remove DNA from total RNA
samples, the DNA-free kit from Ambion was used as recommended by
the supplier.
Strain constructions.
B. subtilis strain
DB104 
pyrDIIcdd was constructed as follows. Plasmid
pMutin was digested with BclI. The 5.9-kb fragment was gel
purified and religated, yielding plasmid pHV502, which lacks the
lacI gene and part of lacZ. A 186-bp fragment
from the cdd gene was amplified from B. subtilis chromosomal DNA by PCR using primers cddHindIII and
cddBamHI (Table 2). The PCR product was
digested with HindIII and BamHI and ligated
to HindIII-BamHI-digested pHV502 to form plasmid pMX.
pMX DNA was introduced into strain DB104 pyrDII by
transformation, selecting for erythromycin resistance. B. subtilis strain DB104 pyrG::cm was
constructed by transforming NdeI-linearized pJH4521 into
DB104, selecting for chloramphenicol resistance, and confirming that
the transformants were auxotrophic for cytidine. The B. subtilis strain DB104 pyrG::erm was
constructed by transforming plasmid pCm::Er into DB104
pyrG::cm.
Construction and integration of pyrG-lacZ fusion
integrant strains.
The integration plasmids listed in Table 1 were
constructed by PCR amplification of fragments containing the desired
region of pyrG using plasmid pJH4133 as the template. The
PCR fragments were digested with EcoRI and BamHI
and ligated into EcoRI- and BamHI-digested pDH32.
The primers used in making each construct are listed in Table 2 as
pyrG-A through pyrG-H. For each of the integration plasmids, the last
two letters of the name (Table 1) indicate the primers from this series
used to construct it. However, pMSANMF was constructed through several
steps. First, 
96 to +20 of the pyrG sequence was amplified
by PCR with primers pyrG-A and pyrG-N. This PCR fragment was digested
with EcoRI and SacI and ligated to
EcoRI-SacI-digested pUC19 to form pUCAN. Then, +30 to +218 of the pyrG sequence was amplified by PCR with
primers pyrG-M and pyrG-F. After digestion with
SacI-BamHI, this PCR product was ligated to
SacI-BamHI digested pUCAN to form pUCANMF.
Because of the overhang and restriction site introduced, nucleotides
+21 to +29 of the pyrG 5' leader sequence were substituted
as shown in Fig. 6. A 0.32-kb EcoRI-BamHI
fragment of pUCANMF was subcloned into pDH32 to form pMSANMF.
pyrG-lacZ fusion integrant strains were constructed as
previously described (33). The integration plasmids were
linearized with ScaI and PstI and transformed
into B. subtilis. Transformants were selected for
chloramphenicol resistance, and disruption of amyE was
verified on 1% starch-LB plates.
Primer extension, Northern hybridization, and RT-PCR.
Primer
extension was performed as described by Saxild et al.
(29). Reverse transcription (RT) reactions with avian
myeloblastosis virus reverse transcriptase (Promega) were incubated at
42°C for 1 h with 10 µg of total RNA and either primer C or
primer B (Table 2). The DNA sequencing ladder used for analysis of
primer extension was generated by the dideoxynucleotide method of
Sanger et al. (27) using the T7 Sequenase v2.0 DNA
sequencing kit (Amersham Life Science) and plasmid pJH4133 as the template.
Northern blot analysis was performed with a modification of the method
of Nygaard et al. (24). RNA was separated on a 1.5% agarose gel and transferred to a BrightStar-Plus positively charged nylon membrane (Ambion). After transfer, RNA was cross-linked to the
membrane by a UV cross-linker. ULTRAhybe hybridization buffer (Ambion)
was used for hybridization. The DNA probes for Northern blot analysis
were generated by standard PCR except that unlabeled dCTP was used at 1 µM and [
-32P]dCTP was added at 0.16 µM
and a total radioactivity of 50 µCi. Plasmid pJH4133 was used as the
template, and pyrG-O and pyrG-P were used as primers for labeling the
pyrG probe. EcoRI-HindIII digested
pFL32 was used as the template, and rpoE-A and rpoE-B were used as
primers for labeling the rpoE probe.
RT-PCR was performed using Superscript II RNase
H
reverse transcriptase (Gibco BRL) for RT as
recommended by the supplier. Total RNA (0.2 µg) treated with DNase I
and 5 ng of primer per µl were used. Two of the 20 µl of RT
reaction mixture was used for PCR. Taq polymerase (Gibco
BRL) was used for PCR. Plasmid pJH4133 was used as the template for the
positive PCR control. Reverse transcriptase was omitted from the normal
reaction to provide a negative control. The primers used for RT-PCR are
listed in Table 2.
Enzymatic assays.
-Galactosidase activity was determined
by a protocol (19) modified from Miller's method
(22). The data in Table 3
are the averages of at least six determinations with the indicated standard deviations. Cells were harvested for assay in the exponential phase of growth at a density of about 90 Klett units, except for slowly
growing derivatives of strain HC11 grown on orotate or QM206 grown with
limiting cytidine, which were harvested at a density of 35 to 65 Klett
units.
Unpublished DNA sequence data.
Preliminary sequence data for
portions of the pyrG genes from several bacteria listed in
Fig. 7 were obtained from The Institute for Genomic Research (TIGR) at
http://www.tigr.org.
| |
RESULTS |
Characterization of pyrG transcripts.
A map of
the B. subtilis genome in the region surrounding
pyrG (16, 32) is shown in Fig.
2A. The pyrG gene lies
immediately downstream of the rpoE gene and is transcribed
in the same direction. The next gene downstream of pyrG is
ywjG, a gene of unknown function transcribed in the opposite
direction. Examination of the DNA sequence of the intercistronic region
between rpoE and pyrG (Fig. 2B) revealed a
reasonable 
A-dependent promoter sequence
upstream of a possible factor-independent transcription terminator
sequence, which presumably functions as the terminator for the
rpoE gene. The site of initiation of pyrG
transcription was determined by primer extension analysis using two
single-stranded deoxyoligonucleotide primers. Primer B was
complementary to mRNA from the pyrG coding region and
downstream from the terminator sequence, whereas primer C was
complementary only to an mRNA sequence upstream of the terminator (Fig.
2B).
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 2.
(A) Structure of the rpoE-pyrG region of
the B. subtilis chromosome. Shaded bars indicate
open reading frames. The genes shown are described in the text. Arrows
indicate the location and direction of transcription of putative
promoters for the genes. (B) Nucleotide sequence of the
rpoE-pyrG intercistronic region. Shaded bars denote the
3' end of the rpoE coding region and the 5' end of the
pyrG coding region. Translation stop and start
codons are underlined, as is the ribosome-binding site (RBS) for
pyrG. 35 and 10 denote the A
recognition sequence for the pyrG promoter. Straight
arrows denote complementary sequences that form a factor-independent
terminator hairpin, and wavy arrows show sequences complementary to
primers B and C used for primer extension experiments in this work (see
Fig. 3).
|
|
Template RNA for primer extension reactions using both primer B and
primer C was extracted from HC11 (pyrB::spec)
cells grown in the presence of 200 µg of cytidine per ml, which
represses pyrG expression, and from HC11 cells grown in the
presence of 100 µg of orotate per ml, a poor pyrimidine source that
causes severe pyrimidine starvation (19). For primer
extension reactions using primer B, RNA was also extracted from DB104
pyrG::erm cells grown in the presence of 10 µg of cytidine per ml plus 50 µg of uridine per ml. As shown below
for experiments with strain QM206 in Table 3, this is a second method
to derepress pyrG without resorting to starvation of a
pyrimidine auxotroph.
In all cases where a primer-extended product was detectable, the
lengths of the extended reverse transcripts from both primers mapped
the major site of transcription initiation to the G residue indicated as +1 in Fig. 2B (Fig. 3). For
primer extension reactions using primer B (located downstream of the
terminator), both methods used to derepress pyrG expression
led to equal amounts of primer-extended product (Fig. 3, right, lanes 1 and 2), whereas RNA extracted from cells grown under conditions that
repress pyrG gave only traces of primer-extended product
(Fig. 3, right, lane 3). This result suggests that repression by
cytidine acts by increasing transcription termination at the terminator
that lies just downstream from the pyrG promoter. For
primer extension reactions using primer C (located upstream
of the terminator), RNA from cells grown under both repressing and
derepressing conditions yielded approximately equal amounts of
primer-extended products (Fig. 3, left, lanes 1 and 2), as expected if
repression is mediated by the terminator downstream from the primer
site.
View larger version (79K):
[in this window]
[in a new window]
|
FIG. 3.
Identification of the start site of pyrG
transcription by primer extension. (Left) Lane 1, 10 µg of total RNA
from strain HC11 grown with 200 µg of cytidine per ml was used; lane
2, 10 µg for total RNA from strain HC11 grown with 100 µg of
orotate per ml was used; lane 3, no primer extension (primer C only was
subjected to electrophoresis). The left side of the panel shows the
results of dideoxy sequencing of the rpoE-pyrG region
with primer C to allow identification of the end of the primer-extended
product. (Right) Lane 1, 10 µg of total RNA from strain DB104
pyrG::erm grown on minimal medium
with 10 µg of cytidine and 50 µg of uridine per ml was used; lane
2, 10 µg of total RNA from strain HC11 grown with 100 µg of orotate
per ml was used; lane 3, 10 µg of total RNA from strain HC11 grown
with 200 µg of cytidine per ml was used. Prematurely terminated
primer extension products are indicated by a, b, and c; product a
probably results from blockage of reverse transcriptase at the
terminator hairpin, and products b and c occur in A- and U-rich regions
of the reverse transcriptase template. The right side of the panel
shows the results of dideoxy sequencing of the rpoE-pyrG
region with primer B.
|
|
For cells grown under derepressing conditions, a ladder of somewhat
longer primer-extended products (1 to 10 nucleotides [nt] longer than
the primary extended product) was clearly observed with primer C
(Fig. 3, left, lane 2) and possibly also with primer B (Fig. 3, right,
lanes 1 and 2). This observation raised the possibility that some
pyrG-containing transcripts might originate from the
upstream rpoE promoter, i.e., that under derepressing conditions some rpoE-pyrG bicistronic
transcripts are formed. Alternatively, the longer
primer-extended products seen with primer C could have resulted
from extension of the extreme 3' end of rpoE transcripts if
they end at the terminator in the pyrG 5' leader. In that
case, the laddering of products would suggest that the rpoE
transcripts are partially degraded.
The length of pyrG and rpoE transcripts was
probed further by Northern hybridization analysis. Bulk RNA was
isolated from HC11 cells grown with cytidine and with orotate, which
are repressing and derepressing conditions for pyrG. Probes
designed to hybridize specifically to transcripts from the
pyrG and rpoE coding regions (Fig. 2A) detected
transcripts that were 1.8 and 0.7 knt in length, respectively (Fig.
4). These are the sizes expected for
separate pyrG and rpoE mRNAs. The abundance of
the rpoE transcripts was not affected by pyrimidine
starvation, but the pyrG transcripts were much more abundant
under these conditions (Fig. 4). This observation indicates that most
or all of the pyrG transcripts whose abundance is regulated
by cytidine originate from the promoter we have mapped to the
rpoE-pyrG intercistronic region. In no case was a transcript
of 2.5 knt, corresponding to a full-length rpoE-pyrG bicistronic transcript, detected. However, the pyrG
transcripts show evidence of considerable degradation, and the presence
of a small amount of hybridizing material larger than 1.8 knt may indicate that an rpoE-pyrG transcript, if one was formed,
could have been degraded under the conditions used.
View larger version (54K):
[in this window]
[in a new window]
|
FIG. 4.
Northern hybridization analysis of rpoE
and pyrG transcripts. Total RNA (10 µg) from strain
HC11 grown on minimal medium with 200 µg of cytidine per ml
(lane 1) or 100 µg of orotate per ml (lane 2) was subjected to
electrophoresis, electroblotted onto nylon membranes, and hybridized
with 32P-labeled deoxyoligonucleotide probes. The positions
of RNA size standards (in kilonucleotides) are on the right of each
panel.
|
|
The question of rpoE-pyrG cotranscription was examined
further by RT-PCR analysis. RNA from derepressed cells (strain HC11 growth with orotate) was used as a source of pyrG mRNA
template for RT-PCR in which primer B or primer C (Fig. 2B) and a
primer complementary to the 3' end of the rpoE coding region
(pyrG-Q) (Table 2) were used for amplification. RT-PCR products of the expected sizes were obtained with primer C but not with primer B (data
not shown). This result indicates that rpoE transcripts terminate at or very close to the terminator located in the
pyrG 5' leader, but no evidence was found for transcripts
that include both rpoE and pyrG coding sequences.
Regulation of pyrG-lacZ expression by
cytidine nucleotides.
We constructed a series of strains in which
the entire rpoE-pyrG intercistronic region was fused to a
lacZ reporter gene and the fusion was integrated in a single
copy into the chromosomes at the amyE locus of several
B. subtilis strains. These fusions allowed us to study
regulation of B. subtilis pyrG expression by
supplementation of the growth medium with pyrimidine nucleotides or
cultivation of the bacteria under conditions of pyrimidine starvation. The lacZ reporter gene permitted
pyrG expression to be readily determined from
-galactosidase assays, whereas we were unable to assay CTP
synthetase activity reliably in crude extracts of B. subtilis cells. When the pyrG-lacZ fusion was
integrated into a wild-type (strain DB104) background and the cells
were grown on minimal medium with or without pyrimidines, a small but statistically reliable repressive effect of cytidine was observed (Table 3). Cells grown with cytidine expressed 25 to 30% lower levels
of -galactosidase than cells grown without supplementation. Uracil
and uridine did not cause significant repression.
When these experiments were repeated with the pyrG-lacZ
fusion integrated into a strain with an in-frame deletion of the
pyrR gene (33), expression levels were reduced
by about 30% compared to expression in wild-type cells and exhibited
only very small repressive effects of cytidine. Since deletion of the
pyrR gene has been shown to lead to very high, constitutive
overexpression of the genes of the pyr operon
(33), the low expression of the pyrG-lacZ
fusion in the pyrR strain indicates that pyrR
is not involved in regulation of pyrG expression. The
reduced level of pyrG expression in the pyrR
strain probably results from very high levels of intracellular
pyrimidine nucleotides (including CTP) in this strain (21,
33).
Regulation of pyrG expression could be demonstrated more
convincingly by integrating the pyrG-lacZ fusion into
strains that could be starved for pyrimidines. A derivative of DB104
with an in-frame deletion of the pyrDII gene grows slowly in
the absence of pyrimidines and exhibits derepressed pyr
genes because of reduced intracellular pyrimidine nucleotide levels
(14). Expression of pyrG-lacZ was elevated
1.7-fold in a pyrDII background (strain QM203) relative
to the wild-type strain QM201 (Table 3). Addition of pyrimidines to
this strain brought about repression of pyrG, twofold
repression by uracil plus uridine, and almost threefold repression by
cytidine. Since the repressive effects of cytidine might be masked by
deamination of this nucleoside by cytidine deaminase (cdd),
pyrG-lacZ expression was also determined in a pyrDIIcdd background (strain QM204). Repression by uracil
plus uridine was not altered by the cdd mutation, but
cytidine became a more effective repressor (seven- to eightfold
repression). These observations suggest that a cytidine nucleotide is
the most effective metabolite for repression of pyrG and
that uridine nucleotides may act only via conversion to cytidine
nucleotides. The largest derepression of pyrG-lacZ
expression observed in our studies was obtained by growth of a
pyrimidine auxotroph, strain QM205
(pyrB::spec) (11), on orotate,
which is taken up slowly by the cells and results in slow growth and
highly derepressed pyr gene expression (19). As
seen in Table 3, pyrG-lacZ was expressed at very high levels
in the pyrB::spec background when the cells
were starved for pyrimidines by growth on orotate and was repressed
20-fold when the cells were grown with cytidine. Growth of strain QM205 with excess uracil plus uridine brought about 12-fold repression.
The foregoing experiments demonstrated repression of pyrG
expression by pyrimidines in the growth medium and suggested that cytidine nucleotides were more effective than uridine nucleotides as
corepressors, but they did not allow the cytidine and uridine nucleotide pools to be manipulated separately. This was accomplished by
studying pyrG-lacZ expression when the fusion was integrated into a strain in which the normal chromosomal pyrG gene was
disrupted by insertion of an erythromycin resistance gene. This strain, QM206, was a cytidine auxotroph; uracil, uridine, and cytosine did not support growth. Interestingly, this strain had a normal cdd gene, which demonstrates that in B. subtilis
unlike enteric bacteria (23)cytidine
deaminase is not sufficiently active to prevent growth of a
pyrG strain on cytidine. Strain QM206 grew more slowly with
10 than with 200 µg of cytidine per ml; cytidine limitation resulted
in derepression of an integrated pyrG-lacZ fusion (Table 3).
Addition of 50 µg of uridine per ml did not increase the slow growth
brought about by 10 µg of cytidine per ml; in fact, it caused the
cells to grow more slowly and resulted in further derepression of
pyrG. This effect can be explained by the fact that cytidine
and uridine are known to share a common uptake protein in B. subtilis, NupC (15, 28). Thus, addition of uridine
results in competitive inhibition of cytidine uptake, decreased pools
of intracellular cytidine nucleotides, and further derepression of
pyrG. Uracil, which is believed to be taken up by the PyrP
carrier in B. subtilis (33), caused modest
inhibition of growth and pyrG derepression. These
experiments indicate that pyrG expression is regulated
largely, if not entirely, by intracellular cytidine nucleotide pools,
because the highest expression was obtained in
pyrG::erm cells that were the most severely
starved for cytidine but would be predicted to have high uridine
nucleotide pools.
Mutational analysis of the pyrG leader indicates an
antitermination mechanism for repression but does not identify an
antiterminator in the pyrG RNA.
Additional insight
into the elements present in the rpoE-pyrG intercistronic
region that are important for pyrG expression and its
repression by cytidine was obtained by characterizing the expression of
a series of deletions of the QM205 pyrG-lacZ fusion that was
studied for Table 3. The parent fusion and the deletions shown
in Fig. 5 were integrated into strain
HC11 (pyrB::spec) and grown under repressing
and derepressing conditions, i.e., in minimal medium with either 200 µg of cytidine or 100 µg of orotate per ml, respectively. Normal
expression and regulation were obtained with all fusion-integrant
strains that contained sequences from the 3' end of rpoE to
+80 in the pyrG leader transcript. However, further
deletion of pyrG leader sequences from +37 to +80, which
specify a putative factor-independent transcription terminator, led to
highly derepressed pyrG expression, which was not reduced by
cytidine in the medium (Fig. 5, strain QM104). The threefold further
increase in expression in cells of this strain that were starved by
growth on orotate may be a nonspecific effect of this extreme
starvation, as has been previously observed for the pyr
operon (19). This conclusion is supported by the observation that an alternative mode of pyrG derepression,
growth of a pyrDIIcdd strain without pyrimidines, yielded
only 1.6-fold derepression in the QM234 strain containing the same
deletion of the terminator but 8-fold derepression in strain QM204
(Fig. 5). When a downstream segment from the rpoE-pyrG
intercistronic region, +57 to +110, was fused to lacZ and
integrated into strain HC11, no detectable -galactosidase was
expressed. These findings support the conclusions from the primer
extension experiments that the only active pyrG promoter
lies upstream of transcription terminator that is predicted to lie
between nt +38 and +70 of the pyrG transcript leader (Fig.
2B). Furthermore, the repression of pyrG expression by
cytidine requires the presence of this terminator. This leads us to
suggest that termination at this site is efficient when cytidine
nucleotides are abundant in the cell and that cytidine starvation
suppresses termination.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 5.
Deletion analysis of pyrG-lacZ fusion
integrants. The portions of the rpoE-pyrG intercistronic
region shown were fused to lacZ in pDH32 and integrated
into the B. subtilis chromosome at
amyE in the indicated strains. The cells were grown
under repressing (+cytidine) or derepressing (+orotate or cytidine)
conditions and harvested during exponential growth. The
-galactosidase activities of the cells were assayed to determine the
degree of pyrG expression.
|
|
What mechanism could account for regulation of termination in the
pyrG 5' leader by cytidine nucleotides? Clearly,
some mechanism for preventing transcription termination must
act when the cytidine nucleotide pool is low. We searched the
sequence of the 5' leader with the MULFOLD program (12, 13,
39) for a possible RNA antiterminator hairpin and identified
only a single, relatively weak alternative RNA structure that would
prevent the more stable terminator hairpin from forming (Fig.
6). However, a mutagenesis experiment in
which nucleotides 21 through 29 were replaced with nucleotides that
cannot base-pair to form the putative antiterminator hairpin in a
pyrG-lacZ fusion integrant gave regulation of
-galactosidase expression by cytidine that was similar to that of
the native pyrG-lacZ fusion integrant (Fig. 6).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 6.
Test of the role of a possible antiterminator hairpin in
the pyrG 5' leader in regulation of pyrG
expression. (Top) Two possible ways of folding the pyrG
5' leader RNA as predicted by MULFOLD. On the left is the more stable
predicted structure, which includes a factor-independent transcription
terminator. On the right is a less stable alternative structure in
which a putative antiterminator hairpin could form. The nucleotides in
boldface are those present in the native sequence (QM205) which were
replaced by the sequence 5'-GAGCUCAUU-3' in the mutant
strain QM301. Sequencing of the mutant DNA confirmed the correct
structure, except that the nucleotide at +5, which is a C in QM205, had
also been replaced by a U in QM301. (Bottom) Results of
-galactosidase assays determining the levels of expression of the
pyrG-lacZ fusions in QM205 and QM301 under repressing
(+cytidine) and derepressing (+orotate) conditions. Cells were grown in
the minimal medium described by Lu et al. (19).
|
|
| |
DISCUSSION |
Prior to the present study, little was known about the regulation
of pyrG expression in B. subtilis. Our
results demonstrate that pyrG transcription is regulated by
cytidine nucleotides, although the repressive effects of cytidine are
very small unless the cells are starved for pyrimidines and are
enhanced when the gene encoding cytidine deaminase is inactivated. This
suggests that the intracellular level of the regulatory metabolite
formed by de novo biosynthesis is high enough to almost fully repress pyrG. Experiments with a strain in which conversion of UTP
to CTP was blocked by a pyrG disruption allowed us to starve
the cells for cytidine nucleotides while providing abundant uridine nucleotides. Under these conditions pyrG was fully
derepressed, which indicated that the cytidine nucleotides are the
repressing metabolites for this gene. While we have not measured
nucleotide pools directly in our experiments, it seems most
probable to us that the actual regulatory metabolite is CTP, since this
nucleotide is the product of the enzyme encoded by pyrG and
CTP is the cytidine nucleotide used directly in biosynthetic pathways.
The most abundant pyrG transcripts found in B. subtilis cells originate from a promoter which we identified in
the rpoE-pyrG intercistronic region, and these became more
abundant in cells that were starved for pyrimidines. Bicistronic
rpoE-pyrG transcripts were not detected, however. Most
rpoE transcripts were the size of monocistronic transcripts,
and their abundance was not altered by starvation for pyrimidines. This
is consistent with the report of López de Saro et al.
(18) that the rpoE promoter is expressed at
substantial levels under all conditions examined.
The most important new finding in our work is that B. subtilis pyrG expression is regulated by a factor-independent
transcription terminator located in the pyrG 5' leader
downstream from the pyrG promoter. Termination at this site
is regulated by cytidine nucleotides over an 8- to 10-fold range if the
effects on pyrG-lacZ fusion integrants can be taken to
reflect levels of pyrG mRNA. Evidence for the role of this
terminator was of two kinds. First, primer-extended transcripts
originating from a primer upstream of the terminator (primer C in Fig.
3) were equally abundant in cells grown under repressing and
derepressing conditions, whereas primer-extended transcripts
originating from a primer downstream of the terminator (primer B in
Fig. 3) were much less abundant in cells grown with excess cytidine.
More direct evidence was provided by deletion analysis of the
pyrG leader region in pyrG-lacZ transcriptional fusions (Fig. 5).
The presence of a regulated transcription terminator upstream of the
coding region of the gene being regulated is the hallmark of an
attenuation system. In two of the best-studied cases of biosynthetic operons regulated by attenuation in B. subtilis, the trp operon
(2) and the pyr operon
(31), a more stable antiterminator hairpin forms in the
leader RNA before the terminator hairpin can form. This condition
prevents termination and allows expression of the downstream genes. In
the presence of an end product of the operon, a regulatory
proteintrp RNA-binding attenuation protein plus
tryptophan in the case of the trp operon and PyrR plus UMP or UTP in the case of the pyr operonbinds
to a specific site in the 5' stem of the antiterminator hairpin and
destabilizes it, allowing formation of the terminator.
In other antitermination systems, the terminator hairpin is the most
stable secondary structure in the leader mRNA, and antitermination is
brought about by the binding of a regulatory protein to sequences upstream of the terminator, which prevents terminator formation. In
some cases, such as the E. coli bgl operon
(1) and the B. subtilis sacB
operon (20, 35), the regulatory protein, BglG or SacY, respectively, binds to an RNA antiterminator hairpin that
overlaps the sequence of the terminator hairpin.
Antitermination in the lambdoid phages is mechanistically
similar but involves direct interactions with RNA polymerase and the
Nus proteins (10). In other cases, such as the
nasF operon of Klebsiella
(4), there is no identifiable antiterminator hairpin that
overlaps the terminator. Instead, the antiterminator protein NasR
appears to bind to a hairpin that lies upstream of the terminator
hairpin and is separated from it by a 9-nt linker (4). The
pyrG leader RNA resembles the latter group of
antitermination systems because the terminator structure is predicted
to be the most stable secondary structure. We suggest that a
hypothetical regulatory protein binds upstream of this terminator when
the level of a cytidine nucleotide, most likely CTP, is low and that
the binding of CTP at high concentrations causes the protein to
dissociate from the pyrG mRNA, allowing transcriptional
termination. The RNA sequences of the pyrG leader upstream
of its terminator have no homology to any of the known antitermination
systems. We searched the sequence of the pyrG 5' leader for
antiterminator hairpin structures that overlap with the terminator and
identified only one candidate (Fig. 6). However, mutation of the
sequence of this region so as to prevent the antiterminator from
forming did not reduce the ability of pyrG to be repressed by cytidine.
A comparison of the pyrG 5' leader sequences from 10 different gram-positive bacteria suggests that the mechanism for
pyrG regulation we have identified is found in these other
bacteria as well (Fig. 7). All of the
leader RNA sequences are capable of forming transcription terminator
structures. Furthermore, three sequence segments are conserved in the
RNA of the pyrG leaders. The first of these, GGGC(U/A)C, is
consistently located at the very 5' end of the pyrG
transcript. In some cases this sequence may form part of the stem of a
hairpin structure, as suggested for the B. subtilis
pyrG leader (Fig. 6), but this did not appear to always be the
case. Two additional conserved segments were observed in each of the
leaders, typically GCUCCC and GGGAGC; these were consistently
base-paired to form the base of the stem of a terminator hairpin, as
shown for B. subtilis pyrG in Fig. 6. Only in the case
of Lactococcus lactis subsp. cremoris is the terminator so truncated that its formation is questionable. The presence of these conserved sequence elements in the gram-positive pyrG leaders provides a hint that they are important for
regulation of expression, either by serving as recognition elements for
a regulatory protein or by participation in a higher-order RNA folding structure that we have not yet identified.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 7.
Conserved sequences in the 5' untranslated leader
regions of the pyrG genes from 10 gram-positive
bacteria. For each sequence the first conserved segment is located 7 nt
downstream from a reasonable 10 sequence for a promoter and is likely
to be at or near the 5' end of the pyrG mRNA. The second
and third conserved sequences are complementary and are predicted to
form part of the stem of a rho-independent transcription terminator
that serves as the pyrG attenuator, which is followed by
several U residues (polyU). Sequences and their sources: Bsu,
B. subtilis (16); Bha, Bacillus
halodurans, GenBank accession no. AP001520; Ban, B.
anthracis, TIGR; Bst, B.
stearothermophilus, TIGR; Efa, E. faecalis,
TIGR; Spy, Streptococcus pyogenes, GenBank accession no.
AE006614; Spn, S. pneumoniae, TIGR; Sau,
Staphylococcus aureus, GenBank accession no. AP003364;
Llac, Lactococcus lactis subsp. lactis,
GenBank accession no. AE006284; Lcre, L. lactis
subsp. cremoris, GenBank accession no. AJ010153.
|
|
An unusual feature of our model is that the pyrG attenuator
also functions as the transcription terminator for the upstream rpoE gene, so that under conditions of cytidine starvation
some rpoE-pyrG bicistronic transcripts should be formed.
Other instances of transcription terminators that appear to also
function as attenuators for downstream genes have been described for
the B. subtilis gltX-cysES (7) and
acsA-tyrS (9) operons, but in the
former case there is not a second promoter immediately upstream of the
terminator. From our observations it appears that the majority of
pyrG transcripts originate from the pyrG
promoter, not the rpoE promoter, and, furthermore, that any
bicistronic transcripts that may be formed are too unstable in vivo to
be detected. Evidence for the cleavage of the B. subtilis
gltX-cysES (25) and acsA-tyrS
(5) transcripts at sites near the intercistronic
terminators has been presented. Such cleavage may be a general feature
of such transcripts.
Our hypothesis for the regulation of pyrG by an
antitermination mechanism obviously needs further experimental testing.
Such work is currently under way in our laboratory.
This research was supported by Public Health Service grant
GM47112 from the National Institute of General Medical Sciences.
| 1.
|
Amster-Choder, O., and A. Wright.
1993.
Transcriptional regulation of the bgl operon of Escherichia coli involves phosphotransferase system-mediated phosphorylation of a transcriptional antiterminator.
J. Cell. Biochem.
51:83-90[CrossRef][Medline].
|
| 2.
|
Antson, A. A.,
E. J. Dodson,
G. Dodson,
R. B. Greaves,
X.-P. Chen, and P. Gollnick.
1999.
Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA.
Nature
401:235-242[CrossRef][Medline].
|
| 3.
|
Asahi, S.,
M. Doi,
Y. Tsunemi, and S. Akiyama.
1989.
Regulation of pyrimidine nucleotide biosynthesis in cytidine deaminase-negative mutants of Bacillus subtilis.
Agric. Biol. Chem.
53:97-102.
|
| 4.
|
Chai, W., and V. Stewart.
1999.
RNA sequence requirements for NasR-mediated, nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5aI nasF operon leader.
J. Mol. Biol.
292:203-216[CrossRef][Medline].
|
| 5.
|
Condon, C.,
H. Putzer, and M. Grunberg-Manago.
1996.
Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.
Proc. Natl. Acad. Sci. USA
93:6992-6997[Abstract/Free Full Text].
|
| 6.
|
Contente, S., and D. Dubnau.
1979.
Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.
Mol. Gen. Genet.
167:251-258[CrossRef][Medline].
|
| 7.
|
Gagnon, Y.,
R. Breton,
H. Putzer,
M. Pelchat,
M. Grunberg-Manago, and J. Lapointe.
1994.
Clustering and co-transcription of the Bacillus subtilis genes encoding the aminoacyl-tRNA synthetases specific for glutamate and for cysteine and the first enzyme for cysteine biosynthesis.
J. Biol. Chem.
269:7473-7482[Abstract/Free Full Text].
|
| 8.
|
Grandoni, J. A.,
S. B. Fulmer,
V. Brizzio,
S. A. Zahler, and J. M. Calvo.
1993.
Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine.
J. Bacteriol.
175:7581-7593[Abstract/Free Full Text].
|
| 9.
|
Grundy, F. J.,
D. A. Waters,
T. Y. Takova, and T. M. Henkin.
1993.
Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis.
Mol. Microbiol.
10:259-271[Medline].
|
| 10.
|
Henkin, T. M.
1996.
Control of transcriptional termination in prokaryotes.
Annu. Rev. Genet.
30:35-57[CrossRef][Medline].
|
| 11.
|
Hu, P., and R. L. Switzer.
1995.
Evidence for substrate stabilization in regulation of the degradation of Bacillus subtilis aspartate transcarbamylase in vivo.
Arch. Biochem. Biophys.
316:260-262[CrossRef][Medline].
|
| 12.
|
Jaeger, J. A.,
D. H. Turner, and M. Zuker.
1989.
Improved predictions of secondary structures for RNA.
Proc. Natl. Acad. Sci. USA
86:7706-7710[Abstract/Free Full Text].
|
| 13.
|
Jaeger, J. A.,
D. H. Turner, and M. Zuker.
1989.
Predicting optimal and suboptimal secondary structure for RNA.
Methods Enzymol.
183:281-306.
|
| 14.
|
Kahler, A. E., and R. L. Switzer.
1996.
Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydrooroate dehydrogenase activity in Bacillus subtilis.
J. Bacteriol.
178:5013-5016[Abstract/Free Full Text].
|
| 15.
|
Kloudová, A., and V. Fucik.
1974.
Transport of nucleoside in Bacillus subtilis: characteristics of cytidine uptake.
Nucleic Acids Res.
1:629-637[Abstract/Free Full Text].
|
| 16.
|
Kunst, F.,
N. Ogasawara,
I. Moszer,
A. M. Albertini,
G. Alloni,
V. Azevedo,
M. G. Bertero,
P. Bessieres,
A. Bolotin,
S. Borchert,
R. Borriss,
L. Boursier,
A. Brans,
M. Braun,
S. C. Brignell,
S. Bron,
S. Brouillet,
C. V. Bruschi,
B. Caldwell,
V. Capuano,
N. M. Carter,
S. K. Choi,
J. J. Codani,
I. F. Connerton,
A. Danchin, et al.
1997.
The complete genome sequence of the Gram-positive bacterium Bacillus subtilis.
Nature
390:249-256[CrossRef][Medline].
|
| 17.
|
López de Saro, F. J.,
A. M. Woody, and J. D. Helmann.
1995.
Structural analysis of the Bacillus subtilis factor : a protein polyanion which displaces RNA from RNA polymerase.
J. Mol. Biol.
252:189-202[CrossRef][Medline].
|
| 18.
|
López de Saro, F. J.,
N. Yoshikawa, and J. D. Helmann.
1999.
Expression, abundance, and RNA polymerase binding properties of the factor of Bacillus subtilis.
J. Biol. Chem.
274:15953-15958[Abstract/Free Full Text].
|
| 19.
|
Lu, Y.,
R. J. Turner, and R. L. Switzer.
1995.
Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression.
J. Bacteriol.
177:1315-1325[Abstract/Free Full Text].
|
| 20.
|
Manival, X.,
Y. Yang,
M. P. Strub,
M. Kochoyan,
M. Steinmetz, and S. Aymerich.
1997.
From genetic to structural characterization of a new class of RNA-binding domain within the SacY/BglG family of antiterminator proteins.
EMBO J.
16:5019-5029[CrossRef][Medline].
|
| 21.
|
Martinussen, J.,
P. Glaser,
P. S. Andersen, and H. H. Saxild.
1995.
Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis.
J. Bacteriol.
177:271-274[Abstract/Free Full Text].
|
| 22.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 23.
|
Neuhard, J., and P. Nygaard.
1987.
Purines and pyrimidines, p. 445-473.
In
F. C. Neidhardt (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C.
|
| 24.
|
Nygaard, P.,
P. Duckert, and H. H. Saxild.
1996.
Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis.
J. Bacteriol.
178:846-853[Abstract/Free Full Text].
|
| 25.
|
Pelchat, M., and J. Lapointe.
1999.
In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase.
RNA
5:281-289[Abstract].
|
| 26.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 27.
|
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 28.
|
Saxild, H. H.,
L. N. Andersen, and K. Hammer.
1996.
dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.
J. Bacteriol.
178:424-434[Abstract/Free Full Text].
|
| 29.
|
Saxild, H. H.,
J. H. Jacobsen, and P. Nygaard.
1995.
Functional analysis of the Bacillus subtilis purT gene encoding formate-dependent glycinamide ribonucleotide transformylase.
Microbiology
141:2211-2218[Abstract].
|
| 30.
|
Steinmetz, M., and R. Richter.
1994.
Plasmids designed to alter the antibiotic resistance expressed by insertion mutations in Bacillus subtilis through in vivo recombination.
Gene
142:79-83[CrossRef][Medline].
|
| 31.
|
Switzer, R. L.,
R. J. Turner, and Y. Lu.
1999.
Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein.
Prog. Nucleic Acids Res. Mol. Biol.
62:329-367[Medline].
|
| 32.
|
Trach, K.,
C. J. W.,
P. L. D. Piggot, and J. A. Hoch.
1988.
Complete sequence and transcriptional analysis of the spo0F region of the Bacillus subtilis chromosome.
J. Bacteriol.
170:4194-4208[Abstract/Free Full Text].
|
| 33.
|
Turner, R. J.,
Y. Lu, and R. L. Switzer.
1994.
Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism.
J. Bacteriol.
176:3708-3722[Abstract/Free Full Text].
|
| 34.
|
Vagner, V.,
E. Dervyn, and S. D. Ehrlich.
1998.
A vector for systematic gene inactivation in Bacillus subtilis.
Microbiology
144:3097-3104[Abstract].
|
| 35.
|
Van Tilbeurgh, H.,
X. Manival,
S. Aymerich,
J.-M. Lhoste,
C. Dumas, and M. Kochoyan.
1997.
Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis.
EMBO J.
16:5030-5036[CrossRef][Medline].
|
| 36.
|
West, T. P., and G. A. O'Donovan.
1982.
Repression of cytidine triphosphate synthetase in Salmonella typhimurium by pyrimidines during uridine nucleotide depletion.
J. Gen. Microbiol
128:895-899[Medline].
|
| 37.
|
Wilson, K.
1990.
Preparation of genomic DNA from bacteria, p. 2.4.1-2.4.2.
In
F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, supplement 9. Greene Publishing Associates and Wiley Interscience, New York, N.Y.
|
| 38.
|
Yanisch-Perron, C.,
J. Vieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene
33:103-119[CrossRef][Medline].
|
| 39.
|
Zuker, M., and P. Stiegler.
1981.
Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information.
Nucleic Acids Res.
9:133-148[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
