From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
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ABSTRACT |
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GerE is a transcription factor produced in the
mother cell compartment of sporulating Bacillus subtilis.
It is a critical regulator of cot genes encoding proteins
that form the spore coat late in development. Most cot
genes, and the gerE gene, are transcribed by
Starvation induces the Gram-positive bacterium Bacillus
subtilis to initiate a series of morphological changes that result in the formation of a dormant spore (1). Early in the sporulation process a septum forms that divides the cell into a larger mother cell
compartment and a smaller forespore compartment. Each compartment contains a copy of the genome, and different genes are expressed in
each compartment. Gene expression drives further morphogenesis, including migration of the septum to engulf the forespore in a double
membrane, deposition of cell wall-like material called cortex between
the membranes, and synthesis in the mother cell of proteins that
assemble on the surface of the forespore to produce a tough shell known
as the coat. The developmental process culminates with lysis of the
mother cell to release a mature spore. When nutrients become available
again, the spore germinates, producing a cell that resumes growth and division.
The program regulating transcription of sporulation genes is
exceptionally well understood (2). It involves the synthesis and
activation of four compartment-specific In addition to positive regulation between one transcription factor and
the next in the mother cell cascade, there is evidence of negative
regulation as well. Overexpression of sigK is not the only defect in
gerE mutant cells. Expression of some cot genes
encoding spore coat proteins is reduced or absent, whereas expression
of other genes is increased. The resulting spores have defective coats
and germinate inefficiently (14). Previously, it was shown that
purified GerE binds to DNA sequences matching the consensus RWWTRGGY-YY
(where R is purine; W is A or T; and Y is pyrimidine) and activates
transcription in vitro from the cotB and
cotC promoters (4) and from three promoters in the
cotVWXYZ cluster (3). The position of binding ranges in the
different promoters from well upstream of the DNase I Footprinting--
DNA fragments labeled at only one end
were prepared as follows. For analysis of the sigK promoter
region, a HindIII-XbaI fragment of pBK16 (15) was
labeled at the 3' ends by the Klenow enzyme fill-in reaction and
[ Construction of a sigK-lacZ Fusion--
DNA between Measurement of Western Blot Analysis--
Cells were induced to sporulate by
nutrient exhaustion in DSM at 37 °C as described previously (20).
Samples (1 ml) were collected at hourly intervals during sporulation,
and whole-cell extracts were prepared as described previously (22).
Protein concentrations were determined by the method of Bradford (23). Proteins (5 µg) were separated by sodium dodecyl sulfate (SDS)-14% Prosieve (FMC BioProducts) polyacrylamide gel electrophoresis with
Tris/Tricine electrode buffer (0.1 M Tris, 0.1 M Tricine, 0.1% SDS) and electroblotted onto Immobilon-P
membranes (Millipore). Blots were incubated as described previously
with polyclonal anti-pro- In Vitro Transcription--
Location of the GerE-binding Site in the sigK Promoter
Region--
Purified GerE was shown previously to strongly inhibit
transcription in vitro of the sigK gene by
Fig. 1 shows that GerE protected a
stretch of DNA from DNase I digestion that included the TSS of
sigK and extended downstream. The protection spanned
positions
We conclude that GerE binds to a site in the sigK promoter
region that overlaps the TSS and extends downstream. Presumably, GerE
binding to this site represses sigK transcription in
vitro (4) by interfering with RNA polymerase binding and/or a
subsequent step in initiation. The location of the GerE-binding site
provides a plausible explanation for the lack of an effect of a
gerE mutation on sigK-lacZ expression reported
previously (13). The sigK portion of the fusion extended
only to +4, so it did not contain the entire GerE-binding site.
GerE Inhibits sigK Expression in Vivo--
To determine whether
GerE affects sigK expression in vivo, we
constructed a new sigK-lacZ transcriptional fusion that
includes the GerE-binding site. DNA between
We also compared the level of sigK gene products in
developing wild-type and gerE mutant cells. The primary
translation product of sigK is pro- GerE Inhibits cotD Transcription in Vitro--
In addition to
possible inhibitory effects of GerE on transcription of genes in the
Location of the GerE-binding Site in the cotD Promoter
Region--
To understand how GerE both positively and negatively
affects cotD transcription, we tested whether GerE binds in
the promoter region by performing DNase I footprinting experiments.
Fig. 5 shows that GerE protected a
stretch of DNA from DNase I digestion that included the The Upstream GerE-binding Site in the cotD Promoter Is Necessary
for Activation of Transcription in Vitro but Not for
Repression--
We hypothesized that GerE bound at the upstream site
in the cotD promoter (i.e. recognizing the
perfect match to the consensus centered at Our results strongly support the model that GerE is a repressor of
sigK transcription which lowers the level of
pro- The negative effect of GerE on the The cotD gene, like many other genes in the Fig. 7 illustrates the regulatory
interactions between the four mother cell-specific transcription
factors (circled) and our model for regulation of
cotD transcription at different times during sporulation.
Early in sporulation, K RNA polymerase. Previously, it was shown that the GerE
protein inhibits transcription in vitro of the
sigK gene encoding
K. Here, we show that
GerE binds near the sigK transcriptional start site, to act
as a repressor. A sigK-lacZ fusion containing the
GerE-binding site in the promoter region was expressed at a 2-fold
lower level during sporulation of wild-type cells than gerE
mutant cells. Likewise, the level of SigK protein (i.e.
pro-
K and
K) was lower in sporulating
wild-type cells than in a gerE mutant. These results
demonstrate that
K-dependent transcription
of gerE initiates a negative feedback loop in which GerE
acts as a repressor to limit production of
K. In
addition, GerE directly represses transcription of particular cot genes. We show that GerE binds to two sites that span
the
35 region of the cotD promoter. A low level of GerE
activated transcription of cotD by
K RNA
polymerase in vitro, but a higher level of GerE repressed cotD transcription. The upstream GerE-binding site was
required for activation but not for repression. These results suggest
that a rising level of GerE in sporulating cells may first activate cotD transcription from the upstream site then repress
transcription as the downstream site becomes occupied. Negative
regulation by GerE, in addition to its positive effects on
transcription, presumably ensures that
K and spore coat
proteins are synthesized at optimal levels to produce a
germination-competent spore.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
subunits of RNA polymerase
(RNAP),1 each of which
directs the enzyme to transcribe a particular set of genes.
F and
G control forespore-specific gene
expression. In the mother cell, activation of
E is
followed by the synthesis and activation of
K. In
addition, two small, DNA-binding proteins, SpoIIID and GerE, activate
or repress transcription of many mother cell-specific genes (3-6). The
mother cell transcription factors form a hierarchical regulatory
cascade in which the synthesis of each factor depends upon the activity
of the prior factor, in the order
E, SpoIIID,
K, and finally GerE (7).
K RNAP initiates a feedback loop
that inhibits transcription of the sigE gene encoding
E (8). Since
E RNAP transcribes the
spoIIID gene (9-11), production of SpoIIID is also
negatively regulated (8, 12). This facilitates the switch from the
early
E- and SpoIIID-directed pattern of gene expression
to the late
K- and GerE-directed pattern. GerE is not a
component of this feedback loop (8); however, there was reason to
believe that GerE might initiate a second feedback loop. GerE was shown
previously to inhibit transcription in vitro of the
sigK gene encoding
K (4). Whether GerE
inhibits sigK transcription in vivo was in doubt,
though, because a sigK-lacZ fusion was not
overexpressed in gerE mutant cells (13). We have resolved
this paradox by mapping a GerE-binding site in the sigK
promoter region. This showed that the sigK-lacZ
fusion examined previously did not contain the entire GerE-binding
site. Here, we demonstrate that GerE represses sigK
expression about 2-fold in vivo, and we discuss the
implications of this negative feedback.
35 region to partially
overlapping it. Here, we show that GerE binds to the cotD
promoter region at a position typical for activation of transcription,
and to a position downstream, which may cause repression of
transcription. Our mapping of GerE-binding sites in the cotD
and sigK promoters provides the first information about how
GerE acts as a transcriptional repressor.
EXPERIMENTAL PROCEDURES
-32P]dCTP or at the 5' ends by treatment with
alkaline phosphatase followed by T4 polynucleotide kinase and
[
-32P]ATP. In both cases, the labeled DNA was digested
with PstI, which cleaved off a small fragment containing the
labeled HindIII end, so it did not interfere with subsequent
DNase I footprinting. The labeled XbaI end is 164 bp
downstream of the sigK transcriptional start site (TSS). For
analysis of the cotD promoter region, pLRK100 (15) was
digested with EcoRI, which cleaves 227 bp upstream of the
cotD TSS, and labeled either at the 3' end by the Klenow enzyme fill-in reaction and [
-32P]dATP or at the 5'
end by treatment with alkaline phosphatase followed by T4
polynucleotide kinase and [
-32P]ATP. In both cases,
the labeled DNA was digested with HindIII, which cleaves
downstream of the cotD promoter, to produce a 443-bp fragment that was purified by elution from an 8% polyacrylamide gel
(16). Labeled DNA fragments were incubated with different amounts of
GerE gel purified from Escherichia coli engineered to
overproduce the protein as described previously (3) and then mildly
digested with DNase I according to method 2 as described previously
(4), except 3 pmol of probe was used and a 7-fold (w/w) excess of
poly(dI-dC) as compared with probe was added as competitor. After DNase
I treatment, the partially digested DNAs were electrophoresed in a 7%
polyacrylamide gel containing 8 M urea alongside a
sequencing ladder generated by subjecting the appropriate end-labeled
DNA to the chemical cleavage reactions of Maxam and Gilbert as
described previously (16).
115 and
+28 relative to the sigK TSS was amplified by the polymerase
chain reaction (PCR) using pBK16 (15) as the template. The upstream
primer was 5' GGGAATTCGATGAAGAATATTTTTAAC 3' and the
downstream primer was 5' GCGAAGCTTCCACAAAAGTATGTA 3'. EcoRI and HindIII restriction sites (underlined)
were designed into the 5' ends of the upstream and downstream primers,
respectively, to allow directional subcloning of the PCR product into
EcoRI-HindIII-digested pTKlac (17).
The resulting plasmid pHI16 was linearized by digestion with
BsaI and transformed into B. subtilis ZB307, in
which marker replacement-type recombination created an
SP
::sigK-lacZ specialized transducing phage as
described previously (18). A phage lysate was prepared by heat
induction and used to transduce B. subtilis SG38
(spo+ trpC2) and 522.2 (gerE36
trpC2) (19) with selection for resistance to chloramphenicol (5 µg/ml) on LB agar as described previously (20).
-Galactosidase Activity--
Sporulation was
induced by nutrient exhaustion in DSM at 37 °C as described
previously (20). Samples (1 ml) were collected at hourly intervals
during sporulation, cells were pelleted, and pellets were stored at
70 °C prior to the assay. The specific activity of
-galactosidase was determined by the method of Miller (21), using
o-nitrophenol-
-D-galactopyranoside as the
substrate. One unit of enzyme hydrolyzes 1 µmol of substrate per min
per unit of initial cell absorbance at 595 nm.
K antibodies that detect both
pro-
K and
K (22). The secondary antibody
was horseradish peroxidase-conjugated anti-rabbit immunoglobulin G
(Bio-Rad). Chemiluminescence detection (ECL) was performed according to
the manufacturer's instructions (Amersham Pharmacia Biotech). The
signal intensities were quantified using a GS 505 Molecular Imager
System (Bio-Rad).
K RNAP was partially
purified from gerE mutant cells as described previously
(15). The enzyme was comparable in protein composition and in
cotD- and sigK-transcribing activities to
fraction 24 shown in Fig. 2 of Kroos et al. (15).
Transcription reactions (45 µl) were performed as described
previously (24), except that RNAP was allowed to bind to the DNA
template for 10 min at 37 °C before the addition of nucleotides (the
labeled nucleotide was [
-32P]CTP). Heparin (6 µg)
was added 2 min after the addition of nucleotides to prevent
reinitiation. After the reactions were stopped, 20 µl of each
reaction mixture was subjected to electrophoresis in a 5%
polyacrylamide gel containing 8 M urea, and transcripts
were detected by autoradiography. The signal intensities were
quantified using a Storm 820 PhosphorImager (Molecular Dynamics). To
prepare a DNA template containing the cotD promoter and
lacking the upstream GerE-binding site, DNA between
44 and +227
relative to the TSS was amplified by the PCR using pLRK100 (15) as the
template. The upstream primer was 5' CGGTTTGCATCAGAACATGT
3' (the underlined portion corresponds to the nontranscribed strand of
the cotD promoter region), and the downstream primer was 5'
GGAAGCTTGCATGCCTGCA 3' (the underlined portion
corresponds to a HindIII site downstream of the
cotD promoter in pLRK100).
RESULTS
K RNAP (4). However, expression of a
sigK-lacZ fusion in vivo was unaffected by a
gerE mutation (13). The discrepancy between the two results
could be explained if the sigK-lacZ fusion did not contain a
binding site for GerE that mediates repression. To see if GerE binds
specifically in the sigK promoter region and, if so, to
determine the position of binding, we performed DNase I footprinting experiments.
4 to +19 on the nontranscribed strand (Fig. 1A)
and positions +1 to +20 on the transcribed strand (Fig. 1B).
Complete protection from DNase I digestion was observed at the lowest
concentration of GerE tested, indicating that GerE binds with
relatively high affinity to this site as compared with other
GerE-binding sites mapped previously (3, 4). Fig. 1C shows
the sequence of the sigK promoter in the region protected by
GerE. Within the protected region are two sequences in inverted orientation that overlap slightly and match the consensus sequence for
GerE binding (Fig. 1D).
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Fig. 1.
GerE footprints in the sigK
promoter region. Radioactive DNA fragments separately
end-labeled on the nontranscribed (A) or transcribed
(B) strand were incubated in separate reactions with a
carrier protein (bovine serum albumin, 310 pmol) only (lane
5) or with 6 pmol (lane 1), 12 pmol (lane
2), 60 pmol (lane 3), or 120 pmol (lane 4)
of gel-purified GerE in addition to the carrier protein and then
subjected to DNase I footprinting in a total volume of 45 µl.
Hatched boxes indicate the region protected from DNase I
digestion by GerE. Arrowheads denote the boundaries of
protection, and numbers to the left refer to positions
relative to the TSS, as deduced from sequencing ladders generated by
chemical cleavage of the respective end-labeled DNA at purines
(lane G over A) or guanines (lane G).
C, position of the GerE-binding site in the sigK
promoter region. The nucleotide sequence of the nontranscribed strand
of the sigK promoter region is shown (13).
Overlining and underlining indicate regions on
the nontranscribed and transcribed strands, respectively, protected by
GerE from DNase I digestion. The TSS is shown by an arrow.
D, nucleotide sequences within the GerE-protected region of
the sigK promoter are aligned with the consensus sequence
for GerE binding (3). Matches to the consensus sequence are shown as
capital letters, and numbers refer to positions
relative to the TSS. Note that the sequence shown for the binding site
between +18 and +7 is from the strand opposite that shown in
C.
115 and +28 relative to
the sigK TSS was fused to lacZ, and the fusion
was recombined into phage SP
. The resulting
SP
::sigK-lacZ phage was transduced into wild-type and gerE mutant B. subtilis, creating
lysogens. Fig. 2 shows the average
-galactosidase activity during sporulation of three isolates of each
type. The average maximum activity was 2-fold higher in gerE
mutant cells than in wild-type cells, demonstrating that GerE inhibits
sigK expression in vivo.
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Fig. 2.
sigK-lacZ expression in wild-type
and gerE mutant cells. sigK-directed
-galactosidase activity was measured at the indicated times during
sporulation of congenic wild-type (SG38,
) and gerE
mutant (522.2,
) strains. Points on the graph are
averages for isolates of each type, and error bars show 1 S.D. of the data.
K, an
inactive precursor that is proteolytically processed to active
K (22). We used anti-pro-
K antibodies to
detect both pro-
K and
K in extracts of
cells subjected to Western blot analysis. Fig. 3 shows that the levels of
pro-
K and
K are higher in gerE
mutant cells than in wild-type cells late in sporulation. Quantitation
of the combined pro-
K plus
K signal for
the experiment shown in Fig. 3 and three additional experiments showed
that the maximum level of SigK gene products in wild-type cells during
sporulation, on average, reached 57% (±6%, 1 S.D.) of the level in
gerE mutant cells. In all four experiments, the level of
both pro-
K and
K was elevated in
T7 and T8 samples from the gerE
mutant compared with wild type. These results show that GerE normally
inhibits the accumulation of pro-
K and
K
during the late stages of sporulation. It is likely that GerE represses
transcription of the sigK gene, reducing the synthesis of
pro-
K and
K. This would explain the
similar 2-fold decrease of sigK-directed
-galactosidase
activity (Fig. 2) and pro-
K plus
K (Fig.
3) in wild-type cells compared with gerE mutant cells. The
alternative explanation that GerE in wild-type cells causes increased
turnover of pro-
K and
K and a similar
increase in turnover of
-galactosidase is unlikely because
-galactosidase activity from a sigK-lacZ fusion lacking the GerE-binding site identified in Fig. 1 is similar during
sporulation of wild-type and gerE mutant cells (13), as is
-galactosidase activity from lacZ fusions to many other
genes.
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Fig. 3.
Levels of
pro- K and
K during sporulation of wild-type and
gerE mutant cells. Whole-cell extracts were
prepared from wild-type (SG38) and gerE mutant (522.2) cells
collected at the indicated number of hours after the onset of
sporulation in DSM. Proteins were fractionated by SDS-PAGE and
subjected to Western blot analysis with anti-pro-
K
antibodies, which detect both pro-
K and
K.
K regulon (due to the inhibition of
K
accumulation by GerE), GerE stimulates expression of certain genes in
the
K regulon (3, 4, 7). Expression of a
cotD-lacZ transcriptional fusion was 7-fold higher in
developing wild-type cells than in gerE mutant cells (7). It
was shown previously that purified GerE stimulates cotD
transcription by
K RNAP 2-3-fold (4). We discovered
that at a higher concentration GerE inhibits cotD
transcription in vitro. Fig. 4
shows the result of an in vitro transcription experiment
with a mixture of DNA templates containing the cotD or
cotC promoter. The cotC promoter was included as
an internal control because GerE has been shown to bind to a site
centered at
68.5 relative to the TSS and to activate transcription by
K RNA polymerase (4). Lower levels of GerE stimulated
cotD transcription about 2-fold (based on quantitation of
signals in the experiment shown and one additional experiment), as
reported previously (4), but higher concentrations of GerE inhibited
cotD transcription. The inhibition was specific to
cotD, since cotC transcription was activated at
the highest concentration of GerE tested.
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Fig. 4.
Effect of GerE on cotD
transcription in vitro. DNA templates (0.2 pmol each template) were transcribed with partially purified
K RNAP (0.2 µg) alone (lane 1) or with 50 (lane 2), 100 (lane 3), 200 (lane 4),
or 400 pmol (lane 5) of gel-purified GerE added immediately
after the
K RNAP. DNA templates were pLRK100 (15)
linearized with HindIII (225-base cotD
transcript) and a HaeIII-EcoRI restriction
fragment from pHI1(4) (196-base cotC transcript). The
positions of run-off transcripts of the expected sizes, as judged from
the migration of end-labeled DNA fragments of MspI-digested
pBR322, are indicated.
35 region of
the cotD promoter and extended upstream. On the
nontranscribed strand, the protection spanned positions
39 to
25
relative to the TSS (Fig. 5A), whereas on the transcribed
strand positions
60 to
23 were protected. We could not be certain
whether protection extends farther upstream on the nontranscribed
strand due to the absence of DNase I cleavage in this region even in
the absence of GerE. GerE appears to bind to the cotD
promoter region with lower affinity than to the sigK promoter region, since a higher concentration of GerE was required to
observe protection from DNase I digestion (compare Figs. 1 and 5). Fig.
5C shows the sequence of the cotD promoter in the region protected by GerE. Typically, GerE protects a stretch of about
20 bp from DNase I digestion (3, 4). The long region of protection
(nearly 40 bp) observed on the transcribed strand in the
cotD promoter region suggests that GerE binds to two sites. Within this region is a perfect match (positions
53 to
42) to the
consensus sequence for GerE binding (Fig. 5D). A second
match (7 out of 10) to the consensus sequence is present at positions
20 to
31, in inverted orientation with respect to the first match
(Fig. 5D), which probably accounts for GerE binding in
this region.
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Fig. 5.
GerE footprints in the cotD
promoter region. Radioactive DNA fragments separately
end-labeled on the nontranscribed (A) or transcribed
(B) strand were incubated in separate reactions with a
carrier protein (bovine serum albumin, 310 pmol) only (lane
1) or with 12 (lane 2), 25 (lane 3), 50 (lane 4), or 100 pmol (lane 5) of gel-purified
GerE in addition to the carrier protein and then subjected to DNase I
footprinting in a total volume of 45 µl. See Fig. 1 legend for
explanation of boxes, arrowheads, and
numbers. C, position of the GerE-binding site in
the cotD promoter region. The nucleotide sequence of the
nontranscribed strand of the cotD promoter region (7) is
aligned with respect to conserved nucleotides found in the 35 regions
of promoters transcribed by
K RNAP (34), shown
above the sequence. Nucleotides in the cotD
35
region that match the consensus are shown as boldface capital
letters. Overlining and underlining indicate
regions on the nontranscribed and transcribed strands, respectively,
protected by GerE from DNase I digestion. The dashed
lines indicate regions of uncertain protection due to a lack
of DNase I digestion in these regions. Numbers refer to
positions relative to the TSS. M indicates A or C. D,
nucleotide sequences within the GerE-protected region of the
cotD promoter are aligned with the consensus sequence for
GerE binding (3). Matches to the consensus sequence are shown as
capital letters, and numbers refer to positions
relative to the TSS. Note that the sequence shown for the binding site
between
20 and
31 is from the strand opposite that shown in
C.
47.5) activates
transcription, because GerE binds at a similar position in the
cotB (4) and cotVWX (3) promoters and activates
transcription. GerE bound at the cotD promoter downstream site (centered at
25.5) might repress transcription, explaining the
inhibition of cotD transcription we observed (Fig. 4). To test these ideas, we repeated the in vitro transcription
experiments with a cotD DNA template lacking the upstream
GerE-binding site. As shown in Fig. 6,
GerE failed to activate transcription of this template. However, GerE
still repressed transcription of this template, under conditions that
permitted GerE to activate cotC transcription. We conclude
that GerE binding to the upstream site in the cotD promoter
is required to activate transcription. GerE binding to the downstream
site probably represses transcription, although we cannot rule out the
possibility that GerE binding farther downstream is also necessary and
was not detected by DNase I footprinting.
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Fig. 6.
Effect of GerE on transcription in
vitro of a cotD template lacking the
upstream GerE-binding site. The amounts of DNA templates,
K RNAP, and GerE were the same as in the Fig. 4 legend.
The cotD template was a 275-bp PCR fragment containing 44 bp
upstream of the TSS (228-base transcript) prepared as described under
"Experimental Procedures." The cotC template was
described in the Fig. 4 legend. The positions of run-off transcripts of
the expected sizes, as judged from the migration of end-labeled DNA
fragments of MspI-digested pBR322, are indicated.
DISCUSSION
K and
K in cells during the late
stages of sporulation. The previous finding that GerE inhibits
sigK transcription in vitro (4) can be explained
by GerE binding near the TSS (Fig. 1) and acting as a repressor. The
previous observation that expression of a sigK-lacZ fusion
lacking the GerE-binding site in the promoter region is unaffected by a
gerE mutation (13), together with our finding that
expression of a sigK-lacZ fusion containing the GerE-binding
site is 2-fold lower in wild-type cells than in gerE mutant
cells (Fig. 2), demonstrates the importance of the GerE-binding site
for sigK regulation in vivo. The lowering of
sigK expression by GerE in wild-type cells appears to result
in a comparable decrease in the level of SigK gene products (Fig. 3).
The simplest interpretation of these results is that GerE represses
sigK transcription, limiting the synthesis of
pro-
K and
K during sporulation.
K level during
sporulation would lower expression of genes for which
K
is the limiting factor for transcription. Four genes in the
K regulon show a lower level of expression in wild-type
cells than in gerE mutant cells. These are cotA
(25), spoVF (26), csk22 (27), and cotM
(28). In the case of cotA, there is evidence of a direct
effect of GerE on transcription. Purified GerE inhibits cotA
transcription in vitro (4). It is unknown how much of the
increased cotA expression observed in a gerE
mutant (25) results from loss of direct inhibition by GerE and how much
results from an elevated level of
K. Also unknown is
whether GerE represses spoVF, csk22, or
cotM directly, or whether these genes are overexpressed in a
gerE mutant because GerE fails to repress sigK.
It should be possible to answer some of these questions and, more
generally, to assess the importance of GerE repression of
sigK during sporulation and germination, by deleting the
GerE-binding site in the sigK promoter.
K
regulon, is positively regulated by GerE. For several genes, GerE has
been shown to bind upstream of the promoter
35 region and stimulate
transcription by
K RNAP in vitro (3, 4). GerE
can increase cotD transcription in vitro as shown
previously (4), but we discovered that a higher concentration of GerE
causes repression (Fig. 4). DNase I footprinting revealed that GerE
protects not only DNA upstream of the
35 region in the
cotD promoter, but the protection extends downstream through
the
35 region (Fig. 5). Binding that extends through the
35 region
has not been observed in other promoters activated by GerE (3, 4). We
reasoned that GerE binding in the cotD
35 region might
repress transcription, and we showed that truncation of cotD
promoter DNA at
44 prevented activation, but allowed repression, by
GerE (Fig. 6). These in vitro results suggest the model that
a rising level of GerE in sporulating cells first activates
cotD transcription by binding upstream of the
35 region
and then represses transcription by binding to a second site just
downstream. It should be straightforward to test the requirement for
the upstream GerE-binding site for activation in vivo, by
making the appropriate 5' deletion (e.g. to
44) and measuring expression of a fusion to a reporter gene. However, testing
the role of the downstream GerE-binding site in repression in
vivo will be more difficult. As noted above, one must be careful to distinguish between direct repressive effects of GerE and indirect effects due to GerE repression of sigK.
E and SpoIIID are active in the
mother cell (Fig. 7A). At this time, sigK is
transcribed by
E RNAP, with SpoIIID serving as an
essential activator (6, 13). As we have shown for GerE (Figs. 5 and 6),
it was shown previously that SpoIIID binds in the
35 region of the
cotD promoter and represses transcription in
vitro (6). Fig. 7B shows the regulatory interactions in
the mother cell slightly later, at about 4 h into the sporulation
process, when the primary product of the sigK gene,
pro-
K, is processed to active
K in
response to a signal from the forespore (22, 29).
K RNAP
initiates a negative feedback loop that inhibits transcription of the
sigE gene encoding
E, which in turn lowers
expression of spoIIID (8, 12). As the levels of
E RNAP and SpoIIID fall, cotD transcription
would no longer be repressed by SpoIIID. At the same time,
K RNAP transcribes gerE (4, 30), and,
according to our model, the GerE produced initially activates
cotD transcription and represses sigK
transcription. For a period, all four mother cell-specific transcription factors probably affect transcription of sigK,
because SpoIIID activates both
E RNAP and
K RNAP to transcribe sigK (6, 13), and GerE
represses sigK transcription (4) (Figs. 1-3). The positive
autoregulatory loop created by
K RNAP transcription of
sigK is kept in check by two negative feedback loops (Fig.
7B). One inhibits transcription of sigE and
therefore inhibits production of
E and SpoIIID (8). The
other leads to synthesis of GerE, which represses sigK
transcription directly (4) (Figs. 1-3). As the level of GerE rises
later in sporulation, GerE may also repress cotD (Fig.
7C). Presumably, the complex regulatory interactions depicted in Fig. 7 ensure that the four transcription factors, as well
as structural proteins like CotD under their control, are made at
optimal levels for formation of the spore cortex and coat.
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Fig. 7.
Regulatory interactions between mother
cell-specific transcription factors and a model for regulation of
cotD transcription at different times during
sporulation. Dashed arrows show gene
(italicized) to product (proteins are circled)
relationships. Solid arrows represent positive regulation of
transcription. Lines with a barred end represent
negative regulation of transcription. A, B, and C
represent early, intermediate, and late stages of sporulation,
respectively, as explained in the text.
Our mapping of GerE-binding sites in the sigK and
cotD promoters and our in vitro transcription
experiments with cotD promoter fragments differing at the 5'
end provide the first insight into how GerE acts as a repressor. In
both the sigK and cotD promoters, GerE binds
within the region likely to be bound by K RNAP, although
the footprints of
K RNAP on these promoters have not yet
been determined. The positions of GerE binding in these promoters
suggest that GerE may repress transcription by interfering with the
initial binding of
K RNAP or a subsequent step in
transcription initiation. It may be possible to distinguish between
these possibilities by measuring the ability of
K RNAP
to bind to these promoters in the presence or absence of GerE. In the
case of cotD, it is unlikely that GerE inhibits elongation of transcripts or inhibits transcription by any other mechanism that
does not depend on sequence-specific DNA binding, because GerE
stimulated cotC transcription in the same reaction mixtures in which it inhibited cotD transcription (Figs. 4 and 6).
These same in vitro transcription experiments, with
cotD promoter fragments differing at the 5' ends, suggest
that at a low GerE concentration, binding to a site upstream of the
35 region activates transcription, and at a higher GerE
concentration, binding to a second site just downstream represses
transcription. In the DNase I footprint experiment shown in Fig. 5,
there is no indication that GerE binds with higher affinity to the
upstream site (a perfect match to the GerE binding consensus sequence)
than the downstream site (a 7 out of 10 match to the consensus).
Although we cannot rule out a slight difference in affinities, another
possible explanation is that
K RNAP competes with GerE
for binding to the downstream site but not the upstream site.
GerE appeared to bind with higher affinity to the sigK
promoter region than to the cotD promoter region (compare
Figs. 1 and 5) or many other GerE-binding sites mapped previously (3,
4). Within the region of the sigK promoter protected by GerE
from DNase I digestion are two sequences in inverted orientation that overlap by 4 bases and match the consensus sequence for GerE binding perfectly or in 7 out of 10 positions. An identical arrangement of
sequences matching the GerE consensus was observed previously just
upstream of the 35 region in the cotYZ promoter, where
GerE appeared to bind with high affinity and activated transcription (3). It is unknown whether more than one molecule of GerE at a time
binds the sigK and cotYZ sites. Also unknown is
whether GerE is monomeric in solution. GerE is predicted to possess a helix-turn-helix DNA-binding motif similar to that in several proteins
whose three-dimensional structure has been determined (31, 32). Some of
these well characterized proteins are dimeric and recognize inverted
repeats in the DNA-binding sites (33). The consensus GerE-binding
sequence is not palindromic, so perhaps GerE can bind as a monomer.
This does not exclude the possibility of interaction between monomers
at sites that exhibit palindromic character, such as the
sigK and cotYZ sites.
GerE appears to be similar to SpoIIID in terms of its DNA sequence recognition characteristics. The SpoIIID-binding site consensus sequence (WWRRACAR-Y) is of similar length and degeneracy as that recognized by GerE (RWWTRGGY-YY) and also is not palindromic (5, 6). Although SpoIIID appears to be monomeric in solution,2 many strong SpoIIID-binding sites exhibit a second good match to the consensus in inverted orientation relative to the best match (6). Mutational analysis will be required to assess the contribution of each consensus match to binding of GerE and SpoIIID at sites with palindromic character and to determine whether two monomers interact (e.g. bind cooperatively) at these sites.
In summary, we have investigated the biochemical basis for negative
regulation by the GerE protein. It appears to act like a classical
repressor, binding in promoter regions at sites that overlap the
position of RNAP binding. GerE repression at the sigK promoter lowers K production during sporulation about
2-fold, potentially regulating the expression of many genes in the
K regulon. In addition, GerE binds in the promoter
regions of certain genes in the
K regulon and directly
represses or activates transcription by
K RNAP. In the
case of cotD, it is likely that GerE binds to a site
upstream of the promoter
35 region and first activates transcription, then, as its level rises in sporulating cells, GerE binds to a site
slightly farther downstream and represses transcription.
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ACKNOWLEDGEMENTS |
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We thank B. Kunkel, R. Losick, P. Zuber, and
C. Moran for providing bacterial strains, phages, and plasmids. We
thank S. Lu for providing anti-pro-K antibodies and B. Zhang for helpful discussions. We are grateful to J. Kaguni for
comments on the manuscript.
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FOOTNOTES |
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* This research was supported by Grant GM43585 from the National Institutes of Health and by the Michigan Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: McArdle Laboratory for Cancer Research,
University of Wisconsin, Madison, WI 53706.
§ To whom correspondence should be addressed. Tel.: 517-355-9726; Fax: 517-353-9334; E-mail: kroos{at}pilot.msu.edu.
2 B. Zhang and L. Kroos, unpublished work.
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ABBREVIATIONS |
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The abbreviations used are: RNAP, RNA polymerase; TSS, transcriptional start site; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; bp, base pair.
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REFERENCES |
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