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Journal of Bacteriology, August 2001, p. 4814-4822, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4814-4822.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Developmental Gene Expression in Bacillus subtilis
crsA47 Mutants Reveals Glucose-Activated Control of the Gene
for the Minor Sigma Factor 
H
Laurie G.
Dixon,1,
Steve
Seredick,1
Martin
Richer,1 and
George B.
Spiegelman1,2,*
Departments of Microbiology and
Immunology1 and Medical
Genetics,2 University of British Columbia,
Vancouver, British Columbia, Canada
Received 22 November 2000/Accepted 30 May 2001
 |
ABSTRACT |
The presence of excess glucose in growth media prevents normal
sporulation of Bacillus subtilis. The crsA47
mutation, located in the gene for the vegetative phase sigma factor
(
A) results in a glucose-resistant sporulation
phenotype. As part of a study of the mechanisms whereby the mutation in
A overcomes glucose repression of sporulation, we
examined the expression of genes involved in sporulation initiation in
the crsA47 background. The crsA47 mutation had
a significant impact on a variety of genes. Changes to stage II gene
expression could be linked to alterations in the expression of the
sinI and sinR genes. In addition, there was a
dramatic increase in the expression of genes dependent on the minor
sigma factor H. This latter change was paralleled by the
pattern of spo0H gene transcription in cells with the
crsA47 mutation. In vitro analysis of RNA polymerase
containing A47 indicated that it did not have unusually
high affinity for the spo0H gene promoter. The in vivo
pattern of spo0H expression is not predicted by the known
regulatory constraints on spo0H and suggests novel
regulation mechanisms that are revealed in the crsA47 background.
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INTRODUCTION |
The activation of genes that
are induced late in the growth cycle of Bacillus subtilis
involves a network of interacting regulatory pathways. These pathways
control the cellular response to conditions that include nutritional
stress and high cell density (reviewed in references 13, 19, 24,
36, and 47). Under the appropriate environmental stimuli,
B. subtilis will differentiate to form dormant endospores.
The key factor in initiating sporulation is the accumulation and
phosphorylation of the response regulator and transcription factor
Spo0A (reviewed in references 13, 19, and 24).
Phosphorylation of Spo0A takes place through a multicomponent pathway
(the phosphorelay) (3) that appears to integrate multiple signals that act positively or negatively to regulate sporulation (24). Once a sufficient level of phosphorylated Spo0A
(Spo0A~P) is reached, a complex series of feedback loops will drive
differentiation forward (19, 24, 36).
One critical component of sporulation is the minor sigma factor,
H, encoded by a gene originally found as a stage zero
sporulation mutant, spo0H (12, 20, 46).
H is required for the transcription of a variety of
sporulation genes, including spo0A, spo0F, and
kinA (20, 41). The activity of
H, which increases as cells enter stationary phase, is
under complex, still-undefined regulatory controls (19, 45,
46). Transcription of the spo0H gene is repressed by
the transition state regulator AbrB (11, 48, 50), and so
the induction of spo0H seen at the transition between log
growth and sporulation is influenced by Spo0A~P repression of the
abrB gene (4, 11). Transcriptional and
translational regulation of spo0H by the presence of
nutrients has also been reported (8, 14).
A variety of experiments have shown that the increase in
H activity as measured by transcription of
H-dependent genes does not match the accumulation of the
H protein, implying the existence of posttranslational
controls (8, 16, 17, 21, 23, 45, 50). Furthermore,
evidence has been presented that external pH (8) and the
activity of the tricarboxylic acid cycle (26) affect
H activity and that H protein levels are
affected by a Lon-type protease during the onset of sporulation and
during stress responses (30). Recently, ClpX has been
implicated in H-dependent transcription activity. In
vivo and in vitro experiments have suggested that ClpX may interact
directly with RNA polymerase containing H and stimulate
transcription (31, 37). The level of
H-dependent transcription increases for 1 to 2 h
after sporulation initiation and then declines (4, 11,
46). The decline of activity has been linked to another Clp
protein, ClpP, and the decline is associated with the loss of
H protein (38).
Excess glucose in the growth medium represses sporulation, and some of
the effects on sporulation have been linked to specific repression of
H activity (1, 7, 8, 16, 17, 50, 52). The
crsA47 mutation renders sporulation resistant to repression
by glucose (49). The molecular basis for this phenotype is
unknown, but the crsA47 mutation is located within the gene
for the A subunit of RNA polymerase (sigA or
rpoD) (27).
In this report, we describe experiments that suggest that the
crsA47 mutation in A affects the expression
of H-dependent genes through its effect on the
expression of spo0H. The effects include overexpression of
the spo0H gene during the onset of sporulation and unusual
extended expression past the time of normal shutoff during
differentiation. As the pattern cannot be explained solely by increased
promoter affinity, these effects suggest novel regulation of the
spo0H gene.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The strains used in this
study are shown in Table 1. The
promoter-lacZ fusions used were created using a B. subtilis amyE integrative vector, with the exception of the
kinA-lacZ fusions that were inserted in the kinA
gene. Promoter-lacZ fusions provided in the JH642 background
were transferred into GBS10 (containing the crsA47 mutation
in the rpoD gene but otherwise isogenic to JH642) by
transformation with chromosomal DNA, and selection for both the
antibiotic resistance and the amyE mutant phenotype was conferred with the acquisition of the construct. The plasmid pGS0H was
created by the ligation of the
EcoRI/BamHI-digested Vent polymerase (New England
Biolabs, Inc.) PCR product generated from chromosomal DNA and the
primer pair 5'-AAGGATCCTGTTTCTGGCGAGTAG-3' and
5'-ACGAATTCGGCACGGACGTTAGAA-3' that targets the
spo0H gene into the EcoRI/BamHI sites
of the B. subtilis integrative vector pDH32
(44). The pDH32-based clone was linearized with
PstI prior to transformation into B. subtilis. Chloramphenicol-resistant transformants generated were confirmed to be
amylase negative by using 1% starch agar plates prior to 
-galactosidase analysis.
Bacterial transformation and growth conditions.
B.
subtilis transformations were performed by the method of Hoch
(24) with 1 to 2 µg of plasmid DNA or 20 to 100 ng of
chromosomal DNA. Transformants were selected on Schaeffer sporulation
agar plates supplemented with 5 µg of chloramphenicol or
kanamycin/ml. Bacillus cultures used to determine
sporulation frequency were grown in Schaeffer spore media (SSM), pH
7.5, supplemented with tryptophan and phenylalanine (each 10 µg/ml),
chloramphenicol or kanamycin (5 µg/ml), and, when appropriate, SSM
with 1% glucose (SSMG). Cells were grown for 22 to 24 h, serially
diluted in SSM, and plated before and after treatment with 0.1 volume
of chloroform to obtain a viable cell count and a spore count.
-Galactosidase assay.
B. subtilis strains used
for analysis of promoter-lacZ activity were inoculated into
SSM, pH 7.5, containing 5 µg of the appropriate antibiotic/ml and
supplemented with tryptophan and phenylalanine (each, 10 µg/ml) and,
when appropriate, 0.2% glucose. Aliquots (0.5 ml) were removed hourly,
the cells were collected by centrifugation, and cell pellets were
frozen at 
70°C until analyzed. -Galactosidase assays and
designation of time zero (T0) in sporulation
were done as previously described (15). Enzyme specific
activity was expressed in Miller units (35). Assays of a
minimum of three independent cultures were performed, and one
representative pattern for each strain is shown. Each point in the
assays is an average of duplicate samples that differed by no more than
5% from the mean.
In vitro transcription assay.
The RNA polymerase
preparations were isolated as described by Dobinson and Spiegelman
(10) from logarithmic-stage cultures of JH642 and GBS10,
except that the heparin-Sepharose column set was eliminated.
Transcription assays used fractions from the glycerol gradient. Two DNA
templates were used. The plasmid pUCA2trpA (6) that
contains the bacteriophage 
29A2 promoter was treated with PvuII, and the 600-bp fragment containing the promoter was
isolated by electrophoresis through agarose and extracted from the
agarose with a GeneClean kit from Qiagen. The spo0H promoter
was isolated by amplifying a DNA fragment using 20 pmol (each) of two
specific primers described above, 800 ng of chromosomal DNA from JH642, and an amplification protocol as follows: preincubation of the template
and primers at 95°C for 5 min, and after the addition of 2.5 U of
Taq polymerase (Promega), 30 cycles of 95°C for 1 min,
61°C for 1 min (with descending annealing temperatures of 0.3°C per
cycle), and 72°C for 1 min. The product was purified by
electrophoresis through 1% agarose and extracted from the gel as
described above for the A2 promoter. The extracted product was digested
with HindIII to provide a fixed endpoint for the transcription assays and then precipitated. The concentrations of
promoter fragments were determined by measuring the absorbance at 260 nM.
The in vitro transcription assay followed published protocols
(
6). In brief, reaction tubes containing 16 µl of
reaction
mixture with template, transcription buffer (
6),
ATP, and [
-
32P]GTP (3 µCi/reaction mixture) were
warmed to 37°C. The polymerase
(2 µl of an appropriate dilution)
was added. Two minutes later,
2 µl of a mixture of heparin (100 µg/ml, final concentration),
UTP, and CTP was added to inactivate
noninitiated RNA polymerase
molecules and allow those that had
initiated to elongate either
to the terminator (in the case of the
29A2 promoter) or to the
end of the DNA fragment (in the case of the
spo0H promoter). After
5 min, a stop buffer containing 7 M
urea was added, and the reaction
products were separated from free
nucleotides on 5% polyacrylamide
gels containing 7 M urea and 0.5×
Tris-borate-EDTA (TBE). The
gels were exposed to a Molecular Dynamics
PhosphorImager screen,
and the data were collected and analyzed with
the ImageQuant 1.0
software on the
instrument.
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RESULTS |
The crsA47 mutation results in overexpression of
H-dependent spo0 genes in glucose-containing
media.
To investigate the mechanism by which the crsA47
mutation resulted in catabolite-resistant sporulation, we examined the
expression of a variety of genes important in the sporulation
initiation pathway in the presence and absence of glucose. Among these
genes, the ones with H-dependent promoters showed very
unusual profiles of activity. Figure 1
shows the expression of the kinA and spoVG
promoter fusions in wild-type (JH642) and crsA47 (GBS10)
backgrounds. The kinA promoter has not been previously shown
to be subject to regulation other than that imposed by H
activity (1, 41). The spoVG gene is preceded by
a H promoter that is repressed during logarithmic growth
by the transition state regulator AbrB (43, 48).
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FIG. 1.
The effect of the crsA47 mutation on
expression of kinA and spoVG. -Galactosidase
activities in strains carrying kinA-lacZ (A) or
spo0VG-lacZ (B) fusions were measured as described in
Materials and Methods. T0 represents the onset
of sporulation. Strains contained the wild-type A gene
(squares) or the crsA47 mutation (diamonds) and were grown
in either SSM (open symbols) or SSMG (filled symbols). (A) JH12664
(kinA-lacZ) and GBS107 (crsA47 kinA-lacZ); (B)
GBS110 (spoVG-lacZ) and GBS109 (crsA47 spoVG-lacZ).
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Three observations were common to the activity of the
kinA
and
spoVG promoters. First, in a wild-type
A
background promoter activity was depressed by the presence of
glucose.
Second, in a
crsA47 background the expression was elevated
by the presence of glucose compared to that seen in the wild type
in
the absence of glucose, with transcription levels persisting
long after
the point of maximum activity in the wild type. Third,
promoter
activity in a
crsA47 background in the absence of glucose
was only marginally affected compared to the effect in the wild
type.
The onset of promoter activity was not affected in the same
way for the
two promoters.
kinA promoter activity (Fig.
1A) in
GBS107
grown in the absence of glucose began earlier than in JH12664,
whereas
the timing of
spoVG transcription activity (Fig.
1B)
appeared
similar in both GBS110 and GBS109. The logarithmic-phase
repression
of
spoVG by AbrB did not differ between wild-type
and
crsA47 mutants.
The suggestion that AbrB regulation was
not altered in the GBS109
was supported by the expression of an
abrB-lacZ fusion which showed
similar patterns in GBS10 and
JH642 (data not
shown).
The expression patterns of
spo0F promoter-
lacZ
fusions are shown in Fig.
2. The
spo0F gene is preceded by dual
A
H promoters, with the
H-dependent
promoter requiring Spo0A~P as a transcription activator
(
28,
41,
51). In JH12862, grown in SSM, expression from
the
spo0F promoter-
lacZ fusion increased during
late-exponential-phase
growth, peaked at roughly
T1, and decreased thereafter. The addition
of
glucose to the media resulted in a decrease in overall expression.
In
GBS105, the expression from
spo0F began earlier and peaked
at higher levels than were seen in JH12862, both with and without
added
glucose. As was seen with both the
kinA and
spoVG
promoters
(Fig.
1), transcription of
spo0F in GBS105 in
cells grown with
added glucose appeared to be stimulated after
T0 compared to levels
seen in cells grown
without glucose.
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FIG. 2.
The effect of the crsA47 mutation on
expression of spo0F. -Galactosidase activities in strains
carrying spo0F-lacZ fusions were measured as described in
Materials and Methods. T0 represents the onset
of sporulation. Strains contained the wild-type A gene
(JH12862; squares) or the crsA47 mutation (GBS105; diamonds)
and were grown in either SSM (open symbols) or SSMG (filled symbols).
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The
crsA47 mutation results in the expression of
spoII genes despite the presence of glucose. The late- and
post-exponential
phase expression of
H-dependent
spo0 genes appeared to be elevated in the cells containing
the
crsA47 mutation (Fig.
1 and
2), suggesting that
H was active in these cells despite the presence of
glucose. The
activation of
H during the transition state
has been shown to be critical for
the initiation of sporulation and is
required for the maximal
expression of genes encoding phosphorelay
proteins and accumulation
of a sufficient level of Spo0A~P to
activate the transcription
of stage II
spo genes. As a test
for whether or not Spo0A~P was
fully activated, we examined the
expression of the stage II genes
spoIIA and
spoIIG (Fig.
3).
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FIG. 3.
The effect of the crsA47 mutation on
expression of spoIIA and spoIIG.
-Galactosidase activities in strains carrying spoIIA-lacZ
(A) or spoIIG-lacZ (B) fusions were measured as described in
Materials and Methods. T0 represents the onset
of sporulation. Strains contained the wild-type A gene
(squares) or the crsA47 mutation (diamonds), and were grown
in either SSM (open symbols) or SSMG (filled symbols). (A) JH16124
(spoIIA-lacZ) and GBS106 (crsA47 spoIIA-lacZ);
(B) JH16304 (spoIIG-lacZ) and GBS101 (crsA47
spoIIG-lacZ).
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In strain JH16124 cells grown in SSM (Fig.
3A), the expression from the
H-dependent
spoIIA promoter-
lacZ
fusion began roughly at
T0 and
peaked at
T2, dropping thereafter. In the presence of
glucose,
spoIIA-lacZ expression was depressed in stationary
phase. In GBS106
grown in SSM (Fig.
3A), transcription activity from
the
spoIIA promoter began to increase at the same time as
that observed in
JH16124 and peaked at levels not substantially
different from
that in JH16124. In the presence of glucose, the
expression of
spoIIA-lacZ in GBS106 began immediately after
the onset of stationary
phase and rose to a level more than twice that
seen in JH16124
grown in the absence of
glucose.
Figure
3B depicts the expression from the
A-dependent
spoIIG promoter-
lacZ fusion in the wild type
(JH16304) and cells containing
the
crsA47 mutation (GBS101).
As seen in Fig.
3A for the
spoIIA-lacZ fusion, promoter
activity from the
spoIIG promoter was repressed
by glucose
in the wild type but not repressed in GBS101. Since
effective
expression of these two stage II operons directly requires
high
Spo0A~P levels, these cells must contain high levels of Spo0A~P.
The
spo0A gene is expressed from two promoters, one
A dependent and one
H dependent. It has
been shown elsewhere that expression for the
H-dependent
promoter is enhanced in
crsA47 mutants (
9).
The presence of the crsA47 mutation results in an
alteration in the pattern of transcription from sinI and
sinR promoters in the presence of glucose.
SinR
inhibits the expression of several spo genes, including
spo0A (34), spoIIG, and
spoIIA (5, 32, 33). The sinR gene is
constitutively expressed from a A-dependent promoter
throughout exponential and post-exponential growth of B. subtilis (18). SinR inhibition of transcription is
negatively regulated by Spo0A~P levels, which stimulate increased transcription of the H-dependent sinI
gene (the gene directly upstream of sinR)
(18). SinI sequesters SinR via protein-protein
interaction preventing SinR-mediated repression of promoter activity
(2, 29). Gaur et al. showed that the presence of excess
glucose in the media inhibits the transcription of the sinI
gene (18). Presumably, inadequate transcription of
sinI results in a SinI/SinR protein ratio insufficient to
fully sequester SinR and relieve repression of sporulation genes
(2, 29).
The data in Fig.
3 showed that
H- and
A-dependent stage II promoters were deregulated in cells
containing the
crsA47 mutation
grown in glucose. Because
SinR is central to regulation of both
the
spoIIG and
spoIIA operons, we examined the regulation of the
sinR and
sinI promoters. The data shown in Fig.
4 indicate that
in cells containing
wild-type
A (GBS114) grown in the absence of glucose,
sinI transcription
levels increased throughout late
logarithmic growth to peak at
T0 (Fig.
4A).
Transcription of
sinR increased throughout late
logarithmic
growth and into stationary phase (GBS116) (Fig.
4B),
presumably in part
due to readthrough from the
sinI promoter (
18)
as well as from the
sinR promoter. The expression patterns
in
Fig.
4 are similar to those observed by others (
18,
33). When
glucose was added,
sinI transcription
remained relatively low
during stationary phase in GBS114 (Fig.
4A),
whereas
sinR transcription
in GBS116 (Fig.
4B) remained
roughly the same as in the absence
of glucose.
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FIG. 4.
The effect of the crsA47 mutation on
expression of sinI and sinR. -Galactosidase
activities in strains carrying sinI-lacZ (A) or
sinR-lacZ (B) fusions were measured as described in
Materials and Methods. T0 represents the onset
of sporulation. Strains contained the wild-type A gene
(squares) or the crsA47 mutation (diamonds) and were grown
in either SSM (open symbols) or SSMG (filled symbols). (A) GBS114
(sinI-lacZ) and GBS113 (crsA47 sinI-lacZ); (B)
GBS116 (sinR-lacZ) and GBS115 (crsA47
sinR-lacZ).
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If transcription of the
sinI and
sinR genes
reflects protein levels, then cells with wild-type
A
grown in the absence of glucose would contain a roughly 20-fold
excess
of SinI over SinR. Since these cells sporulate efficiently,
this ratio
should indicate the level of SinI needed to complex
SinR between
T0 and
T1.5. In the
presence of glucose, expression
of the
sinI promoter in
cells with wild-type
A was reduced, with the implication
that the ratio of SinI to SinR
would not block SinR repression of
sporulation.
sinI promoter activity in cells containing the
crsA47 mutation (GBS113) is also shown in Fig.
4A. In these
cells, the activity
from the
sinI promoter in the absence of
glucose rose slowly during
logarithmic growth to peak at
T0.5 at levels 25 to 30% of that
seen in
GBS114. The
sinI-lacZ activity was also altered in GBS113
grown in the presence of glucose (Fig.
4A), with the observed
pattern
of transcription similar to that seen in other
H-dependent promoters examined in the
crsA47
background. Transcription
from the
sinI promoter rose from
T
1.5 to peak after
T2 at levels five times higher than was seen in cells with wild-type
A.
Figure
4B shows
sinR-lacZ activity in cells containing the
crsA47 mutation (GBS115). Without excess glucose,
transcription
of the
sinR gene was reduced from that seen in
cells containing
wild-type
A (GBS116), peaking at
roughly
T0 and decreasing thereafter. In
the
presence of glucose, transcription rose sharply from
T2 to peak at
T0 at
levels similar to those achieved in GBS116 in
the presence or absence
of
glucose.
Continuing the assumption that the activity of the
sinI and
sinR promoter fusions reflects protein levels, then even
though
SinR and SinI levels were reduced in cells containing the
crsA47 mutation (in media without excess glucose), the ratio
would be
similar to that seen in wild-type cells. Thus, SinR activity
would
be blocked by SinI during early stationary phase. When glucose
was added, the large induction of the
sinI promoter would
further
reduce SinR activity. Thus, unlike cells with wild-type
A where the addition of glucose decreased the ratio of
expression
of
sinI/sinR, in the cells with the
crsA47 mutation the ratio
of
sinI/sinR would
increase with added glucose. This alteration
in
sin operon
transcription in the
crsA47 mutant could contribute
to the
ability of these cells to express the
spoIIA and
spoIIG operons in the presence of
glucose.
The importance of SinR negative regulation in glucose repression of
sporulation was examined by determining the sporulation
efficiency of a
sinR mutant, as shown in Table
2. In the
crsA47 mutant
(GBS10), the sporulation efficiencies of cells grown in
the presence
and absence of glucose were comparable, clearly indicating
a
glucose-resistant sporulation phenotype. In JH642, the addition
of
excess glucose to the medium resulted in a 10
4-fold
decrease in sporulation efficiency. However, in GBS112
(
sigA+ sinR) the sporulation
efficiency in medium with excess glucose
was only three-fold less than
that observed in the absence of
glucose. These results, combined with
those shown in Fig.
2 through
4, suggest that a decrease in SinR
repression whether by
sinR gene deletion or by altering the
ratios of
sinI and
sinR transcription
increased
expression of
spoII genes and contributed to the
glucose-resistant
sporulation phenotype.
The crsA47 mutation results in increased transcription
from the spo0H gene in late- and post-exponential-phase
growth.
The results shown in Fig. 3 and 4 demonstrate increased
expression of both A- and H-dependent
stage II genes in the crsA47 background when the cells were
grown in the presence of excess glucose. The implication from the
analysis of the sinI-sinR operon was that the increase in
stage II gene expression reflected the increase in H
activity, which allowed SinI to inactivate SinR. Thus, overexpression of the spoIIG operon (A dependent) has the
same fundamental mechanism as overexpression of the spoIIA
operon (H dependent); that is, increased activity of
H. However, the reason for the high levels of
stationary-phase transcription from all of the
H-dependent promoters examined in the crsA47
mutant in the presence of glucose was not clear. Previous studies have
shown higher-than-normal H-dependent promoter activity
during the stationary phase either by using an increased copy number of
spo0H or by placing spo0H under the control of
the isopropyl--D-thiogalactopyranoside (IPTG)-inducible PSPAC promoter (21). Thus, the increased
H activity could be due to overexpression of the
spo0H gene in the crsA47 mutant, and this was
examined using a spo0H-lacZ fusion in the presence and
absence of added glucose.
Figure
5 shows the expression patterns of
the
A-dependent
spo0H-lacZ fusion in strains
containing wild-type
sigA (GBS151)
or the
crsA
mutation (GBS150). In GBS151 the activity of the
spo0H promoter began to increase at
T2 to peak at
roughly
T1,
with the maximum activity not
substantially different in the presence
or absence of glucose. This
pattern of transcription is similar
to that seen elsewhere (
1,
50). In GBS150 the timing of transcription
from the
spo0H promoter was the same as in GBS151, but peak activity
was roughly eight times higher in the absence of glucose and 19
times
higher in the presence of glucose than was seen in the GBS151.
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FIG. 5.
Effect of the crsA47 mutation on the
expression of spo0H. -Galactosidase activities in a
strain carrying spo0H-lacZ fusions were measured as
described in Materials and Methods. T0
represents the onset of sporulation. Strains contained the wild-type
A gene (GBS151; squares) or the crsA47
mutation (GBS150; diamonds) and were grown in either SSM (open symbols)
or SSMG (filled symbols).
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The high level of transcription of the
spo0H gene seen in
GBS150 grown in the presence of glucose could lead to excess
H protein and thus explain the unusual patterns of
H-dependent transcription seen in cells containing the
crsA47 mutation.
These data raise the question whether the
extended transcription
from the
A-dependent promoter of
the
spo0H gene reflected a general phenomenon
or whether it
was specific to the
spo0H promoter. The only known
regulator
of the
spo0H promoter is AbrB. We would not expect AbrB
levels to play a role in this regulation of
spo0H beyond
T1 as
the gene is repressed earlier, and this
repression was not changed
in cells containing the
crsA47
mutation (
9). However, it was
possible that the
crsA47 mutation altered the reduction of
A
activity that is normally seen in sporulation (
46).
As part of the characterization of the
crsA47 mutation, we
examined several other
A-dependent promoters. The
rapA-encoded phosphatase removes phosphate
from the
phosphorelay by dephosphorylating phosphorylated Spo0F
(reviewed in
reference
40). Transcription from the
rapA gene
promoter also showed a
crsA47- and excess-glucose-specific
increase
in transcription after
T0 (Fig.
6A). In other work, the same effect
for
the
A-dependent
spo0APv promoter has been
seen (
9). In contrast,
expression of a second
A-dependent gene encoding a phosphatase,
rapB, did not show increased
transcription in strains
containing the
crsA47 mutation and grown
in excess glucose
(Fig.
6B). Thus, the effect of the
crsA47 mutation
was
promoter specific as well as being specific to growth conditions.
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FIG. 6.
Effect of the crsA47 mutation on the
expression of rapA and rapB. -Galactosidase
activities in strains carrying rapA-lacZ (A) or
rapB-lacZ (B) fusions were measured as described in
Materials and Methods. T0 represents the onset
of sporulation. Strains contained the wild-type A gene
(squares) or the crsA47 mutation (diamonds) and were grown
in either SSM (open symbols) or SSMG (filled symbols). (A) JH12961
(rapA-lacZ) and GBS104 (crsA47 rapB-lacZ); (B)
JH12866 (rapB-lacZ) and GBS103 (crsA47
rapB-lacZ).
|
|
In vitro analysis of transcription from the spo0H
promoter by RNA polymerase containing A47.
One
possible explanation for the increase in H activity seen
in strains containing the crsA mutation is that the mutation in A increases the affinity of the polymerase for the
spo0H gene promoter. To directly measure this affinity, we
purified RNA polymerase from wild-type and GBS10 strains and tested
them in vitro in single-round transcription assays (as described in
Materials and Methods) (Fig. 7). As a
means of measuring the specific activity of the RNA polymerase preparations, we compared the activity of the two preparations on a
promoter from bacteriophage 29 that has been cloned into the plasmid
pUCA2trpA (Fig. 7A). Aliquots of the polymerase preparations were mixed
with DNA and initiating nucleotides (ATP plus GTP) and challenged with
a mixture of heparin and CTP plus UTP. The heparin inactivates
noninitiated RNA polymerase molecules while the CTP plus UTP allow
enzymes that have initiated to elongate. Over a range of DNA
concentrations of 0.5 to 8 nM, the wild-type RNA polymerase was three-
to five-fold more active than the preparation from GBS10. We then
repeated this experiment using a DNA fragment containing the
spo0H promoter. This DNA fragment was produced by
amplification of the promoter region. Transcription from this promoter
produced a single transcript (data not shown). The activity of both
polymerase preparations was significantly lower on the spo0H
promoter than on the phage promoter. However, as with the 29A2
promoter, the polymerase containing A47 was three- to
five-fold less active on this template than was the wild-type
polymerase. By this assay, the change in A caused by the
crsA mutation did not increase the affinity of the
polymerase for the spo0H promoter.
View larger version (14K):
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|
FIG. 7.
In vitro transcription activity of RNA polymerase
isolated from JH642 and GBS10. In vitro transcription reactions were
carried out using a control promoter (pUCA2trpA) (A) or a DNA fragment
containing the spo0H gene promoter (B). Reaction mixtures
contained a constant amount of RNA polymerase from either JH642 (wild
type; squares) or GBS10 (crsA; circles) and increasing
amounts of template. Transcription products were separated by
electrophoresis through polyacrylamide gels containing 7 M urea. The
level of product produced was determined using a Molecular Dynamics
PhosphorImager and ImageQuant 1.0 software and is reported in arbitrary
units.
|
|
| |
DISCUSSION |
We began the study of the crsA47 mutation to uncover
the mechanism by which it makes sporulation resistant to the presence of excess glucose in the growth medium. During the course of these studies, we observed the consistent overexpression of
H-dependent genes in strains carrying crsA47
that we have described in this paper. The overexpression occurred after
T0 and only in strains grown in the presence of
excess glucose. The overexpression peaked 2 to 3 h later than the
normal peak of H-dependent expression and then declined.
This combination of features suggests a novel feature of
H transcription regulation that is revealed in the
crsA47 genetic background.
The regulation of H activity appears to be complex.
There is a low level of transcription of the spo0H gene
during vegetative growth, and at least a few H-dependent
genes are transcribed during this time (19, 42). In
wild-type cells, transcription dependent on H is induced
during the transition stage, and it normally peaks within 1 to 2 h
after the onset of sporulation and decays after that time
(46). Comparison of the levels of H protein
with H-dependent transcription in vegetative growth and
in early sporulation indicates the presence of posttranscriptional
regulation of H (1, 8, 16, 17, 21, 23, 42,
50). Later in sporulation, the decrease in H
activity is associated with loss of H protein due to the
activity of ClpC protease (38). Another Clp family
protein, ClpX, influences the induction of activity of H
during early sporulation, although not through changes in the level of
H protein. It has been reported that ClpX stimulates
H-dependent transcription by direct interaction with the
polymerase containing H (31, 37). It is
known that H activity is regulated at transcription,
since spo0H gene transcription is repressed by AbrB
(11, 43). It has been recently shown that
H-dependent expression of a limited set of genes is
enhanced by amino acid starvation and that this enhancement requires
the relA gene product (14). While this finding
is yet another illustration of the complexity of controls over
H activity, it is probably not related to our findings,
which appeared to be specific to the presence of glucose.
It seems likely to us that the overexpression of
H-dependent genes in GBS10 and its derivatives grown in
the presence of glucose cannot be explained by loss of repression by
AbrB, since AbrB repression was normal in GBS10 (9), and
H-dependent transcription continues to increase after
the time when AbrB is repressed (9). Given the similarity
of the expression patterns of the spo0H gene (Fig. 5) and
the H-dependent genes (Fig. 1 and 3), we suggest that
overexpression of spo0H in cells containing the
crsA47 mutation is a sufficient explanation for the
unusually high transcription of H-dependent genes.
In a direct in vitro test of the activity of RNA polymerase isolated
from GBS10 and wild-type cells (Fig. 7), there was no evidence that the
crsA47 mutation increased the activity at the spo0H promoter. Furthermore, several lines of in vivo
evidence support the finding that the crsA47 mutation does
not simply increase the affinity of the polymerase for the
spoH promoter. First, GBS10 does not grow unusually slowly,
as might be expected if the crsA47 mutation altered the
polymerase specificity. Second, the overexpression of the
spo0H promoter in GBS10 happened only in stationary phase and only in the presence of excess glucose in the growth medium, suggesting a specific regulatory mechanism. Third, two other
A-dependent promoters studied (for the abrB
and rapB genes) did not show unusual expression patterns in
GBS10 (9). We note in passing that the regulation of AbrB,
which is known to control expression of a number of genes
(48), must be particularly important to the overall
physiology of cells with a crsA47 mutation, because it was
found that a crsA47 abrB double mutant grew so poorly even in rich media that regulation in the strain could not be studied (9).
Our results indicate that there must be a regulator of the
spo0H promoter (and possibly other similar promoters) whose
activity is changed in GBS10 grown in excess glucose. We cannot provide any indication whether the regulator is an activator of the
spo0H promoter that is hyperactive or a repressor whose
activity is reduced. The latter seems more likely, since the extended
high expression can be viewed as a lack of shutoff of spo0H
transcription. The existence of such a regulator implies that other
controls of H are yet to be discovered.
A consequence of H overexpression in the presence of
glucose was the change in the SinI-to-SinR ratio, with the predicted result that SinR repression of stage 0 and stage II sporulation genes
would be reduced. There is little question that reduction of SinR in
the cell would increase the glucose resistance of sporulation (32). This is likely to be a major contributor to the
glucose-resistant sporulation in GBS10 and illustrates the redundant
pathways that regulate entry into sporulation.
| |
ACKNOWLEDGEMENTS |
We thank M. Perego, J. A. Hoch, and I. Smith for their
continuous generosity in providing strains and insights during the course of this study.
The research was supported by grants from the Natural Sciences and
Engineering Research Council of Canada and the Canadian Institutes of
Health Research to G.B.S.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 6174 University
Blvd., Vancouver, British Columbia, Canada V6T 1Z3. Phone: (604)
822-2036. Fax: (604) 822-6041. E-mail:
spie{at}interchange.ubc.ca.
Present address: Department of Microbiology and Immunology,
Stanford University, Stanford, Calif.
| |
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Journal of Bacteriology, August 2001, p. 4814-4822, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4814-4822.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
