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Journal of Bacteriology, June 2000, p. 3446-3451, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Control of Sporulation Gene Expression in Bacillus
subtilis by the Chromosome Partitioning Proteins Soj
(ParA) and Spo0J (ParB)
John D.
Quisel and
Alan D.
Grossman*
Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139
Received 7 February 2000/Accepted 24 March 2000
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ABSTRACT |
Two chromosome partitioning proteins, Soj (ParA) and Spo0J (ParB),
regulate the initiation of sporulation in Bacillus
subtilis. In a spo0J null mutant, sporulation is
inhibited by the action of Soj. Soj negatively regulates expression of
several sporulation genes by binding to the promoter regions and
inhibiting transcription. All of the genes known to be inhibited by Soj
are also activated by the phosphorylated form of the transcription
factor Spo0A (Spo0A~P). We found that, in a spo0J null
mutant, Soj affected sporulation, in part, by decreasing the level of
Spo0A protein. Soj negatively regulated transcription of
spo0A and associated with the spo0A promoter
region in vivo. Expression of spo0A from a heterologous promoter in a spo0J null mutant restored Spo0A levels and
partly bypassed the sporulation and gene expression defects. Soj did not appear to significantly affect phosphorylation of Spo0A. Thus, in
the absence of Spo0J, Soj inhibits sporulation and sporulation gene
expression by inhibiting accumulation of the activator protein Spo0A
and by acting downstream of Spo0A to inhibit gene expression directly.
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INTRODUCTION |
Bacillus subtilis can
sporulate in response to starvation and high cell density.
Sporulation involves an asymmetric cell division and
requires DNA replication and chromosome segregation. Spo0A is a
critical transcription factor in early sporulation. Cells initiate
sporulation when they accumulate a threshold amount of phosphorylated Spo0A (Spo0A~P) (3, 6, 12). Many signals that regulate sporulation do so by affecting the accumulation of
Spo0A~P. Spo0A obtains phosphate from a set of proteins called the
phosphorelay (2). A balance of kinases and phosphatases regulates phosphate input into the phosphorelay and the phosphorylation state of Spo0A (3, 30-32).
Two proteins involved in chromosome partitioning, Soj and Spo0J, also
regulate the initiation of sporulation, perhaps in response to
chromosome structure or partitioning (4, 14, 28). Soj and
Spo0J are members of the ParA-SopA and ParB-SopB protein
families, respectively. Many ParA- and ParB-type proteins are encoded
on low-copy-number plasmids and are required for accurate plasmid partitioning (29, 42). Chromosome-encoded family members
have also been identified, several of which function in chromosome partitioning (14, 22, 26). Members of the ParA-SopA families can act as transcriptional repressors and are themselves often regulated by members of the ParB-SopB family (7, 9, 24, 27,
36).
Spo0J is required both for accurate chromosome partitioning and for
sporulation (14). Spo0J binds to a series of sites
(parS sites) in the origin-proximal 20% of the chromosome
(22), clustering them into a large focus (10, 21,
23). Defects in spo0J perturb chromosome partitioning.
Cultures of spo0J null mutant cells produce approximately
1% anucleate cells, a frequency approximately 20- to 100-fold greater
than that of wild-type cells (14).
A null mutation in spo0J also causes an approximately
300-fold defect in sporulation relative to wild-type cells (14,
28, 34). spo0J null mutants are blocked early in
sporulation (stage 0) and have decreased expression of at least three
Spo0A~P-activated, sporulation genes (14, 28). The defects
in sporulation and gene expression caused by 
spo0J are
completely suppressed by the deletion of soj. Deletion of
soj alone has little effect on sporulation. These results
suggest that Soj is a negative regulator of sporulation and is itself
antagonized by Spo0J (14). This may represent a signaling
pathway that regulates sporulation in response to aspects of chromosome
organization or partitioning.
Soj negatively regulates transcription of several early
sporulation genes. Histidine-tagged Soj inhibits
transcription from the spoIIG promoter in vitro
(4). In addition, formaldehyde crosslinking has shown
that Soj associates with several Spo0A~P-dependent promoters in
vivo, including spoIIA, spoIIE, and
spoIIG, and that this association correlates with
decreased gene expression (36).
We have found that Soj also affects sporulation and
Spo0A~P-dependent gene expression by decreasing levels of Spo0A
protein. Soj negatively regulated transcription of spo0A and
associated with the spo0A promoter region in vivo.
Expression of spo0A from a heterologous promoter restored
Spo0A levels and partly bypassed the sporulation and gene expression
defects of a spo0J null mutant.
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MATERIALS AND METHODS |
Strains.
The B. subtilis strains used are listed
in Table 1. All strains are derivatives
of JH642 (33), which is referred to as wild type. Standard
procedures were used for transformations and strain construction
(11). The spo0J::spc and
(soj-spo0J)::spc deletion-insertions
were previously described (14). The
Pspac-spo0A+ and
Pspac-spo0Asad67 constructs were previously
described (15). The spoIIG-lacZ fusion
(17) is located in the specialized transducing phage SP
.
A cat marker ~90% linked (by transformation) to
spo0A (13) was used to transfer the
rvtA11 mutation (38).
The transcriptional fusion of the
spo0A sporulation
promoter Ps to
lacZ
[
spo0As(
129)
lacZ] was
constructed in the
amyE vector
pKS2 and was integrated into
the
B. subtilis chromosome by double
crossover at the
amyE locus. The endpoints of the fusion were
129 bp upstream
of the Ps transcription start site and the
BglII
restriction
site within the
spo0A coding
region.
Routine growth and maintenance of
E. coli and
B. subtilis was done in Luria-Bertani (LB) medium. For sporulation
experiments,
the rich sporulation broth, 2×SG (
20) was
used. IPTG (isopropyl-
-
D-thiogalactopyranoside)
was used
at 0.5 mM to induce expression of
spo0A from Pspac.
Antibiotics
were used at the following concentrations: ampicillin at
100 µg/ml,
chloramphenicol (Cm) at 5 µg/ml, spectinomycin (Spc) at
100 µg/ml,
neomycin (Neo) at 5 µg/ml, and erythromycin and
lincomycin together
(MLS) at 0.5 and 12.5 µg/ml,
respectively.
Protein extracts and Western blot analysis.
Cells were grown
in 2×SG, and samples of 1 to 5 ml were taken 2 h after the end of
exponential growth and centrifuged to collect cells.
Pspac-spo0A expression was induced with 0.5 mM IPTG three to
four doublings before the end of exponential growth. Cell pellets were
frozen and stored at 70°C. Pellets were resuspended in ice cold
buffer (10 mM Tris, pH 8; 100 mM NaCl; 10 mM KCl) with 1 mM phenazine
methosulfate (PMSF) and lysed by sonication on ice for four to six
pulses of 10 s each (Branson Sonifier, Power 4, duty cycle 100%).
Cell debris was pelleted by centrifugation in a microcentrifuge (10 min, 13,000 rpm, 4°C), and the supernatant was collected for further
use. Total soluble protein was determined by the Bio-Rad Protein Assay
(Bio-Rad) using bovine serum albumin as the standard. Sodium dodecyl
sulfate (SDS) sample buffer was added, and the extracts were vortexed
for 20 s and briefly centrifuged prior to electrophoresis on
SDS-12% polyacrylamide gels.
Proteins were transferred to polyvinylidene difluoride membrane
(Schleicher and Schuell) for 45 min at 450 mA using a semidry
electroblotter (Owl Scientific). Membranes were blocked by incubation
in Tris-buffered saline with Tween 20 (TBST [
37]) plus
5% dried
milk (Carnation). The primary antibody was a polyclonal
rabbit
antisera raised against purified Spo0A tagged with six
histidines.
The antibody was diluted 1:1,000 in blocking solution. The
secondary
antibody was
125I-labeled goat anti-rabbit
immunoglobulin G (DuPont-New England
Nuclear), used at 0.3 µCi/ml.
The
125I signal was detected using a
PhosphorImager (Molecular Dynamics)
and quantified using
ImageQuant software (Molecular
Dynamics).
-Galactosidase assays.
Cells were grown and treated as
described in the figure legends. -Galactosidase activity was
determined as described previously (16, 25). The specific
activity is calculated as the change in A420 per
minute per milliliter of culture per unit of optical density at 600 nm
times 1,000.
Spore assays.
Cells were grown in 2×SG at 37°C, and
spores were assayed 20 to 24 h after the end of exponential
growth. The number of viable cells per milliliter of culture was
determined as the total number of CFU on LB plates. The number of
spores per milliliter of culture was determined as the number of CFU on
plates after heat treatment (80°C for 20 min). The percent
sporulation is 100 times the ratio of spores per milliliter to viable
cells per milliliter.
Formaldehyde cross-linking and immunoprecipitations.
Two
hours after the end of exponential growth in 2×SG, 10-ml samples of
culture were taken for analysis. Generally, cross-linking and sample
preparations were performed as described earlier (22, 36)
and are based on previously published chromatin immunoprecipitation assays (8, 39, 40). Samples were treated with sodium
phosphate (10 mM final concentration) and formaldehyde (1% final
concentration) for 3 min at room temperature. Cells were pelleted and
washed twice with 10 ml of phosphate-buffered saline (pH 7.3)
(1), resuspended in 500 µl of solution A (10 mM Tris [pH
8], 20% sucrose, 50 mM NaCl, 10 mM EDTA) containing 20 mg of lysozyme
per ml, and incubated at 37°C for 30 min. Then, 0.5 ml of 2× IP
buffer (100 mM Tris, pH 7; 300 mM NaCl; 2% Triton X-100) and PMSF (to
final concentration of 1 mM) were added, and the cells were incubated for 10 min at 37°C. DNA was sheared to an average size of 500 to
1,000 bp by sonication. Insoluble debris was removed by centrifugation, and the supernatant was transferred to a fresh microfuge tube. To
determine the amount of DNA immunoprecipitated relative to the amount
of total DNA prior to immunoprecipitation, 100 µl of supernatant was
removed and saved for later analysis ("total" DNA control).
Protein and protein-DNA complexes were immunoprecipitated (overnight,
4°C) by incubation with affinity-purified polyclonal
anti-Soj
antibodies followed by incubation with 30 µl of a 50%
protein
A-Sepharose slurry (1 h, room temperature). Complexes
were collected by
centrifugation and washed seven times with 1×
IP buffer (twice-diluted
2× IP buffer described above) and twice
with 1 ml of TE (10 mM Tris,
pH 8; 0.1 mM EDTA). The slurry was
resuspended in 100 µl of TE. The
100 µl of "total" DNA control
was mixed with 100 µl of TE and
brought to a final concentration
of 0.1% SDS. Formaldehyde crosslinks
of both immunoprecipitated
and total DNA samples were reversed by
incubation at 65°C for
6 h. Samples were used for PCR without
further
treatment.
PCR was performed with Vent DNA polymerase (New England Biolabs) using
serial dilutions of the immunoprecipitate and the total
DNA as
templates. Oligonucleotide primers were typically 20 to
25 bases in
length, and the amplified fragments ranged from ~250
to 550 bp in
size. Sequences of all primers are available upon
request. Relative
amounts of Soj-DNA complexes were determined
by comparing the intensity
of bands in the linear range of the
PCR from both the immunoprecipitate
and the "total" DNA control.
Gels were photographed onto Polaroid
665 film, and the negatives
were scanned and processed using Adobe
Photoshop
software.
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RESULTS AND DISCUSSION |
Soj decreases the level of Spo0A protein in a spo0J
null mutant.
Deletion of spo0J causes a decrease in
expression of at least three early sporulation genes that require the
Spo0A transcription factor for activation (14). We found
that Spo0A protein levels were lower in the spo0J null
mutant. We measured the amount of Spo0A protein by quantitative Western
blots (see Materials and Methods). A spo0J null mutant had
approximately 45% less Spo0A protein than the wild-type cells (Fig.
1A). The amount of Spo0A was restored to
wild-type levels in a (soj-spo0J) double mutant. This
indicates that the decrease in Spo0A protein levels in the spo0J mutant was dependent on soj, that Soj
negatively regulates accumulation of Spo0A protein, and that this
effect of Soj is antagonized by Spo0J.
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FIG. 1.
Quantitative 125I Western blot of Spo0A in
strains containing mutations in spo0J and soj.
Strains were grown in 2×SG medium. Samples were harvested 2 h
after the end of exponential growth. Three different amounts of protein
were loaded for each sample to show the linearity of the detection
system. Quantitation of Spo0A protein levels compiled from independent
experiments is shown below the gel. Numbers are the mean ± the
standard error of the mean, normalized such that wild-type cells have 1 unit of Spo0A protein. (A) Spo0A expressed from the native promoter.
Proteins in each lane were isolated from the following strains. Lanes 1 to 3, AG1113 (wild type); lanes 4 to 6, KI1778 (spo0J);
lanes 7 to 9, KI1798 [(soj-spo0J)]. The total amounts
of protein loaded were 2 (lanes 1, 4, and 7), 4 (lanes 2, 5, and 8) and
6 (lanes 3, 6, and 9) µg. (B) Spo0A expressed from the Pspac
promoter. Proteins in each lane are isolated from the following
strains. Lanes 1 to 3, DZR160 (Pspac-spo0A); lanes 4 to 6, JQ280 (Pspac-spo0A spo0J); lanes 7 to 9, JQ279
[Pspac-spo0A (soj-spo0J)]. The total amounts
of protein loaded were 3 (lanes 1, 4, and 7), 6 (lanes 2, 5, and 8),
and 9 (lanes 3, 6, and 9) µg.
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The effect of
spo0J and
soj on Spo0A protein
levels was dependent on the
spo0A promoter. We placed
expression of
spo0A under
control of the
LacI-repressible-IPTG-inducible promoter, Pspac
(
43), in a
strain otherwise null for
spo0A. Expression of
spo0A from Pspac bypassed the effect of Spo0J and Soj on
Spo0A protein
levels. In the presence of IPTG, Pspac-
spo0A
permitted production
of similar levels of Spo0A in
spo0J+,
spo0J, and
soj-spo0J strains (Fig.
1B). Furthermore, the amount
of
Spo0A present in early sporulation was similar to that found
in
wild-type cells expressing
spo0A from its native promoters.
These data indicate that Soj regulates the accumulation of Spo0A
protein when
spo0A is expressed from its own promoter and
that
Soj might affect transcription of
spo0A. Results
discussed below
indicate that the effect of Soj on Spo0A protein levels
contributes
to the sporulation defect in the
spo0J mutant.
Soj cross-links to the spo0A promoter region in a
Spo0J-regulated manner.
Soj associates with several sporulation
promoters in vivo (36) and at least one of these promoters
in vitro (4). We tested for the association of Soj with the
spo0A promoter region in vivo by using a
cross-linking-immunoprecipitation assay (see Materials and Methods).
Briefly, formaldehyde was added to cultures to cross-link proteins and
DNA. Cells were lysed, and the DNA was sheared into fragments with an
average size of 500 to 1,000 bp. We used affinity-purified polyclonal
antibodies to immunoprecipitate Soj and DNA associated with Soj. After
thorough washing, the cross-links were reversed and the presence of a
particular region of the chromosome in the immunoprecipitate was
detected by PCR.
We found that Soj was associated with the
spo0A promoter
region in wild-type cells and in a
spo0J null mutant (Fig.
2). Cross-linking
was done 2 h after
the end of stationary phase, a time when the
effect of Soj on
transcription of sporulation genes is readily
apparent. In the
experiments shown (Fig.
2), dilutions of total
DNA (before
immunoprecipitation) and dilutions of DNA from the
immunoprecipitates
were used for PCR. This allowed for the comparison
of relative amounts
of specific regions in the immunoprecipitate
from wild-type and
spo0J mutant cells (
36). There was approximately
fourfold-more specific DNA in the immunoprecipitates from the
spo0J null mutant than from wild-type cells (Fig.
2). This
difference
was not caused by differences in the amount of DNA present
in
the immunoprecipitation nor in the amount of Soj present in the
cells. All the immunoprecipitations were done from approximately
equal
amounts of sheared DNA (Fig.
2, righthand panels), and there
is
slightly less Soj protein present in
spo0J null mutants than
in wild-type cells (S. Venkatasubramanyam and A. D. Grossman,
unpublished results). The presence of DNA in the immunoprecipitate
was
dependent on Soj because little or no DNA was detected in
immunoprecipitates from a
(
soj-spo0J) double mutant (Fig.
2).
These results indicate that Soj specifically associates with DNA
near the
spo0A promoter and that this association increases
in
the absence of Spo0J.
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FIG. 2.
Association of Soj with the spo0A promoter
region in vivo. Wild-type (JH642), spo0J (AG1468), and
(soj-spo0J) (AG1505) strains were grown in 2×SG
medium at 37°C. Two hours after the end of exponential growth,
formaldehyde was added to cross-link the protein and DNA. Protein-DNA
complexes were immunoprecipitated by using anti-Soj antibodies. The
presence of a given promoter region was assayed by PCR amplification
with primers designed to amplify the promoter regions of
spo0A, and codV and recA were used as
controls. Lanes labeled IP DNA are DNA products from PCR assays
performed on a dilution series (4, 2, 1, 0.5, and 0.25 µl) of the
immunoprecipitated material. Lanes labeled Total DNA are DNA products
from PCR assays performed on a dilution series (the equivalent of
1/250, 1/500, 1/1,000, 1/2,000, and 1/4,000 µl) of sample DNA taken
prior to immunoprecipitation.
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These results are consistent with previous analysis of the in vivo
association of Soj with the promoter regions of the
spoIIA,
spoIIE, and
spoIIG loci (
36). In all
cases, the association
of Soj with DNA increases in the absence of
Spo0J and correlates
with decreased gene expression. All of these
promoters are also
activated by Spo0A~P, but association of Soj with
the promoter
regions in vivo was independent of
spo0A (data
not shown). It
is possible that Soj interacts with a binding site
similar to
that for Spo0A, but in vitro experiments have indicated that
Soj
binding to DNA is complicated and that Soj is not simply
recognizing
the same sequences as Spo0A (
4).
Soj inhibits transcription of the sigma-H-dependent promoter of
spo0A.
To determine whether Soj affects transcription of
spo0A, we examined expression of spo0A-lacZ
fusions. Transcription of spo0A is directed from two
promoters (Fig. 3A): a sigma-A-dependent promoter (Pv) expressed during growth and a sigma-H and
Spo0A~P-dependent promoter (Ps) expressed early during sporulation
(5, 35, 41). Transcription from spo0A Ps was
reduced approximately 60% in the spo0J mutant and was fully
restored in the (soj-spo0J) double mutant (Fig. 3B).
Expression from the Pv promoter was not affected by Spo0J or Soj (data
not shown). These results show that, in a spo0J
background, Soj inhibits transcription from the
spo0APs promoter.
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FIG. 3.
spo0J and soj regulate the
expression of the spo0A sporulation promoter. (A)
spo0A is expressed from two promoters: sigma-A-activated Pv
and sigma-H-activated Ps. (B) -Galactosidase activity expressed from
the spo0A-Ps-129-lacZ fusion. Strains were grown in 2×SG
medium, and samples were taken for the determination of
-galactosidase specific activity. Time zero indicates the time
at which the culture left exponential growth. Symbols:  , wild-type
(AG1332);  , spo0J (JQ266);  ,
(soj-spo0J) (JQ270).
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Previous results indicated that mutations in
spo0J and
soj had little if any effect on transcription of
spo0A from the complete
Pv+Ps promoter region
(
4). We found that expression of
spo0A(
Pv+Ps)-
lacZ was reduced
approximately 25% in a
spo0J null mutant (data not
shown).
This effect is quite small and is consistent with approximately
equal
contributions from each promoter to the total amount of
spo0A transcript (
5). Since the effect of
spo0A protein levels
appears larger than the effect on
transcription, we suspect that
transcripts initiating from Ps might be
translated more efficiently
than those initiating from
Pv.
Restoring Spo0A protein to wild-type levels in a
spo0J background partly restores sporulation and early
sporulation gene expression.
We have shown that in the absence of
Spo0J, Soj causes an ~45% decrease in Spo0A protein levels. It is
possible that this decrease causes part, none, or all of the
sporulation defect in spo0J cells. To distinguish between
these possibilities, we examined whether restoring Spo0A protein to
near wild-type levels, by expressing it from Pspac, could also restore
sporulation in a spo0J null mutant.
Expressing
spo0A from Pspac partly rescued sporulation in
spo0J cells, increasing sporulation approximately 10-fold
relative
to
spo0J cells expressing
spo0A from
the endogenous promoters
(Table
2). In
wild-type or
(
soj-spo0J) backgrounds,
Pspac-
spo0A+ did not affect sporulation.
The amount of Spo0A protein that
accumulated in
Pspac-
spo0A+ strains varied from ~15% less
than to ~40% greater than wild
type, without affecting either the
degree of sporulation rescue
or the kinetics of Spo0A-activated gene
expression. Therefore,
it is unlikely that the 10-fold rescue of
sporulation was caused
by an overexpression of
spo0A. Thus,
the decrease in Spo0A levels
observed in
spo0J cells
causes part, but not all, of the sporulation
defect in these cells.
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TABLE 2.
Expression of spo0A from the Pspac promoter
partially rescues the sporulation defect of a spo0J
null mutant
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Transcription of
spo0A from Pspac also partially restored
expression of
spoIIE and
spoIIG in the
spo0J mutant.
spoIIA,
spoIIE,
and
spoIIG are all transcribed early in sporulation from
Spo0A~P-activated
promoters and code for proteins that are essential
for spore formation.
In a
spo0J mutant,
Pspac-
spo0A+ caused a three- to fourfold
increase in expression (as judged
by fusions to
lacZ) of
spoIIG (Fig.
4) and
spoIIE (data not shown)
relative to a strain expressing
spo0A from its native promoters.
Pspac-
spo0A+ had no detectable effect on
spoIIA promoter activity (data not
shown). In wild-type or
(
soj-spo0J) backgrounds,
Pspac-
spo0A+ had no effect on the timing or
level of activity from any of
the
spoII promoters tested.
These results show that, in
spo0J cells, Soj represses
spoIIE and
spoIIG expression partly through
its
effect on Spo0A levels. However, it is clear from these results
that
Soj also inhibits transcription of
spoIIA,
spoIIE, and
spoIIG in a manner independent of its
effect on Spo0A levels. At least
part of this inhibition appears
to be direct as Soj associates
with these promoter regions in vivo
(
36) and inhibits transcription
from the
spoIIG
promoter in vitro (
4).
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FIG. 4.
Expression of spoIIG-lacZ in strains
expressing spo0A from the native promoters or the Pspac
promoter. Strains were grown in 2×SG medium, and samples were taken
for the determination of -galactosidase specific activity. Strains
expressing spo0A from Pspac were treated with 0.5 mM IPTG 3 to 4 h before the end of exponential growth. Time zero indicates
the time at which the culture left exponential growth. Symbols:
, wild-type (JQ284); , spo0J (JQ302);  ,
Pspac-spo0A (JQ304);  , Pspac-spo0A spo0J
(JQ305).
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A constitutively active form of Spo0A does not fully bypass the
sporulation defect of the spo0J mutant.
Soj inhibits
expression from at least four sporulation promoters
(spo0APs, spoIIA, spoIIE, and
spoIIG), and part of this effect appears to be
independent of its effect on Spo0A protein levels. Many signals that
regulate the initiation of sporulation act by affecting phosphorylation
of Spo0A (3, 12). We used a constitutively active mutant
form of Spo0A to show that Soj does not significantly impair
phosphorylation of Spo0A.
If Soj regulates sporulation by decreasing Spo0A phosphorylation, then
a mutant form of Spo0A (called Spo0A
Sad67) (
15)
that can activate gene expression and sporulation in
an
unphosphorylated state ought to bypass the sporulation defect
of a
spo0J mutant. Expression of
spo0Asad67 from the Pspac promoter permits
wild-type sporulation levels
in cells that cannot phosphorylate Spo0A
and allows Spo0A~P-activated
gene expression in the absence of the
regulatory signals that
are normally required (
15).
We compared sporulation in
spo0J cells carrying either
Pspac-
spo0A+ or
Pspac-
spo0Asad67. In the presence of IPTG,
both Pspac-
spo0A and
Pspac-
spo0Asad67 rescued sporulation to the same
degree, i.e., about sevenfold
(Table
3).
Pspac-
spo0Asad67 was no more efficient at
bypassing the effects of
spo0J on sporulation
than was
Pspac-
spo0A+. This result strongly
indicates that Soj does not inhibit sporulation
by decreasing the
phosphorylation state of Spo0A.
rvtA11 allele of spo0A and sporulation
defect of spo0J cells.
The notion that Soj and
Spo0J do not affect the phosphorylation state of Spo0A appears to
contradict previous findings that indicated Soj and Spo0J might
affect the pathway leading to phosphorylation of Spo0A
(14). In wild-type cells, the phosphorylation pathway consists of at least three histidine kinases (KinA, KinB, and KinC),
Spo0F, Spo0B, and Spo0A (2). The kinases autophosphorylate and donate phosphate to Spo0F. Phosphate is then transferred from Spo0F
to Spo0B and finally from Spo0B to Spo0A. Mutations in spo0F and/or spo0B abolish sporulation (34), indicating
that the kinases normally cannot directly phosphorylate Spo0A
sufficiently to activate sporulation. A mutation in spo0A,
rvtA11, suppresses the sporulation defect caused by
spo0F and spo0B mutations (38), and
this suppression depends on kinC (Table
4) (18, 19), indicating that
Spo0ArvtA11 can obtain phosphate directly from KinC.
Previously we showed that
rvtA11 significantly bypasses the
sporulation defect caused by a null mutation in
spo0J
(
14),
indicating that
spo0J might block
sporulation by decreasing activity
of the phosphorelay. The implication
that
spo0J affects sporulation
by decreasing
phosphorylation of Spo0A is contradictory to results
with the
spo0Asad67 mutants described
above.
To explore this apparent contradiction, we determined if
kinC is required for the
rvtA11 bypass of
spo0J. If the phosphorelay
is inhibited in the
spo0J mutant, then the bypass in sporulation
caused by
rvtA11 should depend on
kinC. We found that
even in
a
kinC null mutant,
rvtA11 was able to
partly suppress the sporulation
defect of the
spo0J mutant
(Table
4). In the absence of
kinC,
the sporulation
efficiency of the
rvtA11 spo0J strain decreased
threefold, indicating that the
spo0J null mutation causes a
small
effect on the phosphorelay but that most of the suppression by
rvtA11 was independent of KinC. These results are consistent
with
those obtained with the
spo0Asad67 mutation
and reinforce the conclusion that the Soj-dependent
decrease in
sporulation observed in
spo0J cells is not caused
by a decrease in phosphorylation of Spo0A. We do not know how
rvtA11 suppresses the sporulation defect of a
spo0J mutant better
than the constitutively active
Pspac-
spo0Asad67.
Summary.
We have used genetic methods to clarify the role of
the partitioning protein, Soj, in regulating the initiation of
sporulation. Our current view of the effects of Soj on sporulation gene
expression is summarized in Fig. 5. In
addition to its apparently direct effect on transcription of several
spoII genes (4, 36), Soj also negatively
regulates expression of spo0A, which is itself required to
activate transcription of the spoII genes. Restoring Spo0A to wild-type levels partially bypasses the sporulation defect in
spo0J cells. Soj does not appear to regulate
sporulation significantly by affecting phosphorylation of Spo0A. These
results are consistent with Soj acting as a transcriptional repressor
of several Spo0A~P-activated genes, including spo0A,
spoIIA, spoIIE, and spoIIG (Fig. 5).
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|
FIG. 5.
A model for the regulation of gene expression by Spo0J,
Soj, and Spo0A. Soj negatively regulates expression of spo0A
Ps, spoIIA, spoIIE, and spoIIG.
Spo0A~P stimulates expression from these same promoters. Soj acts
directly and indirectly through its effect on spo0APs. Spo0J
antagonizes Soj.
|
|
Spo0J probably antagonizes the activity of Soj by maintaining Soj
localization at the cell poles and away from the promoter
regions of
Spo0A~P-activated genes (
24,
36). This sequestration
appears to be related to the putative ATPase activity of Soj.
To date,
it has not been possible to detect Soj ATPase activity
in vitro, either
in the presence or in the absence of Spo0J (
4,
36). We
suspect that the activity of Spo0J is somehow associated
with its role
in organizing the origin region of the chromosome
(
22,
23)
and/or its function in chromosome partitioning (
14).
It will
be challenging to determine how this regulation
occurs.
| |
ACKNOWLEDGMENTS |
We thank K. Siranosian for constructing the spo0A-lacZ
fusions, K. Ireton for several strains and for doing preliminary
experiments, and K. Bacon for comments on the manuscript. J.D.Q. was
supported, in part, by an NSF predoctoral fellowship. This work was
also supported, in part, by NIH Public Health Services grant GM41934 to
A.D.G.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Bldg. 68-530, Massachusetts Institute of Technology,
Cambridge, MA 02139. Phone: (617) 253-1515. Fax: (617) 253-2643. E-mail: adg{at}mit.edu.
| |
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Abstract
Introduction
