Sir William Dunn School of Pathology,
University of Oxford, Oxford OX1 3RE, United Kingdom
 |
INTRODUCTION |
During sporulation in Bacillus
subtilis, differential gene expression in the prespore and the
mother cell compartments is governed by four different,
sporulation-specific sigma factors, whose activities are tightly
regulated both temporally and spatially (9, 11, 18, 30). Two
sigma factors are active specifically in the prespore
first
F and then later Gwhile
E and then K control transcription in the
mother cell.
In the mother cell, the expression of both the E and
K regulons has been shown to be subject to extra levels
of control by small DNA-binding transcriptional regulators, SpoIIID and
GerE (reviewed recently in reference 18). Similarly,
in the prespore, the SpoVT protein has been identified to control
G-dependent gene expression (3). A number of
genes are now known to be regulated by F, including
various genes required for sporulation (gpr
[33], spoIIIG [32],
spoIIR [16, 23], and spoIIQ
[22]), a catalase (katX
[2]), a DD-carboxypeptidase
(dacF [28]), and several genes of unknown
function (6), but so far, no factor capable of modulating
F-dependent transcription has been found. Here we report
the identification and characterization of a new prespore-specific
regulatory gene, rsfA, which codes for a protein with
similarity to regulatory leucine zipper proteins. We show that the
expression of rsfA is dependent on both F and
G. Disruption of rsfA had different effects
on the expression of F-dependent genes. Consistent with
its role as a transcriptional regulator, an RsfA-green fluorescent
protein (GFP) fusion protein was shown to colocalize with chromosomal
DNA. Limited experiments with a paralogue of rsfA, called
ylbO, suggest that it may play an analogous role to
rsfA in the control of mother cell gene expression.
| |
MATERIALS AND METHODS |
Bacterial strains.
The B. subtilis strains and
plasmids used in this study are described in Table
1. The Escherichia coli strain
used was DH5
[F
endA1 hsdR17
(rK mK+) supE44
thi-1 recA1 gyrA96 (Nalr)
relA1 
(lacZYA-argF)U169
80dlac(lacZ)M15; Gibco-BRL].
General methods.
B. subtilis cells were made competent
for transformation with DNA by the method of Anagnostopoulos and
Spizizen (1) as modified by Jenkinson (15).
B. subtilis chromosomal DNA was prepared by a scaled-down
method based on the one described by Errington (8). DNA
manipulations and E. coli transformations were carried out
by standard methods (27).
The solid medium used for growing B. subtilis was nutrient
agar (Oxoid). Chloramphenicol (5 µg ml1), kanamycin (5 µg ml1), tetracycline (12 µg ml1),
erythromycin (1 µg ml1) and lincomycin (25 µg
ml1), and 0.01% X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) were
added as required. The media used for growing E. coli were 2× TY (tryptone-yeast extract) (27) and nutrient agar
(Oxoid) supplemented with ampicillin (100 µg ml1) as required.
PCR primers.
Detailed information on the primers used is
available upon request.
Construction of the deletion strains 1224 [(ywhD-rsfA)] and 1225 (rsfA) by
double-crossover homologous recombination.
The 1,513-bp DNA
fragment containing the C-terminal coding region of ywhE and
the 1,454-bp DNA fragment containing ywfM and part of
ywfL were amplified by PCR with Pfu DNA
polymerase with primers ywhE-F and ywhE-R and ywfM-F and ywfL-R,
respectively. The two PCR products were then digested with
SalI, ligated to an XhoI-XhoI fragment
containing the tetracycline resistance gene (isolated from plasmid
pSG4906). The ligation reaction was transformed into a wild-type
B. subtilis strain, 168, with selection for tetracycline resistance. One of the transformants was examined by PCR to confirm that it had contained the deletion, and the strain was designated 1224. The deleted region was 10 kb long and contained genes
ywhD-A, thrZ, mmr, ywgBA,
ywfO, ywzC, and rsfA.
Strain 1225 (rsfA) was constructed in a similar way, with
primers ywfO-F2 and ywfN-R5 and ywfN-F2 and ywfL-R. The PCR products were digested with SstI and PstI, respectively,
and ligated to the tetracycline resistance gene isolated from pBEST309
(digested with SstI and PstI). The ligation
reaction was transformed into strain 168 directly, and one of the
transformants was examined by PCR to confirm the deletion.
Identification of the gene responsible for the phenotype of the
deletion in strain 1224.
DNA fragments containing the
ywhD-to-mmr (6 kb) and
mmr-to-rsfA (6.9 kb) regions were amplified by
long-range PCR with primers ywhD-F and mmr-R and thrZ-F and ywfM-R,
respectively. The PCR products were then digested with XhoI,
ligated to SalI-digested pPS1326, and transformed directly
into strain 1224. The transformants were examined by long-range PCR to
confirm the structure of the insertions at the amyE locus.
Complementation analysis with small overlapping DNA fragments (of
approximately 1 to 1.2 kb) in the mmr-rsfA region was
performed in the same way with different primers.
Insertion of rsfA at amyE.
A 1,240-bp DNA
fragment containing the rsfA gene (and its putative
promoter) was amplified by PCR with primers ywzC-F and ywfM-R and
Pfu DNA polymerase. The PCR product was digested with restriction enzyme SalI, ligated to SalI-digested
pSG4903, and then transformed directly into strain 1225, with selection
for kanamycin resistance. Several Spo+-appearing
transformants were first checked for an Amy phenotype and
then were examined by PCR and with restriction enzyme digestions to
confirm the site of the insertion and its orientation relative to
amyE. Strain 1237 had rsfA inserted in the
opposite orientation relative to amyE.
Induction of sporulation.
B. subtilis cells were grown
in hydrolyzed casein growth medium (CH) at 37°C and induced to
sporulate by the resuspension method of Sterlini and Mandelstam
(29), as specified by Partridge and Errington
(25). Times (minutes) after resuspension of cells in the
starvation medium were denoted t0,
t60, etc. Spore heat resistance (85°C, 15 min)
was measured 9 h after the initiation of sporulation and expressed
as the percentage of viable cells.
Induction of sigma factors during vegetative growth.
Overnight cultures grown at 30°C were diluted in fresh warm medium to
an optical density at 570 nm (OD570) of 0.03 and grown at
37°C. For each strain, the culture was divided into two portions at
an OD570 of 0.2, and IPTG
(isopropyl--D-thiogalactopyranoside [1 mM, final
concentration]) was added to one of the two cultures. The incubation
of both cultures was continued and samples were removed at intervals
for monitoring -galactosidase and OD570.
Assay for -galactosidase activity.
-Galactosidase
activity was measured either with ONPG
(o-nitrophenyl--D-galactopyranoside) and
expressed as Miller units as described by Daniel et al. (5)
or with MUG (4-methylumbelliferyl--D-galactopyranoside) and expressed as MUG units by the method of Errington and Mandelstam (10). One MUG unit is the amount of enzyme that catalyzes
the production of 1 nmol of 4-methylumbelliferone per min.
Live cell fluorescence microscopy.
Live cell examination of
GFP and DAPI (4,6-diamidino-2-phenylindole) fluorescence and image
capture were done as described previously (36).
| |
RESULTS |
Identification of the rsfA gene.
In the course of
conducting a screen for chromosome segregation mutants, we noticed that
deletion of a 10-kb fragment from the chromosome (3,851 to 3,861 kb),
containing genes ywhD to ywfN, reduced the level
of expression of a prespore-specific reporter gene, gpr-lacZ
(data not shown). When introduced into wild-type strain 168, the
deletion caused a reduction in sporulation frequency of about 30%
(strain 1224). To identify the gene(s) or DNA sequence(s) responsible
for this phenotype, we reintroduced into strain 1224 segments of DNA
from the deleted region at the amyE locus, in two
overlapping fragments of about 6 kb each (ywhD to
mmr and mmr to ywfN). Only the
fragment containing genes mmr to ywfN
complemented the Spo phenotype of strain 1224. We then divided this
region into seven overlapping fragments and again introduced the
individual fragments into strain 1224 at the amyE locus.
Only one of the fragments, the one containing the intact
ywfN gene, showed complementation of the Spo phenotype. We
next constructed a ywfN::tet null
mutant (strain 1225) and confirmed that the mutant showed a decrease in
gpr-lacZ expression (Fig. 1)
and reduction in frequency of heat resistance similar to that of strain
1224. We renamed the ywfN gene rsfA (for
regulator of sigma F-dependent gene expression [see below]).
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FIG. 1.
Disruption of rsfA reduces the expression of
a F-dependent reporter gene, gpr-lacZ.
Wild-type ( ) and the rsfA::tet
mutant ( ) strains containing a gpr-lacZ fusion were
induced to sporulate, and samples were taken at intervals for assay of
-galactosidase activity. Time zero is the time of initiation of
sporulation.
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In the course of these experiments, an rsfA
(ywfN) mutant became available via the European
Community-sponsored Functional Analysis Project on B. subtilis. The BFA1335 mutant contains an insertion of the
nonreplicating pMUTIN4 plasmid (34), which disrupts
rsfA and fuses its promoter to lacZ. Again, this
mutant showed a reduced sporulation frequency similar to that of strain 1224.
The rsfA gene encodes a protein 258 amino acids long with an
acidic isoelectric point (5.1). The deduced amino acid sequence showed
some features of eukaryotic gene regulatory proteins, including a
stretch of basic amino acids and a possible leucine zipper motif, suggesting that the protein forms dimers or multimers by means of a
coiled-coil structure (Fig. 2).
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FIG. 2.
The deduced amino acid sequence of RsfA. The stretch of
basic amino acids is underlined. The heptad repeat of leucine residues
characteristic of "leucine zippers" is indicated by underlined
boldface type.
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The expression of rsfA is prespore specific and is
dependent on both F and G.
To
monitor the expression of the rsfA gene, strain BFA1335 was
grown in CH medium and then induced to sporulate, and samples were
taken for analysis of -galactosidase expression during both vegetative growth and sporulation. No expression of
rsfA-lacZ could be detected during vegetative growth (data
not shown). During sporulation, expression was induced from about
t75 (75 min after the initiation of sporulation)
(Fig. 3). Expression was abolished in a
spoIIAC mutant (in which F is not formed),
and it was approximately doubled in a spoIIGA mutant (in
which E is not activated) (Fig. 3A), which is
characteristic of several F-dependent genes (16,
21-23).
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FIG. 3.
Effects of mutations in genes encoding
sporulation-specific sigma factors on expression of
rsfA-lacZ. Strains containing an rsfA-lacZ fusion
in a wild-type background ( [strain BFA1335]) or in the presence
of mutations in spoIIAC ( [strain 1226]) and
spoIIGA ( [strain 1227]) (A) or spoIIIG ( [strain 1228]) (B) were induced to sporulate at time zero, and
samples were taken at intervals for assay of -galactosidase
activity.
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The expression of rsfA-lacZ was reduced slightly at later
times in a spoIIIG mutant (Fig. 3B), suggesting that
expression of rsfA is driven not only by EF,
but also by EG. This was confirmed by the strong
induction of rsfA-lacZ during exponential growth in cells
engineered to synthesize either F or G
(Fig. 4).
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FIG. 4.
Induction of rsfA-lacZ in vegetative cells
producing F or G. Strains 1229 (,  [producing F under Pspac
control]), 1230 ( ,  [producing G under
Pspac control]), and BFA1335 (, [an
isogenic strain without the Pspac construct])
were grown in 2× TY, and samples from cultures with (solid symbols) or
without (open symbols) IPTG were taken for assay of -galactosidase
activity.
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Effects of an rsfA null mutation on prespore-specific
gene expression.
The structural similarity of RsfA to leucine
zipper proteins and the existence of the basic amino acid stretch
suggested that the protein might function as a transcriptional
regulator. Since rsfA was expressed long before
G becomes active, it was likely that RsfA mainly
regulates F-dependent transcription. To test this, we
examined the expression of lacZ fusions to several
F-dependent genes in the
rsfA::tet null mutant. To eliminate
possible secondary effects on G (because the recognition
specificities of F and G overlap
[31, 32]), the strains used also carried a
spoIIIG deletion. The rsfA mutation had small but
reproducible effects on the levels of transcription from all of the
promoters tested, except that of spoIIQ, which was
unaffected (typical results are shown in Fig.
5). Expression of fusions to the
katX, spoIIIG, and sspE-2G promoters
was, like that of gpr, reduced in an rsfA mutant
background compared with that of the wild type. Interestingly, in
contrast, the spoIIR gene showed increased expression,
suggesting that it is negatively regulated by RsfA (Fig. 5D). Similar
results were obtained in a spoIIIG+ background
(data not shown), in accordance with the idea that RsfA mainly affects
transcription directed by EF.
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FIG. 5.
Contrasting effects of an rsfA null mutation
on expression of lacZ fusions to various prespore-specific
genes. Isogenic strains containing a spoIIIG mutation and
lacZ fusions to katX (A [strains 1234 and
1245]), spoIIIG (B [strains 1235 and 1246]),
sspE-2G (C [strains 1236 and 1247]), spoIIR (D
[strains 1232 and 1243]), spoIIQ (E [strains 1233 and
1244]), or rsfA (F [see below]) were induced to sporulate
at time zero, and samples were taken at intervals for assay of
-galactosidase activity. In panels A to E, results are compared for
the rsfA+ strain (solid symbols) and the
isogenic rsfA::tet derivative (open
symbols). In panel F, the open symbols correspond to strain BFA1335
(rsfA::pMUTIN4 rsfA-lacZ), and the
solid symbols correspond to a derivative carrying a wild-type copy of
rsfA inserted into the amyE locus (strain
1239).
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Negative autoregulation of rsfA expression.
To
test whether rsfA was autoregulated, we introduced a
wild-type copy of rsfA at the amyE locus into the
strain BFA1335. The expression of rsfA-lacZ in the resulting
strain, 1239, was then examined and compared with that of BFA1335. As
shown in Fig. 5F, the presence of a wild-type copy of rsfA
reduced the expression of rsfA-lacZ at later time points. It
thus, appeared that rsfA exerts negative regulation on its
own promoter, as on the spoIIR promoter.
An RsfA-GFP fusion colocalizes with chromosomal DNA.
The
different effects of the rsfA null mutation on the
expression of prespore-specific genes suggested that the protein acts directly on DNA, rather than by affecting the stability or activity of
F. As one approach to testing whether the protein
associates with DNA in vivo, we examined the subcellular localization
of RsfA by making a fusion of the GFP from Aequorea victoria
to the C terminus of RsfA. The strain, 1242, containing the
rsfA-gfp fusion construct (and a truncated rsfA
gene) produced an amount of heat-resistant spores similar to that of
wild-type strain 168 (not shown), suggesting that the fusion protein is
functional. As shown in Fig. 6A, the RsfA-GFP fusion protein was localized to the prespore in wild-type strain 1242, as expected for a F-dependent gene. To see
more clearly the relative locations of the fusion protein and DNA, we
used a spoIIIE36 mutant, in which only 30% of the prespore
chromosome is present in the prespore compartment (35).
Figure 6B shows that in the spoIIIE36 mutant, the RsfA-GFP
fusion protein colocalized with the prespore chromosome.
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FIG. 6.
Fluorescence microscopic localization of RsfA-GFP in
sporulating cells or in vegetative cells engineered to produce
F. (A and B) Sporulating cells
(t180) of the wild type (A [strain 1242]) or
spoIIIE36 mutant (B [strain 1254]) containing the
rsfA-gfp fusion. (C and D). Cells of strains containing an
IPTG-inducible spoIIAC gene allowing ectopic synthesis of
F in vegetative cells and either the rsfA-gfp
fusion (C [strain 1253]) or a Pgpr-gfp fusion
(D [strain 1252]) 120 min after the addition of IPTG. (Left panels)
GFP signals showing the localization of the GFP fusions. (Middle
panels) DAPI signals showing the nucleoids. (Right panels) Overlays of
the GFP and corresponding DAPI images. Arrows point to some of the
prespore compartments.
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The affinity of RsfA for DNA was further demonstrated by the
colocalization of the RsfA-GFP fusion to chromosomal DNA when the
expression of rsfA-gfp was induced during vegetative growth by the synthesis of F, as shown in Fig. 6C. Under the
same conditions, the control Pgpr-GFP fusion
(also F-dependent) showed a uniformly distributed GFP
signal throughout the whole cell (Fig. 6D), confirming that the
localization pattern of RsfA-GFP was not caused by the GFP moiety of
the fusion protein.
Interestingly, when rsfA was placed under the control of a
xylose-inducible Pxyl promoter and strain 1241 was induced to synthesize RsfA during vegetative growth, the cells
showed impaired chromosome distribution and segregation and eventually
lysed (data not shown). The molecular basis for this effect is not
clear, but it could be consistent with the notion that RsfA acts
directly on DNA.
The ylbO gene encodes a protein with a high degree of
similarity to RsfA.
A search of the B. subtilis genome
DNA sequence revealed that the ylbO gene located at 1,575.5 kb on the chromosome encodes a 193-amino-acid protein with high
similarity to RsfA, particularly over the N- and C-terminal regions of
the proteins (Fig. 7).
To test whether the function of ylbO was related to that of
rsfA, we examined the phenotype of strain BFA3241, in which
ylbO has been disrupted and its promoter fused to
lacZ. This strain showed no obvious phenotype, and it
produced amounts of heat-resistant spores similar to those produced by
wild-type strain 168 (not shown). Nevertheless, expression of the
ylbO-lacZ fusion was induced to a rather high level during
sporulation (Fig. 8). In contrast to
rsfA, the expression of ylbO-lacZ was completely
abolished by a mutation in the spoIIGA gene required for
activation of E (Fig. 8). In view of this dependence and
the relatively early timing of expression during sporulation,
ylbO is almost certainly a mother cell-specific gene under
the control of E (9), but its function
remains unknown.
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FIG. 8.
Expression of ylbO-lacZ during sporulation.
Isogenic strains containing a ylbO-lacZ fusion were induced
to sporulate at time zero, and samples were taken at intervals for
assay of -galactosidase activity. The strains used were otherwise
wild type ( [strain BFA3241]) or carried mutations in the
spoIIAC ( [strain 1255]) or spoIIGA ( [strain 1256]) gene.
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| |
DISCUSSION |
We have discovered the first accessory regulator for
F-dependent transcription, RsfA. Its gene was initially
identified as being required for maximal expression of the
prespore-specific gpr-lacZ fusion in a spoIIIE36
mutant. We then found that rsfA disruption affects the
expression of most genes in the F regulon, suggesting
that it is a global regulator of transcription. Comparison of previous
published results on expression of genes in the F
regulon reveals a range of subtle differences in their levels of
expression and kinetics, and this is well illustrated by the time
courses shown in Fig. 1 and 5. Some of the differences in kinetics are
likely to be due to differential dependence of the promoters on
EF and EG. However, RsfA can now also be
implicated in these effects. Inspection of the data shown in Fig. 1 and
5 reveals that the genes that are expressed independently of RsfA
(spoIIQ) or are negatively regulated by it
(spoIIR and rsfA itself) show relatively early and steep expression kinetics (at least as judged by the
lacZ fusion data in Fig. 5D to F). This would be consistent
with their promoters being recognizable by EF
independent of accessory factors. In contrast, those genes that are
positively regulated by RsfA (gpr, katX,
spoIIIG, and sspE-2G) all show later kinetics,
and their rate of rise to maximal expression is slower. This could
reflect the need for the RsfA product to accumulate in the cell before
these genes become fully expressed, as we suggested previously for the
action of SpoIIID on some genes in the E regulon
(12). Thus, although RsfA is not essential for sporulation, it appears to improve the efficiency of sporulation by fine-tuning the
expression of genes in the F regulon, particularly the
timing of their expression. It is not yet clear whether RsfA also
modulates the expression of G-dependent genes. However,
since rsfA can also be transcribed by EG, as
for many other F-dependent genes (e.g., gpr
and spoIIIG), it would not be surprising if RsfA also played
a role in the regulation of some G-dependent genes. The
deleterious effects of expression of rsfA in vegetative
cells were surprising. It seems possible that one role of this protein
could be to shut down expression of some genes that are needed for
vegetative growth, but not in the developing prespore. However, it is
also possible that this effect is an artifact due to overproduction of RsfA.
The RsfA protein has a leucine zipper motif (Fig. 2), which is present
in many eukaryotic gene regulatory proteins, such as the Jun/AP1 family
of transcription factors (24) and the yeast general control
protein GCN4 (7). It has been proposed that the leucine side
chains extending from one -helix interact with those from a similar
-helix of a second polypeptide, forming a coiled-coil which
facilitates formation of dimers or multimers. RsfA also contains a
stretch of basic amino acids (Fig. 2), which could serve to bind to
phosphate groups on DNA. As one way of testing for an association with
DNA, we examined the localization of an RsfA-GFP fusion. As shown in
Fig. 6, the protein was closely associated with DNA, both in
sporulating cells and when ectopically expressed during vegetative
growth. Alignment of the promoter regions of genes affected by
rsfA did not reveal common sequence motifs that might
represent a recognition site for binding of the protein. In some
respects, its modest, though global, effects on gene expression are
reminiscent of proteins such as H-NS of E. coli, which bind
fairly nonspecifically to the nucleoid and regulate the expression of
many genes. Biochemical studies are now needed to establish whether
RsfA binds directly to DNA to bring about its regulatory effects,
whether the DNA binding is sequence specific, and how the positive and
negative effects are achieved.
RsfA had a clear paralogue, in the form of the ylbO gene,
identified in the complete genome sequence (19). Although a
ylbO disruption mutant had no obvious phenotypic effect, the
gene turned out to be expressed specifically during sporulation and at
quite a high level. The limited experiments we have done (time course and dependence on spoIIGA) suggest that it is expressed
within the E regulon. It will be interesting to
determine whether ylbO has a role in the regulation of gene
expression in the mother cell, paralleling that of RsfA in the prespore.
We thank M. Arnaud, R. Daniel, D. Foulger, N. Fransden, A. Moir,
J. P. Rawlins, P. Setlow, and P. Stragier for strains and plasmids.
This work was supported by grants from the Biotechnology and Biological
Sciences Research Council.
| 1.
|
Anagnostopoulos, C., and J. Spizizen.
1961.
Requirements for transformation in Bacillus subtilis.
J. Bacteriol.
81:741-746[Free Full Text].
|
| 2.
|
Bagyan, I.,
L. Casillas-Martinez, and P. Setlow.
1998.
The katX gene, which codes for the catalase in spores of Bacillus subtilis, is a forespore-specific gene controlled by F, and KatX is essential for hydrogen peroxide resistance of the germinating spore.
J. Bacteriol.
180:2057-2062[Abstract/Free Full Text].
|
| 3.
|
Bagyan, I.,
J. Hobot, and S. Cutting.
1996.
A compartmentalized regulator of developmental gene expression in Bacillus subtilis.
J. Bacteriol.
178:4500-4507[Abstract/Free Full Text].
|
| 4.
|
Cormack, B. P.,
R. H. Valdivia, and S. Falkow.
1996.
FACS-optimized mutants of the green fluorescent protein (GFP).
Gene
173:33-38[CrossRef][Medline].
|
| 5.
|
Daniel, R. A.,
A. M. Williams, and J. Errington.
1996.
A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis.
J. Bacteriol.
178:2343-2350[Abstract/Free Full Text].
|
| 6.
|
Decatur, A., and R. Losick.
1996.
Identification of additional genes under the control of the transcription factor F of Bacillus subtilis.
J. Bacteriol.
178:5039-5041[Abstract/Free Full Text].
|
| 7.
|
Ellenberger, T. E.,
C. J. Brandl,
K. Struhl, and S. C. Harrison.
1992.
The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex.
Cell
71:1223-1237[CrossRef][Medline].
|
| 8.
|
Errington, J.
1984.
Efficient Bacillus subtilis cloning system using bacteriophage vector 105J9.
J. Gen. Microbiol.
130:2615-2628[Medline].
|
| 9.
|
Errington, J.
1993.
Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis.
Microbiol. Rev.
57:1-33[Abstract/Free Full Text].
|
| 10.
|
Errington, J., and J. Mandelstam.
1986.
Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis.
J. Gen. Microbiol.
132:2967-2976[Medline].
|
| 11.
|
Haldenwang, W. G.
1995.
The sigma factors of Bacillus subtilis.
Microbiol. Rev.
59:1-30[Abstract/Free Full Text].
|
| 12.
|
Illing, N., and J. Errington.
1991.
The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother-cell-specific sporulation genes under the control of the E form of RNA polymerase.
Mol. Microbiol.
5:1927-1940[Medline].
|
| 13.
|
Itaya, M.
1992.
Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome.
Biosci. Biotechnol. Biochem.
56:685-686[Medline].
|
| 14.
|
Itaya, M.,
K. Kondo, and T. Tanaka.
1989.
A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome.
Nucleic Acids Res.
17:4410[Free Full Text].
|
| 15.
|
Jenkinson, H. F.
1983.
Altered arrangement of proteins in the spore coat of a germination mutant of Bacillus subtilis.
J. Gen. Microbiol.
129:1945-1958[Medline].
|
| 16.
|
Karow, M. L.,
P. Glaser, and P. J. Piggot.
1995.
Identification of a gene, spoIIR, that links the activation of E to the transcriptional activity of F during sporulation in Bacillus subtilis.
Proc. Natl. Acad. Sci. USA
92:2012-2016[Abstract/Free Full Text].
|
| 17.
|
Karow, M. L., and P. Piggot.
1995.
Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation.
Gene
163:69-74[CrossRef][Medline].
|
| 18.
|
Kroos, L.,
B. Zhang,
H. Ichikawa, and Y.-T. N. Yu.
1999.
Control of factor activity during Bacillus subtilis sporulation.
Mol. Microbiol.
31:1285-1294[CrossRef][Medline].
|
| 19.
|
Kunst, F., et al.
1997.
The complete genome sequence of the Gram positive bacterium Bacillus subtilis.
Nature
390:249-256[CrossRef][Medline].
|
| 20.
|
Lewis, P. J., and A. L. Marston.
1999.
GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis.
Gene
227:101-109[CrossRef][Medline].
|
| 21.
|
Lewis, P. J.,
S. R. Partridge, and J. Errington.
1994.
factors, asymmetry, and the determination of cell fate in Bacillus subtilis.
Proc. Natl. Acad. Sci. USA
91:3849-3853[Abstract/Free Full Text].
|
| 22.
|
Londoño-Vallejo, J.-A.,
C. Frehel, and P. Stragier.
1997.
spoIIQ, a forespore-expressed gene required for engulfment in Bacillus subtilis.
Mol. Microbiol.
24:29-39[CrossRef][Medline].
|
| 23.
|
Londoño-Vallejo, J.-A., and P. Stragier.
1995.
Cell-cell signalling pathway activating a developmental transcription factor in Bacillus subtilis.
Genes Dev.
9:503-508[Abstract/Free Full Text].
|
| 24.
|
Neuberg, M.,
J. Adamkiewicz,
J. B. Hunter, and R. Muller.
1989.
A Fos protein containing the Jun leucine zipper forms a homodimer which binds to the AP1 binding site.
Nature
341:243-245[CrossRef][Medline].
|
| 25.
|
Partridge, S. R., and J. Errington.
1993.
The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation in Bacillus subtilis.
Mol. Microbiol.
8:945-955[Medline].
|
| 26.
|
Partridge, S. R.,
D. Foulger, and J. Errington.
1991.
The role of F in prespore-specific transcription in Bacillus subtilis.
Mol. Microbiol.
5:757-767[CrossRef][Medline].
|
| 27.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 28.
|
Schuch, R., and P. J. Piggot.
1994.
The dacF-spoIIA operon of Bacillus subtilis, encoding F, is autoregulated.
J. Bacteriol.
176:4104-4110[Abstract/Free Full Text].
|
| 29.
|
Sterlini, J. M., and J. Mandelstam.
1969.
Commitment to sporulation in Bacillus subtilis and its relationship to the development of actinomycin resistance.
Biochem. J.
113:29-37[Medline].
|
| 30.
|
Stragier, P., and R. Losick.
1996.
Molecular genetics of sporulation in Bacillus subtilis.
Annu. Rev. Genet.
30:297-341[CrossRef][Medline].
|
| 31.
|
Sun, D.,
P. Fajardo-Cavazos,
M. D. Sussman,
F. Tovar-Rojo,
R.-M. Cabrera-Martinez, and P. Setlow.
1991.
Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by EF: identification of features of good EF-dependent promoters.
J. Bacteriol.
173:7867-7874[Abstract/Free Full Text].
|
| 32.
|
Sun, D.,
P. Stragier, and P. Setlow.
1989.
Identification of a new -factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.
Genes Dev.
3:141-149[Abstract/Free Full Text].
|
| 33.
|
Sussman, M. D., and P. Setlow.
1991.
Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination.
J. Bacteriol.
173:291-300[Abstract/Free Full Text].
|
| 34.
|
Vagner, V.,
E. Dervyn, and S. D. Ehrlich.
1998.
A vector for systematic gene inactivation in Bacillus subtilis.
Microbiology
144:3097-3104[Abstract].
|
| 35.
|
Wu, L. J., and J. Errington.
1994.
Bacillus subtilis SpoIIIE protein required for DNA segregation during asymmetric cell division.
Science
264:572-575[Abstract/Free Full Text].
|
| 36.
|
Wu, L. J.,
A. Feucht, and J. Errington.
1998.
Prespore-specific gene expression in Bacillus subtilis is driven by sequestration of SpoIIE phosphatase to the prespore side of the asymmetric septum.
Genes Dev.
12:1371-1380[Abstract/Free Full Text].
|
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Abstract
Introduction
