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Journal of Bacteriology, August 1999, p. 4986-4994, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of the yrbA Gene of
Bacillus subtilis, Involved in Resistance and Germination
of Spores
Hiromu
Takamatsu,1
Takeko
Kodama,1
Tatsuo
Nakayama,2 and
Kazuhito
Watabe1,*
Faculty of Pharmaceutical Sciences, Setsunan
University, Osaka,1 and Department of
Biochemistry, Miyazaki Medical College,
Miyazaki,2 Japan
Received 28 December 1998/Accepted 1 June 1999
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ABSTRACT |
Insertional inactivation of the yrbA gene of
Bacillus subtilis reduced the resistance of the mutant
spores to lysozyme. The yrbA mutant spores lost their
optical density at the same rate as the wild-type spores upon
incubation with L-alanine but became only phase gray and
did not swell. The response of the mutant spores to a combination of
asparagine, glucose, fructose, and KCl was also extremely poor; in this
medium yrbA spores exhibited only a small loss in optical
density and gave a mixture of phase-bright, -gray, and -dark spores.
Northern blot analysis of yrbA transcripts in various
sig mutants indicated that yrbA was transcribed
by RNA polymerase with 
E beginning at 2 h after the
start of sporulation. The yrbA promoter was localized by
primer extension analysis, and the sequences of the 
35 (TCATAAC)
and 10 (CATATGT) regions were similar to the
consensus sequences of genes recognized by E. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins
solubilized from intact yrbA mutant spores showed an
alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some
other minor changes. Electron microscopic examination of
yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the
appearance of an inner coat layer-like structure around the outside of
the coat. This abnormal coat structure was also observed on the outside
of the developing forespores of the yrbA mutant. These
results suggest that YrbA is involved in assembly of some coat proteins
which have roles in both spore lysozyme resistance and germination.
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INTRODUCTION |
Many gram-positive soil
microorganisms, such as Bacillus subtilis, develop dormant
spores when nutrients are exhausted. Spore formation is the result of a
complex process of macromolecular assembly that is controlled at
different stages of sporulation. For example, RNA polymerase sigma
factors are activated sequentially in the mother cell or forespore
compartment and regulate the expression of sporulation-related genes
(10, 38). The bacterial spores are metabolically dormant and
have a unique thick protein shell known as the spore coat
(3). The coat is composed of dozens of proteins
(38) arranged in an electron-dense thick outer layer and a
thinner, lamella inner layer (6). These layers provide a
protective barrier against bactericidal enzymes and chemicals, such as
lysozyme and organic solvents (9). SpoIVA is synthesized from 2 h after the start of sporulation (T2) in the
mother cell compartment and assembles around the outer membrane of the
forespore in B. subtilis (39). This protein is
thought to be required for the formation of a basement layer on which
spore coat proteins assemble (8, 29, 39). One of the coat
protein components, CotE, is also a morphogenic protein required for
the assembly of the outer coat (47). cotE mutant
spores are refractile and resistant to heat and chemicals but are
lysozyme sensitive and germinate slower and less efficiently than
wild-type spores (47). The CotT protein of B. subtilis is synthesized as a 10.1-kDa precursor, which is
processed to a coat polypeptide of 7.8 kDa, and insertional inactivation of the cotT gene resulted in spores with an
altered appearance of the inner coat layers and slow germination in
response to a solution containing fructose, glucose, and asparagine
(4). Thus, the coat components may play an important role in
responding to germinants and also in preventing access of lysozyme to
the peptidoglycan of the spore cortex.
We identified a DNA fragment containing three deduced open reading
frames, orf1, orf2, and orf3
(42) (DDBJ accession no. D50551) near the nadA
gene in the 243° region (nadC, nadA, yrbA, yrbB, and yrbC, in that order);
orf1, orf2, and orf3 in our work
correspond to yrbA, yrbB, and yrbC,
respectively, as determined by the B. subtilis genome
sequencing project (19). In a previous paper
(42), we demonstrated by immunoelectron microscopy that the
YrbB protein was located in spores, primarily in the cortex layer. In
this work, we have analyzed the function and expression of
yrbA and found that yrbA expression was dependent on E-containing RNA polymerase. The yrbA
mutant spores had abnormal coat layers, had lost their response to a
germination solution containing asparagine, glucose, fructose, and KCl
(AGFK) and resistance to lysozyme, and were deficient in some coat proteins.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, media, and general techniques.
The B. subtilis strains used in this study are listed in
Table 1 and were all grown in DS medium
at 37°C (34). Escherichia coli was grown in
Luria-Bertani medium. The conditions for sporulation of B. subtilis and the method for purification of mature spores have
been described previously (2, 41). Recombinant DNA methods were as described by Sambrook et al. (33). Methods for
preparing competent cells for transformation and for preparing
chromosomal DNA from B. subtilis were as described by
Cutting and Vander Horn (5).
Preparation and purification of spores.
B. subtilis
spores prepared in DS medium were harvested after incubation for
48 h and washed several times with deionized water. The spores
obtained were then purified by a urografin gradient procedure as
described by Nicholson and Setlow (27).
Construction of yrbA and yrbB
mutants.
Oligonucleotide primers YRBA158
(5'-CGTCTAGAAAAGAGCCAAAAGCGG-3') and YRBA562R
(5'-TTAGATCTTCTACACCGCCTACCT-3') were used to amplify a DNA
fragment from nucleotide (nt) +158 to +562 of yrbA, and
primers CRB1 (5'-AATCTAGAGAACTGACACGCTTAA-3') and CRB2
(5'-TAGATCTGTGGTGTTTCGGTTACC-3') were used to amplify a DNA
fragment from nt 93 to +234 of yrbB. The PCR products were
cleaved at the XbaI and BglII sites and inserted
between the XbaI and BglII sites of pDH88
(12) to construct plasmids pYRBA5 and pCOXA5, respectively.
The resulting plasmids were introduced into strain 168 by a single
crossover with selection for resistance to chloramphenicol (5 µg/ml)
to give strains TB713 (yrbA mutant) and TB711
(yrbB mutant).
RNA preparation and Northern analysis.
B. subtilis
cells were grown in DS medium, and 20-ml samples were harvested every
hour throughout sporulation. RNA for Northern blots was then prepared
by a modification of the procedure described by Igo and Losick
(15). Aliquots (10 µg) of the RNA preparation were
analyzed by size fractionation through a 1% (wt/vol) agarose gel
containing 2.2 M formaldehyde and were transferred to a positively charged Hybond-N+ membrane (Amersham). The membrane was
stained with 0.04% methylene blue in 0.5 M sodium acetate (pH 5.2) to
measure the concentrations of 16S and 23S RNAs in the preparations as
described previously (13). The RNA on the membrane was
hybridized to probe 1 and probe 2 DNAs, which are specific for
yrbA and yrbB, respectively. The 0.5-kb probe 1, corresponding to nt 22 to 526 downstream of the putative translation
initiation codon of yrbA, was prepared by PCR with primers
COXRA10 (5'-AAAGGCGATTCGCTCTGG-3') and YRBA500T (5' - TAATACGACTCAC TATAGGGCGAGGGCATAT TAGGCATAT TCGG -3').
The 0.4-kb probe 2, corresponding to nt 170 to +216
relative to the putative translation initiation codon of
yrbB, was prepared by PCR with primers CRB1
(5'-AATCTAGAGAACTGACACGCTTAA-3') and COX200T (5'-TAATACGACTCACTATAGGGCGAGTTCCGTCAGTTGCCAAAGG-3').
The underlined regions in the primers represent the T7 promoter
sequence. The RNA probes were prepared by using the Boehringer Mannheim
digoxigenin labeling system, and hybridization was performed by the
procedure recommended by Boehringer Mannheim.
Mapping of the 5' terminus of yrbA mRNA.
Cells
were grown in DS medium, and 20-ml samples were harvested at
T5 of sporulation. RNA for primer extension
analyses were prepared by a modification of the procedure described by
Igo and Losick (15) and Cutting et al. (7).
Primer extension was performed with a cDNA synthesis kit (Pharmacia
Biotech) and a 5'-end digoxigenin-labeled primer, COXPM1R
(5'-TTCCCCTCCTATGCAAAACG-3'), which was complementary to
nt+5 to nt+24 downstream of the translational start point of
yrbA. The reaction was carried out as recommended by
Boehringer Mannheim, except that the reaction mixture was incubated at
42°C. Oligonucleotide primers COXRAM400
(5'-TCGACACAATCAACCAGGCT-3') and COXRA320R
(5'-ACATCAGCTTCAGGGTACAC-3') were used to amplify a DNA
fragment from nt 394 to +374 of yrbA. The 5'-end-labeled primer was also used to generate a sequence ladder by the dideoxy chain
termination method with the DNA fragment as a template. The products of
primer extension were then subjected to electrophoresis in a 5%
(wt/vol) polyacrylamide slab gel containing 8 M urea, and products were
detected as recommended by Boehringer Mannheim.
Spore germination.
Purified spores were heat activated at
65°C for 15 min and suspended in 50 mM Tris-HCl (pH 7.5) buffer at an
optical density at 660 nm of 0.5. Either 10 mM L-alanine,
AGFK (3.3 mM L-asparagine, 5.6 mM D-glucose,
5.6 mM D-fructose, and 10 mM KCl), or 10 mM Tris-HCl (pH
7.5) was then added. Germination was monitored by measurement of the
decrease in absorbance (660 nm) of the spore suspension at 37°C for
up to 90 min as described previously (2).
Spore resistance.
Cells were grown in DS medium at 37°C
for 18 h after the end of exponential growth (to
T18), and spore resistance was assayed as
follows. The cultures were either heated at 80°C for 30 min, treated
with lysozyme (250 µg/ml [final concentration]) at 37°C for 10 min, or treated with 10% (vol/vol) chloroform at room temperature for
10 min as described previously (27). After the cultures were
serially diluted 100-fold in distilled water, appropriate volumes of
the dilutions were spread on Luria-Bertani agar plates, which were
incubated overnight at 37°C. The proportion of survivors was
determined by counting the colonies.
Solubilization of proteins from spores.
Cultures (5 ml) were
harvested at T18 of sporulation and washed with
10 mM sodium phosphate buffer (pH 7.2) containing 0.5 M sodium
chloride. The pellets were suspended in 0.1 ml of lysozyme solution (10 mM sodium acetate [pH 7.2], 1% lysozyme) and incubated for 15 min at
37°C. After addition of 1.0 ml of 10 mM sodium phosphate buffer (pH
7.2) containing 0.5 M sodium chloride, the suspensions were centrifuged
to remove soluble proteins from mother cells and spores. The spores in
the pellet fraction were suspended in 100 µl of buffer containing 2%
(wt/vol) sodium dodecyl sulfate (SDS), 5% (vol/vol) 2-mercaptoethanol,
10% (vol/vol) glycerol, 62.5 mM Tris-HCl (pH 6.8), and 0.05% (wt/vol)
bromophenol blue and boiled for 5 min.
SDS-PAGE and immunoblotting.
Protein samples were analyzed
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (12% acrylamide)
as described previously (1). For immunoblotting, proteins
were transferred onto a polyvinylidene difluoride membrane (0.45-µm
pore size) (Immobilon; Millipore) and detected by using rabbit
immunoglobulin G against YrbB (42) as the first antibody and
donkey anti-rabbit immunoglobulin G-horseradish peroxidase conjugate
as the second antibody (Amersham).
NH2-terminal sequence analysis.
Samples were
subjected to SDS-PAGE, electroblotted onto a polyvinylidene difluoride
membrane as described above, and briefly stained with Coomassie
brilliant blue. After extensive washing, the protein bands of interest
were excised and applied to a Procise 492 gas-phase sequencer (Applied
Biosystems Division, Perkin-Elmer), and sequences of
NH2-terminal amino acids were determined as described previously (22).
Electron microscopy.
Purified spores and sporulating cells
were fixed with 2.5% glutaraldehyde and then 2% OsO4 as
described by Ryter et al. (31) and embedded in Quetol 653 by
the method of Kushida (20). Thin sections of spores and
sporulating cells were observed with a JEM-1200EX electron microscope
operating at 80 kV.
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RESULTS |
Expression of yrbA during growth and sporulation.
The yrbA-yrbC region contains at least three complete open
reading frames, designated yrbA, yrbB, and
yrbC. We first determined the sizes and times of appearance
of yrbA and yrbB mRNAs during growth and
sporulation by Northern blot analysis of samples containing essentially
the same amounts of 16S and 23S RNAs (Fig.
1). Both 1.2- and 2.0-kb transcripts
containing yrbA mRNA were detected beginning at
T2 of sporulation by using probe 1 (Fig. 1A). By using probe 2, which is specific for yrbB mRNA, a 0.7-kb
mRNA was found to be transcribed from yrbB beginning at
T3 of sporulation (Fig. 1B, lane 4), while the
2.0-kb mRNA found with the yrbA probe also hybridized to the
yrbB probe. The origins of these mRNAs were confirmed by
additional Northern blot analysis of RNAs from yrbA or
yrbB mutants. As shown in Fig. 1B, only the 0.7-kb
yrbB mRNA was present in the yrbA mutant (TB713
cells), while this mRNA was not found in the yrbB mutant
(TB711 cells); the 2.0-kb mRNA was not detected in either mutant. These
results suggest that at least two putative promoters are used for
expression of these genes and that the 2.0-kb mRNA is most probably a
read-through product from yrbA to yrbB.
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FIG. 1.
Northern blot analysis of yrbA mRNA. The gene
products of B. subtilis 168 trpC2 (wild-type)
were analyzed by using probes specific for yrbA (probe 1)
(A) and yrbB (probe 2) (B). The loci, probes used in these
experiments, and prospective regions of promoters for yrbA
and yrbB are shown in panel C. Total RNA (10 µg) was
prepared from cells of strains TB713 (yrbA) and TB711
(yrbB) at T5 of sporulation and
analyzed by hybridization with probe 2 (lanes 7 and 8 in panel B).
Arrowheads indicate the positions of mRNAs hybridized with the probes.
The number of hours after the end of the exponential phase of growth is
shown at the top.
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We then examined dependency of
yrbA expression on various
sigma factors. After the onset of sporulation of
B. subtilis,
E is the first of the sigma factors to
appear in the mother cell,
with
F as its counterpart in
the forespore;
F is essential for the activation of
pro-
E (
17,
21,
40). Northern blot analysis
showed that RNA from
either
sigE or
sigF mutant
cells failed to hybridize with the
yrbA-specific probe,
whereas RNA from
sigG or
sigK mutant cells
gave
the same two hybridizing bands as did wild-type cells when
analyzed at
T4 of sporulation (Fig.
2A). The results in Fig.
2B
allow a
similar conclusion; probe 2 hybridized to a 2.0-kb mRNA
species in the
RNAs from
sigG and
sigK mutants, while the 0.7-kb
mRNA was detectable only in the sample prepared from
sigK
cells
(Fig.
2B). SpoIIID was also not essential for the transcription
of
yrbA (data not shown). These results strongly suggest
that
expression of
yrbA starts at
T2
and is dependent on E-
E RNA polymerase in the mother
cell compartment and that the 0.7-kb
yrbB mRNA was
transcribed by RNA polymerase containing the forespore-specific
G.
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FIG. 2.
Northern blot analysis of yrbA and
yrbB transcripts in various sigma factor-deficient cells.
RNAs (10 µg) of cells of spoIIG41 (sigE),
spoIIAC1 (sigF), spoIIIG 1
(sigG), and spoIIIC94 (sigK) mutant
strains were subjected to Northern blot analysis with probes specific
for yrbA (A) and yrbB (B). The times of harvest
of the cells are shown at the top. Arrowheads indicate the positions of
RNAs hybridized with the probes.
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Location of the yrbA promoter.
To further analyze
the dependence of yrbA expression on sigma factors, the
start point of yrbA transcription was mapped by primer
extension (Fig. 3). No extension product
was detected when RNA of yrbA mutant cells at
T5 of sporulation was analyzed; in contrast, a
single extension product was seen with RNA isolated from the wild-type
cells at T5 of sporulation. The size of this transcript indicated that transcription of yrbA starts at a
G residue 56 nt upstream from the TTG translation initiation codon (Fig. 4A), and regions centered 10 and 35 bp upstream of the apparent yrbA transcription start site
are very similar to 10 and 35 regions of promoters utilized by
E-containing RNA polymerase (Fig. 4B).
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FIG. 3.
Identification of the yrbA promoter. RNAs (20 µg) extracted from wild-type (168) (lane 5) or yrbA (lane
6) cells at T5 of sporulation were used for
primer extension. The sizes of the extended products were compared with
a DNA sequencing ladder of the adjacent sequence of the 5' region of
yrbA. The yrbA transcription start site is shown
by an arrowhead and an asterisk on the sequence.
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FIG. 4.
Genomic structure of the region upstream of
yrbA. (A) Nucleotide sequence of the yrbA
promoter, showing putative 35 and 10 regions and the transcription
start site (+1). The nucleotide sequence complementary to the synthetic
oligonucleotide used in primer extension is underlined. The asterisk
indicates a translation stop codon. (B) Sequences near the
transcription start sites of genes transcribed by RNA polymerase
containing E. The underlined nucleotides denote the transcription
start site. The consensus sequence proposed by Roels et al.
(29) is shown at the top (K = G or T; m = C or A;
r = G or A). References for the sequences of these promoters are
as follows: spoIID, 30;
spoIIID, 18 and 43;
spoIVF 7; spoIIIA,
8; spoIVA P1 and spoIVA P2,
29; and cotE P1, 48.
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Properties of mutant spores.
The resistance of yrbA
spores was also examined to learn whether YrbA played any role in spore
properties. The yrbA mutation had no effect on vegetative
growth (data not shown) or on spore resistance to heat and chloroform
(Table 2). However, the yrbA mutation reduced spore resistance to lysozyme. The sensitivity of
yrbA spores to lysozyme was confirmed by observation by
phase-contrast microscopy, as lysozyme treatment generated lysed
yrbA spores (data not shown).
The
yrbA mutant spores lost most of their optical density in
germination with
L-alanine (Fig.
5), and dipicolinic acid was
released
from the
yrbA spores to almost the same extent as from
wild-type spores after incubation with
L-alanine for 90 min
(data
not shown). However, microscopic observation revealed that the
germinated wild-type spores were phase dark and swollen, while
the
mutant spores were only phase gray and not swollen (Fig.
6C
and D). Dormant-spore properties
disappear sequentially during
germination, with dipicolinic acid
released in the first minutes
of germination, followed by loss of spore
refractility (
35,
37,
44). Consequently, our results suggest
that in
L-alanine,
yrbA mutant spores have a
defect at a late stage of spore germination.
The most notable property
of the
yrbA spores was their germination
response to AGFK.
With AGFK the
yrbA spores showed only an extremely
small
change in optical density (Fig.
5), and after 90 min the
population had
some phase-bright spores, some phase-gray spores,
and some phase-dark
spores (Fig.
6F). Since
yrbA spores lost no
significant heat
resistance or dipicolinic acid content after
incubation with AGFK for
90 min (data not shown), this suggests
that spores with the
yrbA defect have an additional early defect
in germination
with AGFK.
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FIG. 5.
Germination of wild-type and yrbA mutant
spores. Wild-type (A) and TB713 (yrbA) (B) spores were heat
activated at 65°C for 15 min and either germinated in 10 mM
L-alanine ( ) or AGFK ( ) or incubated with 10 mM
Tris-HCl ( ) at 37°C.
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FIG. 6.
Phase-contrast microscopy of wild-type and
yrbA spores. Wild-type spores (A, C, and E) and TB713
(yrbA) spores (B, D, and F) were incubated with 10 mM
Tris-HCl (pH 7.5) (A and B), 10 mM L-alanine (C and D), or
AGFK (E and F) at 37°C for 90 min.
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Since the reduction in lysozyme resistance of the
yrbA
spores implied that
yrbA is involved in spore coat
morphogenesis, we
analyzed coat proteins by SDS-PAGE. The coat protein
profile of
the
yrbA (TB713) mutant spores on SDS-PAGE was
significantly different
from that of the wild-type spores (Fig.
7A), as proteins of 24
(P24), 25 (P25),
26 (P26), 31 (P31), and 36 (P36) kDa were absent
from
yrbA
spores. Analysis of the NH
2-terminal sequence of protein
P36 gave HYSHYDIEEAV, corresponding to the sequence from His-3
to
Val-13 of CotG (
32), while the NH
2-terminal
sequence of P31
was MENANYPNM, corresponding to the sequence from
Met-164 to Met-172
of YrbA. YrbA is deduced to be a 43-kDa protein from
its nucleotide
sequence; therefore, we assume that P31 was generated by
proteolysis
of YrbA. We could not determine a unique N-terminal
sequence for
P24, P25, or P26 because these bands contained several
different
polypeptides.
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FIG. 7.
SDS-PAGE analysis of proteins solubilized from spores.
Spores were prepared from T18 sporulating cells.
The protein samples were solubilized from the spores by boiling with
SDS and 2-mercaptoethanol and analyzed by SDS-PAGE (12% gel). (A)
Coomassie brilliant blue stain; (B) immunoblotting with anti-YrbB
antibody. The protein samples were from wild-type spores (lanes 1),
TB711 (yrbB) spores (lanes 2), and TB713 (yrbA)
spores (lanes 3). The arrowhead in panel B indicates the migration
position of YrbB.
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Since
yrbA is upstream of
yrbB, it was possible
that the
yrbA mutation might affect YrbB synthesis. However,
YrbB is present
in
yrbA mutant spores (Fig.
7B), and no
visible difference between
the coat protein samples prepared from
wild-type and
yrbB spores
was seen upon SDS-PAGE (Fig.
7A,
lanes 1 and
2).
Morphology of yrbA mutant spores.
Given the spore
coat defects in yrbA spores, we analyzed the ultrastructure
of these spores by electron microscopy (Fig.
8). The coat of the wild-type spores has
two major layers, a highly electron-dense and thicker outer coat layer
and a fine lamellar inner coat layer (Fig. 8A). Some changes in coat
morphology were observed in the yrbA mutant spores (Fig. 8B,
C, and D); the outer layer was less electron dense and less thick (Fig.
8B and C), and a separate layer-like structure loosely surrounded the
outside of the coat (Fig. 8D). The abnormal coat structure was also
observed outside the developing forespores of the mutant at
T8 of sporulation, and the spore coat layer(s)
was partially detached from the surface of the developing forespores
(Fig. 8E and F). An inner coat layer-like structure was found around
the outside of an electron-dense layer of the developing forespores
(Fig. 8E and F).
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FIG. 8.
Ultrastructure of yrbA spores. Wild-type (A)
and yrbA (B, C, and D) spores were collected at
T18 of sporulation and analyzed by electron
microscopy as described in Materials and Methods. Sporulating cells of
the yrbA mutant were also collected at
T8 of sporulation and analyzed similarly (E and
F). Arrowheads indicate abnormal spore coat. Bars, 0.2 µm.
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DISCUSSION |
The control of yrbA by E is supported by
two types of experiments. First, Northern blot analysis showed that
transcription of yrbA mRNA was dependent on E
and F but not on G or K
(Fig. 2). Since activation of E is regulated by
F during sporulation, expression of yrbA is
under E or F control. Second, examination
of the yrbA promoter revealed 35 (TCATAAC) and
10 (CATATGT) sequences separated by 14 bp; these sequences
conform well to the 10 and 35 consensus sequences of many
E-dependent promoters (7, 11, 18, 26, 28-30, 43,
48) (Fig. 3 and 4). These results indicated that transcription of yrbA is most likely regulated by RNA polymerase with
E in the mother cell compartment and also suggest that
YrbA may be a coat protein made in the mother cell. However, it is
possible that RNA polymerase with K also recognizes the
yrbA promoter to a small degree, because the yrbA
promoter is similar to those of the K-dependent genes
(Fig. 4C).
In order to assess YrbA function, we prepared an insertional
yrbA mutant and purified the mutant spores by our standard
method with lysozyme digestion to remove vegetative cell debris.
However, during the lysozyme treatment, the yrbA spores lost
their refractility, becoming phase gray after the treatment (data not
shown). Lysozyme's target site in spores is the cortex, and digestion
of this structure results in loss of spore refractility. However,
dormant spores generally resist lysozyme digestion because of
impermeability of their complex coat structure, which is exterior to
the cortex. Consequently, the lysozyme sensitivity of the
yrbA spores suggested that YrbA has some effect on
expression or assembly of some coat components. This was confirmed by
SDS-PAGE analysis of yrbA spores (Fig. 7), as
yrbA spores lacked not only YrbA but also CotG, even though
Northern blot analysis showed that cotG was transcribed normally from T4 of sporulation in
yrbA cells (data not shown). These data suggest that YrbA is
involved in spore coat assembly but not in regulation of cot
gene transcription. CotE is another protein required for morphogenesis
of the coat layer of B. subtilis, and a mutation in the
cotE gene results in the loss of some proteins from spore
coats, with a resultant decrease of spore resistance to lysozyme
(47). Electron microscopic observation also revealed that
the coat layers of yrbA spores were less electron dense and less thick than coat layers in wild-type spores, and a coat layer(s) was partially detached from the surface of yrbA mutant
forespores and extended into the mother cell compartment (Fig. 8). This
morphological change is somewhat similar to that of a spoIVA
mutant, in which abnormally assembled coat layers develop in the mother
cell compartment (8, 29, 39). These results further imply
that YrbA is a morphogenetic protein that is required for the assembly
of protein components into the spore coats and the development of
lysozyme resistance.
The yrbA mutant spores were also defective in some late step
of L-alanine-induced germination and had a defect early in
germination with AGFK (Fig. 5). The process of spore germination in
B. subtilis requires the action of a germinant on a trigger
site within the spores. B. subtilis spores germinate with
L-alanine alone or with AGFK, none of whose components is
germinative on its own (45, 46). Spores of mutants with
mutations in the gerA operon are defective specifically in
the response to L-alanine but germinate normally in AGFK
(24). In contrast gerB, gerK, and
fruB mutant spores germinate normally in
L-alanine but are defective in germination in AGFK
(16, 24). This difference suggests that the spore has two
different systems for detecting these two germination signals
(25). Both cotE and gerE spores also
lack some spore coat proteins and have defects in spore germination, as
do yrbA mutant spores (23, 47). CotE is required
for the assembly of outer coat proteins, and GerE is a DNA binding
protein essential for expression of some cot genes (14,
47, 49), but these proteins are thought to be neither germinant
receptors nor factors which directly control the process of spore
germination. We assume that YrbA is also involved in assembly of some
spore coat components required for L-alanine- or
AGFK-stimulated germination.
| |
ACKNOWLEDGMENTS |
We are grateful to Anne Moir for critical review and discussions,
and we thank Michael G. Bramucci for critical reading of the
manuscript. We thank JEOL Datum Co. (Tokyo, Japan) for technical support for electron microscopy.
This work was supported by grant JPSP-RFTF96L00105 from the Japan
Society for the Promotion of Science.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan. Phone and fax: (81) 720-66-3112 or -3114. E-mail:
watabe{at}pharm.setsunan.ac.jp.
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Journal of Bacteriology, August 1999, p. 4986-4994, Vol. 181, No. 16
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Abstract
Introduction
