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J Bacteriol, March 1998, p. 1375-1380, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Regulation and Characterization of a Newly Deduced
Cell Wall Hydrolase Gene (cwlJ) Which Affects
Germination of Bacillus subtilis Spores
Shu
Ishikawa,1
Kunio
Yamane,2 and
Junichi
Sekiguchi1,*
Department of Applied Biology, Faculty of
Textile Science and Technology, Shinshu University, Ueda-shi, Nagano
386,1 and
Institute of Biological
Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki
305,2 Japan
Received 14 November 1997/Accepted 3 January 1998
 |
ABSTRACT |
The predicted amino acid sequence of Bacillus subtilis
ycbQ (renamed cwlJ) exhibits high similarity to those
of the deduced C-terminal catalytic domain of SleBs, the specific
cortex-hydrolyzing enzyme of B. cereus and the deduced one
of B. subtilis. We constructed a
cwlJ::lacZ fusion in the B. subtilis
chromosome. The 
-galactosidase activity and results of Northern
hybridization and primer extension analyses of the cwlJ
gene indicated that it is transcribed by E
E RNA
polymerase. cwlJ-deficient spores responded to both
L-alanine and AGFK, the A580 values
of spore suspensions decreased more slowly than in the case of the
wild-type strain, and the mutant spores released less dipicolinic acid
than did those of the wild-type strain during germination. However, the
mutant spores released only slightly less hexosamine than did the
wild-type spores. In contrast, B. subtilis sleB spores did
not release hexosamine at a significant level. While cwlJ
and sleB spores were able to germinate, CJSB (cwlJ
sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in
A580 and slow release of dipicolinic acid. CJSB
spores became slightly gray after 6 h in the germinant, but their
refractility was much greater than that of sleB mutant
spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.
| |
INTRODUCTION |
During sporulation and germination
of Bacillus subtilis, the action of autolysins is assumed to
be required for asymmetric septum peptidoglycan hydrolysis, engulfment,
cortex maturation, mother cell lysis, and cortex hydrolysis during
germination (28, 33). Mother cell lysis depends on the
compensatory effect of cell wall hydrolases CwlB (LytC) and CwlC
(11, 13, 34). For cortex maturation, a defect in the
cwlD gene leads to a lack of germination and blocking of the
formation of muramic acid lactam structure in the cortex (2, 26,
31). Recently, Makino and colleagues reported that the B. cereus sleB gene encodes a 24-kDa mature germination-specific
N-acetylmuramoyl-L-alanine amidase which
degrades decoated spores from various organisms (18, 22). B. subtilis sleB is homologous to B. cereus sleB,
and B. subtilis sleB mutant spores are able to germinate and
form colonies. However, B. subtilis SleB showed no activity
against degraded decoated spores or other substrates (21).
Our work on the B. subtilis genome sequencing project has
revealed the ycbQ gene, which is homologous with the
cortex-hydrolyzing sleB genes (22, 25). In this
study, we describe the regulation and function of the cwlJ
(ycbQ) gene and the compensatory effect of the CwlJ and
B. subtilis SleB proteins on germination.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The strains of B. subtilis and Escherichia coli and the plasmids used in
this study are described in Table 1.
B. subtilis was grown on nutrient agar medium (Difco) at
30°C for about 10 h, then inoculated into DSM (Schaeffer medium)
(30), and shaken at 37°C. If necessary, erythromycin and
spectinomycin were added to the medium to final concentrations of 0.3 and 50 µg/ml, respectively. E. coli was grown in LB medium
(29) at 37°C. If necessary, ampicillin was added to a
final concentration of 50 or 100 µg/ml.
Plasmid construction.
To construct a B. subtilis
cwlJ mutant, an internal fragment of the cwlJ gene was
amplified by PCR using two primers, forward primer cbQHF
(5'-GCCGAAGCTTG10TGAGAGCAACGAGTGC26;
the internal sequence of the cwlJ region is
italicized, numbering is with respect to the first A of the
translational start codon of cwlJ, and the HindIII site is underlined) and reverse primer cbQBR
(5'-GCGCGGATCCT212ATCCATGAGTCACAGCC195; the sequence complementary to the internal region of
cwlJ is italicized, and the BamHI site is
underlined), with B. subtilis 168 DNA as a template. The PCR
fragment was digested with HindIII and BamHI. To remove an extra cloning site region, pGEM-3zf(+) was digested with
EcoRI and SmaI, blunt ended with mung bean
nuclease, and then self-ligated. The resulting plasmid, pGEM
ES, and
pMUTin2 were digested with HindIII and BamHI
and then ligated to the digested PCR fragment, followed by
transformation of E. coli JM109. The resulting plasmid,
pGEMcJ, was used to synthesize an RNA probe, and pMUTin2cJ was
used for the transformation of E. coli C600 to produce
concatemeric DNAs (3).
To construct a B. subtilis sleB mutant, the entire
sleB region was amplified by PCR using two primers, forward
primer soPF (5'-GCCGCTGCAGC
165
GTTCCGTTAATATGATGC147; the upstream sequence of the sleB gene is italicized, numbering is with
respect to the first A of the translational start codon of
sleB, and the PstI site is underlined) and
reverse primer soER
(5'-GCGCGAATTCC2383GCATTCAATATACTCACG2365; the sequence complementary to the internal region of
sleB is italicized, and the EcoRI site is
underlined), with B. subtilis 168 DNA as a template.
The PCR fragment was digested with PstI and
EcoRI, followed by ligation into the corresponding sites of
pUC118. The resultant plasmid, pUCSO, was digested with
HincII and SmaI and then ligated into the
HincII-StuI fragment of the spectinomycin resistance (Spr) cassette plasmid pDG1727 (7).
The resultant plasmid, pUCSOSP, was used to construct a B. subtilis sleB mutant.
Mutant construction.
A cwlJ-deficient mutant,
cbQ, was constructed by transformation of B. subtilis 168 with pMUTin2cJ. Disruption of the cwlJ gene by means of
Campbell-type recombination was confirmed by PCR. Thus, the cbQ mutant
was a cwlJ-lacZ transcriptional fusion strain.
sleB and sleB cwlJ mutants 168SB and CJSB,
respectively, were constructed by transformation of B. subtilis with ScaI-digested pUCSOSP DNA. The
double-crossover recombination event was confirmed by PCR using primers
soPF and soER.
Transformation of E. coli and B. subtilis.
E. coli transformation was performed as described by
Sambrook et al. (29), and B. subtilis
transformation was performed by the competent cell method
(1).
Spore germination.
B. subtilis 168, cbQ, 168SB, and
CJSB were cultured in DSM for 2 days at 37°C. Spores were suspended
in deionized water and then washed by centrifugation as described by
Nicholson and Setlow (23) until all cell debris and
vegetative cells had been removed. The spores were heat activated at
80°C for 20 min, unless otherwise noted, and then diluted with a 10 mM Tris-HCl buffer (pH 8.4). Germination was initiated by the addition
of L-alanine to 10 mM or AGFK (L-asparagine,
D-glucose, D-fructose, KCl) to 10 mM each ingredient. At appropriate times, the A580 of
the mixture was measured and an 11-ml sample was taken and centrifuged
in a microcentrifuge. The supernatant (1 ml) was used for the
measurement of released dipicolinic acid as described by Nicholson and
Setlow (23). The rest of the supernatant was dried with a
concentrator (model CC-180; TOMY), followed by measurement of the
released reducing groups by a modification (35) of the
method of Park and Johnson, with N-acetylglucosamine as a
standard. Dipicolinic acid in sporulating cells was determined by the
method of Jannsen et al. as described by Nicholson and Setlow
(23).
-Galactosidase assay.
The -galactosidase assay was
performed basically as described by Shimotsu and Henner
(32). One unit of -galactosidase activity was defined as
the amount of enzyme necessary to release 1 nmol of 2-nitrophenol from
2-nitrophenyl--D-galactopyranoside in 1 min.
Northern blot and primer extension analyses.
Cells (15 units
of optical density at 600 nm) cultured in DSM were harvested and then
suspended in 1 ml of chilled killing buffer (36). After
centrifugation at 12,000 × g for 1 min at 4°C, the
pellet was suspended in 1 ml of SET buffer containing lysozyme (final
concentration, 6 mg/ml). After incubation for 10 min at 0°C, the
suspension was centrifuged at 12,000 × g for 1 min at
4°C. The pellet was used for RNA preparation with Isogen (Nippon
Gene) according to the manufacturer's instructions.
Agarose-formaldehyde gel electrophoresis was performed as described by
Sambrook et al. (29). The transfer of RNAs onto a nylon
membrane (Magnagraph, Micron Separations) was performed with a vacuum
blotter (model BE-600; BIOCRAFT). The DNA fragment used for preparing
an RNA probe was amplified by PCR with M13(
21) and M13RV (Takara) as primers and with pGEMcJ DNA, containing the internal region of cwlJ, as a template. The amplified fragment was digested
with HindIII, and then fragments were purified by phenol
and chloroform treatment, followed by precipitation with ethanol. The
RNA probe was prepared with a DIG RNA labeling kit (Boehringer
Mannheim), and Northern (RNA) hybridization was performed according to
the manufacturer's instructions. Primer extension analysis was
performed as described previously (31), using primer PEXcbQ
(5'-G44CCATCAAATCGACATCC27; 5' and
3' ends correspond to the complementary nucleotides at positions 44 and
27 with respect to the 5' end of the cwlJ gene).
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RESULTS |
The B. subtilis genome project has revealed the
existence of many cell wall hydrolase homologs, one of which is the
product of the ycbQ gene located at 24° on the B. subtilis chromosome map. The amino acid sequence of YcbQ (renamed
CwlJ) exhibits 28 and 30% identity with those of the deduced catalytic
domains of B. cereus SleB and B. subtilis SleB
(Fig. 1). The SleB proteins contain
signal sequences and direct repeated sequences in their N-terminal
regions, but CwlJ lacks both signal and repeated sequences.
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FIG. 1.
Alignment of the deduced amino acid sequences of
B. subtilis CwlJ (Bs-CwlJ; YcbQ) (25), B. subtilis (Bs-SleB; deduced B. subtilis
cortex-hydrolyzing amidase) (22), and B. cereus
SleB (Bc-SleB; B. cereus cortex-hydrolyzing amidase)
(21). Amino acid identities are indicated by shading, and
amino acids identical in the three proteins are indicated by asterisks.
Amino acids are numbered from the N termini of the proteins, and dashes
indicate the introduction of gaps in the alignment. The arrowhead and
arrows indicate a signal sequence cleavage site and repeated sequences,
respectively. The nucleotide sequence G256GC258
(numbering with respect to the first A of the translational start codon
of cwlJ) under GSDB, EMBL, DDBJ, and NCBI accession no.
D30808 should be corrected to CGG; thus, the corresponding amino acid,
G86 (numbered with respect to the N-terminal amino acid)
should be corrected to R.
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Expression of the cwlJ gene.
The
cwlJ-lacZ fusion strain, cbQ, was cultured in DSM, and
-galactosidase activity was determined. The activity was first detected after t2 (2 h after the onset of
sporulation) and was maximal at around t7 (Fig.
2A), which indicates that the
cwlJ expression is regulated by a sporulation-specific sigma
factor. RNAs from four sigma factor-deficient strains, 1S86
(SigF), 1S60 (SigE), SpoIIIG1
(SigG), and 1S38 (SigK), were analyzed by
Northern blotting using an RNA probe containing the internal region of
the cwlJ gene. Hybridizing bands for the 168, SigG, and SigK RNAs were detected at 0.47 kb, but no band was observed for the SigF and
SigE RNAs (Fig. 2B). The cwlJ gene consists of
426 nucleotide residues followed by a deduced rho-independent
terminator (G = 21.6 kcal/mol) (Fig.
3) (25). Therefore, these
results indicate that the cwlJ gene is transcribed by
EE RNA polymerase as a monocistronic mRNA. To determine
the promoter region, primer extension analysis was performed with 168, SigF, SigE, SigG, and
SigK RNAs along with primer PEXcbQ. Extended products
were found at t4.5 and/or
t7.5 for 168, SigG, and
SigK RNAs, but no products were observed at
t4.5 for the SigF and
SigE RNAs (Fig. 3A). The products started at A, G, and A
(residues 23, 22, and 21 bp upstream, respectively, of the
translational start codon) (Fig. 3). The upstream sequences
TCATcAc and aATAtgAT (capital letters denote
consensus sequence), with their spacing of 14 bp, were very similar to
the 35 and 10 consensus sequences of E-dependent
promoters ([T/G][A/C]ATA[A/T][A/T] and CATACA(A/C)T, respectively, with a spacing of 14 bp) (8).
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FIG. 2.
Time course of the production of the
cwlJ-lacZ fusion protein (A) and Northern blot analysis of
the cwlJ region (B). (A) cwlJ-directed
-galactosidase activity of strain cbQ was determined at the
indicated times after the onset of sporulation. Squares, cell growth at
an optical density of 600 nm (OD600); diamonds,
-galactosidase activity. (B) Northern hybridization performed with
the cwlJ-specific RNA probe as described in Materials and
Methods. The lanes contained 5 µg of RNA from B. subtilis
168 at t2 (2), t0
(0), t2 (2), or t4 (4),
and B. subtilis 1S86 (SigF), 1S60
(SigE), SpoIIIG1 (SigG), and 1S38
(SigK) at t1.5 (1.5),
t3 (3), t4.5 (4.5), or
t6 (6). 0.47 indicates the size (in kilobases)
of the hybridizing RNA in comparison with the migration of 23S and 16S
RNAs.
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FIG. 3.
Determination of the transcriptional start sites by
primer extension analysis (A) and nucleotide sequence of the upstream
region of cwlJ (B). (A) RNA (5 µg) from B. subtilis 168 at t1.5 (lane 1),
t4.5 (lane 2), or t7.5
(lane 3), 1S86 (SigF) at t4.5
(lane 4), 1S60 (SigE) at t4.5
(lane 5), SpoIIIG1 (SigG) at
t4.5 (lane 6), or 1S38 (SigK) at
t4.5 (lane 7) or t7.5
(lane 8) was hybridized with a labeled cbQPEX1 primer, which is
complementary to nucleotides 27 to 44 in the sequence in panel B. The
primer-extended products obtained with reverse transcriptase were
subjected to electrophoresis in 12% (wt/vol) polyacrylamide sequencing
gels, followed by autoradiography with an imaging plate (BAS-MP; Fuji).
The dideoxy-DNA sequencing reaction mixtures with the cbQPEX1 primer
were electrophoresed in parallel (lanes G, A, T, and C). The positions
of the products are indicated by arrowheads. The boxed area indicates
the 10 region of the E promoter. In panel B, the
promoter regions (35 and 10) of E and the
transcriptional start position are indicated by underlines and
arrowheads, respectively. The deduced rho-independent terminator is
indicated by opposing arrows.
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Construction and characterization of cwlJ,
sleB, and cwlJ sleB disruptants.
cbQ
(cwlJ), 168SB (sleB), and CJSB (cwlJ
sleB) disruptants were constructed as described in Materials and
Methods. The cbQ strain showed normal cell growth, cell separation, and
motility and produced highly refractile spores. A previous report
indicated that B. subtilis sleB-deficient spores did not
complete darkening and released less dipicolinic acid during
germination but still produced viable colonies after germination
(21). Since CwlJ exhibits high amino acid sequence
similarity with the C-terminal region of B. subtilis SleB
(Fig. 1), germination was compared among 168, cbQ, 168SB, and CJSB
spores. During germination in L-alanine-containing buffer,
the 168SB spores showed a smaller decrease in
A580 than the wild-type 168 spores. The decrease
in absorbance of cbQ spores exhibited a different pattern: there were
large differences between cbQ spores and 168 and 168SB spores 30 min
after the onset of germination. Levels of release of dipicolinic acid
by these three strains during spore germination were also different, as
168SB and cbQ spores released dipicolinic acid in slightly lower and
much lower amounts, respectively, than did 168 spores. CJSB spores
exhibited only a 25% reduction in A580 at
6 h after germination and a slower decrease in absorbance at 30 min after the onset of germination. CJSB spores released dipicolinic acid more slowly than the cbQ spores (Fig.
4). The cbQ spores released only slightly
less hexosamine than those of the wild type, but the 168SB and CJSB
spores did not release significant amounts of hexosamine. These results
suggest that SleB is a major germination-specific cortex hydrolase and
CwlJ is a minor one. The absorbance profiles of the spores in AGFK as a
germinant were very similar to those in L-alanine as a
germinant (9). Therefore, these effects were common to these
germination pathways.
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FIG. 4.
Spore germination of B. subtilis 168, cbQ
(cwlJ), 168SB (sleB), and CJSB (cwlJ
sleB). The germination of spores of the B. subtilis
strains was monitored at A580 at the indicated
times after the addition of L-alanine and is expressed as
relative absorbance. The released dipicolinic acid and reducing groups
in the supernatants of the spore suspensions were also measured.
Squares, diamonds, circles, and triangles indicate B. subtilis 168, cbQ, 168SB, and CJSB, respectively.
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Germination frequencies and microscopic observation of the mutant
spores.
Table 2 shows the
germination frequencies of the mutant spores with or without heat
activation at 80°C for 20 min. These results indicate that the cbQ
and 168SB spores are similar with respect to heat resistance and
viability to spores of 168 under the conditions used. Spores of strain
CJSB did not produce colonies on LB agar even without heat activation.
Thus, this defect was not caused by heat sensitivity of the CJSB
spores. Phase-contrast microscopy of the wild-type and mutant spores is
shown in Fig. 5. Dormant spores of the
mutants and the wild type were bright, and it was difficult to observe
a difference in refractility. The refractility of cbQ spores changed
from bright to dark after 6 h under the germination conditions,
whereas 168SB spores became dark gray and CJSB spores became weakly
bright under the same conditions. These phenomena show good agreement
with the germination frequencies (Table 2). Therefore, these results
indicate that both CwlJ and SleB are necessary for normal germination
behavior of B. subtilis spores.
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FIG. 5.
Phase-contrast microscopy of B. subtilis 168, cbQ, 168SB, and CJSB spores treated with L-alanine. Spores
were germinated at 37°C for 6 h as described for Fig. 4, with
the addition of germination buffer (10 mM L-alanine, 10 mM
Tris-HCl [pH 8.4]), and then observed by phase-contrast microscopy.
Bar, 5 µm.
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| |
DISCUSSION |
B. cereus SleB was extracted from dormant spores, and
the protein, which had been processed by signal peptidase, was active against the decoated spores of various bacilli (18, 22).
However, the gene product of B. subtilis sleB, which had
been cloned by colony hybridization with the B. cereus sleB
fragment as a probe, was not detected in the germination exudate of the
B. subtilis spores (21). B. subtilis
CwlJ did not contain a signal sequence or direct repeated sequences in
its N-terminal region (Fig. 1). Since the cwlJ gene was
transcribed by EE RNA polymerase (Fig. 3), expression of
cwlJ is mother cell specific, and a signal sequence may not
be necessary for CwlJ to localize in the spore cortex. Direct repeated
sequences have been found in the noncatalytic domains of many cell wall
hydrolases (e.g., CwlA, CwlB [LytC], CwlC, LytD [CwlG], and XlyA
from B. subtilis [11-13, 15, 17, 19, 27]
and CwlL, CwlM, and CwlX from B. licheniformis and a cell
wall hydrolase from Bacillus species [14, 16,
24]). In the case of CwlM, the noncatalytic domain containing
direct repeats was related to the substrate specificity (14). In the case of the major autolysin, CwlB, the
truncated protein comprising only the noncatalytic domain, bound
tightly to cell walls (12). Therefore, CwlJ, lacking the
noncatalytic domain, may have a wide substrate specificity and/or bind
weakly to the B. subtilis cortex.
In Clostridium perfringens, two cortex-hydrolyzing enzymes
(spore cortex lytic amidase SleC and cortical fragment-lytic muramidase SleM) play roles in cortex hydrolysis during spore germination (4,
20). Foster and Johnstone proposed a germination mechanism for
B. megaterium (6), and Foster recently proposed a
germination mechanism in B. subtilis in which three types of
enzymes with different substrate specificities (amidase, endopeptidase,
and transglycosylase) were required for cortex hydrolysis during
germination in that order (5). Since the
sleB-deficient mutant 168SB did not show significant
hexosamine release during germination, SleB plays a major role in
cortex hydrolysis during germination. Therefore, SleB probably
corresponds to the amidase proposed by Foster. However, the 168SB
spores did form colonies on LB agar plates. Therefore, there should be
another lytic enzyme (probably an amidase) that plays a role in cortex
hydrolysis at the early stage of germination. CwlJ is such a candidate,
because spores of the cwlJ-deficient mutant cbQ exhibited a
slow decrease in the A580 by 30 min after the
onset of germination, and its dipicolinic acid release rate was lower
than that of 168SB; cbQ also released slightly less hexosamine than did
the wild-type 168 (Fig. 4). Moreover, the combination of these two
proteins was essential for normal spore germination, because the
cwlJ sleB double-mutant (CJSB) spores did not form colonies
on LB agar plates (Table 2) and showed a change in refractility from
only bright to faint gray (Fig. 5).
Obviously CwlJ plays a different role from SleB in spore germination.
In addition to the foregoing results, the initial response of
germination was severely affected by the cwlJ sleB double
mutation, there being a smaller decrease in the
A580 value. The absorbance curve seems to be the
combined pattern of the single mutations (Fig. 4). Therefore, CwlJ may
be a minor cortex-lytic enzyme during germination. But another
possibility, that CwlJ is a cortex maturation enzyme, cannot be
eliminated at this stage. Previously, spores of the
cwlD-deficient mutant ADD1, which lacks muramic acid
lactam-forming activity, were found not to germinate (2, 26,
31). However, the cbQ spores germinated at the normal level
(Table 2). Therefore, the latter possibility presupposes the presence
of a new cortex hydrolase that acts at the early stage of germination
and is unable to digest the cortex of cbQ spores.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Applied Biology, Faculty of Textile Science and Technology, Shinshu
University, 3-15-1 Tokida, Ueda-shi, Nagano 386, Japan. Phone:
81-268-21-5344. Fax: 81-268-21-5331. E-mail:
jsekigu{at}giptc.shinshu-u.ac.jp.
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REFERENCES |
| 1.
|
Anagnostopoulos, C., and J. Spizizen.
1961.
Requirements for transformation in Bacillus subtilis.
J. Bacteriol.
81:741-746[Free Full Text].
|
| 2.
|
Atrih, A.,
P. Zöllner,
G. Allmaier, and S. J. Foster.
1996.
Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.
J. Bacteriol.
178:6173-6183[Abstract/Free Full Text].
|
| 3.
|
Canosi, U.,
G. Morelli, and T. A. Trautner.
1978.
The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis.
Mol. Gen. Genet.
166:259-267[Medline].
|
| 4.
|
Chen, Y.,
S. Miyata,
S. Makino, and R. Moriyama.
1997.
Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene.
J. Bacteriol.
179:3181-3187[Abstract/Free Full Text].
|
| 5.
|
Foster, S. J.
1997.
The role of cortex hydrolysis during spore germination, p. 42.
Abstracts of the 9th International Conference on Bacilli.
Universite de Lausanne, Lausanne, Switzerland.
|
| 6.
|
Foster, S. J., and K. Johnstone.
1990.
Pulling the trigger: the mechanism of bacterial spore germination.
Mol. Microbiol.
4:137-141[Medline].
|
| 7.
|
Guerout-Fleury, A. M.,
K. Shazand,
N. Frandsen, and P. Stragier.
1995.
Antibiotic-resistance cassettes for Bacillus subtilis.
Gene
167:335-336[Medline].
|
| 8.
|
Haldenwang, W. G.
1995.
The sigma factors of Bacillus subtilis.
Microbiol. Rev.
59:1-30[Abstract/Free Full Text].
|
| 9.
| Ishikawa, S., and J. Sekiguchi. Unpublished data.
|
| 10.
|
Karmazyn-Campelli, C.,
C. Bonamy,
B. Savelli, and P. Stragier.
1989.
Tandem genes encoding -factors for consecutive steps of development in Bacillus subtilis.
Genes Dev.
3:150-157[Abstract/Free Full Text].
|
| 11.
|
Kuroda, A.,
Y. Asami, and J. Sekiguchi.
1993.
Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis.
J. Bacteriol.
175:6260-6268[Abstract/Free Full Text].
|
| 12.
|
Kuroda, A., and J. Sekiguchi.
1990.
Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene.
J. Gen. Microbiol.
136:2209-2216[Abstract/Free Full Text].
|
| 13.
|
Kuroda, A., and J. Sekiguchi.
1991.
Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene.
J. Bacteriol.
173:7304-7312[Abstract/Free Full Text].
|
| 14.
|
Kuroda, A.,
Y. Sugimoto,
T. Funahashi, and J. Sekiguchi.
1992.
Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.
Mol. Gen. Genet.
234:129-137[Medline].
|
| 15.
|
Lazarevic, V.,
P. Margot,
B. Soldo, and D. Karamata.
1992.
Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier.
J. Gen. Microbiol.
138:1949-1961[Abstract/Free Full Text].
|
| 16.
|
Lee, J. K.,
C. W. Edwards, and F. M. Hulett.
1991.
Identification of four unique clones encoding 10 kDa proteins from Bacillus that cause phenotypic complementation of a phoA mutant strain of Escherichia coli.
J. Gen. Microbiol.
137:667-677[Abstract/Free Full Text].
|
| 17.
|
Longchamp, P. F.,
C. Mauël, and D. Karamata.
1994.
Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX.
Microbiology
140:1855-1867[Abstract/Free Full Text].
|
| 18.
|
Makino, S.,
N. Ito,
T. Inoue,
S. Miyata, and R. Moriyama.
1994.
A spore-lytic enzyme released from Bacillus cereus spores during germination.
Microbiology
140:1403-1410[Abstract/Free Full Text].
|
| 19.
|
Margot, P.,
C. Mauël, and D. Karamata.
1994.
The gene of the N-acetylglucosaminidase, a Bacillus subtilis cell wall hydrolase not involved in vegetative cell autolysis.
Mol. Microbiol.
12:535-545[Medline].
|
| 20.
|
Miyata, S.,
R. Moriyama,
N. Miyahara, and S. Makino.
1995.
A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization.
Microbiology
141:2643-2650[Abstract/Free Full Text].
|
| 21.
|
Moriyama, R.,
A. Hattori,
S. Miyata,
S. Kudoh, and S. Makino.
1996.
A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.
J. Bacteriol.
178:6059-6063[Abstract/Free Full Text].
|
| 22.
|
Moriyama, R.,
S. Kudoh,
S. Miyata,
S. Nonobe,
A. Hattori, and S. Makino.
1996.
A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme.
J. Bacteriol.
178:5330-5332[Abstract/Free Full Text].
|
| 23.
|
Nicholson, W. L., and P. Setlow.
1990.
Sporulation, germination and outgrowth, p. 391-450. In
C. R. Harwood, and S. M. Cutting (ed.), Molecular biological methods for Bacillus.
John Wiley & Sons, Ltd., Chichester, United Kingdom.
|
| 24.
|
Oda, Y.,
R. Nakayama,
A. Kuroda, and J. Sekiguchi.
1993.
Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis.
Mol. Gen. Genet.
241:380-388[Medline].
|
| 25.
|
Ogawa, K.,
E. Akagawa,
K. Nakamura, and K. Yamane.
1995.
Determination of a 21548 bp nucleotide sequence around the 24° region of the Bacillus subtilis chromosome.
Microbiology
141:269-275[Abstract/Free Full Text].
|
| 26.
|
Popham, D. L.,
J. Helin,
C. E. Costello, and P. Setlow.
1996.
Muramic lactam in peptidoglycan of B. subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance.
Proc. Natl. Acad. Sci. USA
93:15405-15410[Abstract/Free Full Text].
|
| 27.
|
Rashid, M. H.,
M. Mori, and J. Sekiguchi.
1995.
Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization.
Microbiology
141:2391-2404[Abstract/Free Full Text].
|
| 28.
|
Rogers, H. J.,
H. R. Perkins, and J. B. Ward.
1980.
.
Microbial cell walls and membranes.
Chapman and Hall, London, United Kingdom.
|
| 29.
|
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.
|
| 30.
|
Schaeffer, P.,
J. Millet, and J. P. Aubert.
1965.
Catabolite repression of bacterial sporulation.
Proc. Natl. Acad. Sci. USA
54:704-711[Free Full Text].
|
| 31.
|
Sekiguchi, J.,
K. Akeo,
H. Yamamoto,
F. K. Khasanov,
J. C. Alonso, and A. Kuroda.
1995.
Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis.
J. Bacteriol.
177:5582-5589[Abstract/Free Full Text].
|
| 32.
|
Shimotsu, H., and D. J. Henner.
1986.
Modulation in Bacillus subtilis levansucrase gene expression by sucrose, and regulation of the steady-state mRNA level by sacU and sacQ genes.
J. Bacteriol.
168:380-388[Abstract/Free Full Text].
|
| 33.
|
Shockman, G. D., and J.-V. Höltje.
1994.
Microbial peptidoglycan (murein) hydrolases, p. 131-166. In
J.-M. Ghuysen, and R. Hakenbeck (ed.), Bacterial cell wall.
Elsevier, Amsterdam, The Netherlands.
|
| 34.
|
Smith, T. J., and S. J. Foster.
1995.
Characterization of the involvement of two compensatory autolysins in mother cell lysis during sporulation of Bacillus subtilis 168.
J. Bacteriol.
177:3855-3862[Abstract/Free Full Text].
|
| 35.
|
Thompson, J. S., and G. D. Shockman.
1968.
A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls.
Anal. Biochem.
22:260-268[Medline].
|
| 36.
|
Völker, U.,
S. Engelmann,
B. Maul,
S. Riethdorf,
A. Völker,
R. Schmid,
H. Mach, and M. Hecker.
1994.
Analysis of the induction of general stress proteins of Bacillus subtilis.
Microbiology
140:741-752[Abstract/Free Full Text].
|
J Bacteriol, March 1998, p. 1375-1380, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
