Hydrolase Gene of Bacillus subtilis

JOURNAL OF BACTERIOLOGY, Oct. 1993, p. 6260-6268
0021-9193/93/196260-09$02.00/0
Copyright X 1993, American Society for Microbiology
Vol. 175, No. 19
Molecular Cloning of a Sporulation-Specific Cell Wall
Hydrolase Gene of Bacillus subtilis
AKIO KURODA, YASUO ASAMI, AND JUNICHI SEKIGUCHI*
Department ofApplied Biology, Faculty of Textile Science and Technology,
Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386, Japan
Received 24 May 1993/Accepted 27 July 1993
Southern hybridization analysis of Bacillus subtilis 168S chromosomal DNA with a Bacilus licheniformis cell
wall hydrolase gene, cwlM, as a probe indicated the presence of a cwlMf homolog in B. subtils. DNA sequencing
of the cwLM homologous region showed that a gene encoding a polypeptide of 255 amino acids with a molecular
mass of 27,146 Da is located 625 bp upstream and in the opposite direction of spoVJ. The deduced amino acid
sequence of this gene (tentatively designated as cwlC) showed an overall identity of 73% with that of cwlM and
of 40% with the C-terminal half of the B. subtiUis vegetative autolysin, CwIB. The construction of an in-frame
cwlC-lacZ fusion gene in the B. subtils chromosome indicated that cwlC is induced at 6 to 7 h after sporulation
(t6 to t7). The spoIHIC (d~") mutation and earlier sporulation mutations greatly reduced the expression of the
cwlC-lacZ fusion gene. Northern hybridization analysis using oligonucleotide probes of the cwlC region
indicated that a unique cwlC transcript appeared at t7*. and t9. Transcriptional start points determined by
primer extension analysis suggested that the -10 region is very similar to the consensus sequence for the
oK-dependent promoter. Insertional inactivation of the cwlC gene in the B. subtUis chromosome caused the
disappearance of a 31-kDa protein lytic for Micrococcus cell walls, which is mainly located within the
cytoplasmic and membrane fractions of cells at t9. The CwIC protein hydrolyzed both B. subtilis vegetative cell
walls and spore peptidoglycan.
From Bacillus subtilis, two vegetative autolysins, a 50kDa N-acetylmuramoyl-L-alanine amidase (amidase) and a
90-kDa endo-3-N-acetylglucosaminidase (glucosaminidase),
have been purified and characterized (16, 38). The former
occurs in large amounts (16, 24), and the gene, cwlB, has
recently been cloned and studied at the molecular level (24,
29). The cwlB operon consists of three genes encoding a
putative lipoprotein (LppX), a modifier protein (CwbA) that
stimulates amidase activities, and CwlB, in that order (22,
24, 26, 29). The transcription of the cwlB operon mainly
depends on expression of the eD protein, which is responsible for cell motility and chemotaxis (26, 32). Other cell wall
hydrolases of B. subtilis have been described previously by
us (23) and Foster (11, 12). A 30-kDa amidase gene, cwl4,
has been cloned, but its expression is very low under normal
growth conditions (11, 23). Foster demonstrated novel cell
wall lytic activities of A3 (34-kDa) and A4 (30-kDa), which
increases during sporulation, and also sporulation-specific
activities of A5 (23-kDa) and A6 (41-kDa) by means of
renaturing gel electrophoresis in substrate-containing gels
(12). The physiological roles of these multiple autolysins,
i.e., cell wall turnover (2, 5), cell separation (5, 10, 37),
flagellation (10), and competence (3), have been suggested
by many investigators, but there was little evidence because
they used regulatory mutants [flaD (sin, lyt) and sacU (Hy)]
with reduced autolysin levels (26). Asymmetric septum
peptidoglycan hydrolysis (17, 30), which is a morphogenic
transition between sporulation stages II and III, cortex
maturation (12, 15), mother-cell lysis (12), and germination
(12) would be subject to the actions of these autolysins.
However, the specific functions of individual autolysins of
B. subtilis remained unknown except that vegetative cell
wall turnover is apparently decreased in a cwlB(lytC)*
deficient mutant (31). Therefore, it is important to determine
the roles of individual autolysin genes during growth and
differentiation, including their transcriptional regulation.
Previously, we cloned the cwlM gene encoding a 28-kDa
Bacillus lichenifornis cell wall lytic amidase (27). In this
paper, we report the cloning of a sporulation-specific cell
wall hydrolase gene, cwlC, of B. subtilis, taking advantage of
its homology with cwlM. We also constructed and characterized an in vitro-derived deletion mutant of cwlC.
MATERIALS AND METHODS
Bacterial strains, phages and plasmids. The strains of B.
subtilis used in this study are shown in Table 1. Escherichia
coli JM109 [recAI A(lac-proAB) endAI gyrA96 thi-1 hsdR17
supE44 relAI (F':traD36 proAB lacIcZAM15)] (Takara
Shuzo Co., Kyoto, Japan) and plasmid pUC119 (Takara
Shuzo Co.) were used for cloning, and E. coli JM103 [endA
A(lac-proAB) thi strA supE hsdR4 sbcB15 (F':traD36proAB
laclVZAM15)] (33) and phages M13mpl8 and M13mpl9
(Takara Shuzo Co.) were used for sequencing. pUD1 (42)
contains a chloramphenicol acetyltransferase gene (cat). B.
subtilis and E. coli were grown in modified Luria-Bertani
medium (5 g of yeast extract, 10 g of polypeptone, 10 g of
NaCl per liter [pH 7.2]) at 37°C. When necessary, ampicillin,
tetracycline, and chloramphenicol were added to final concentrations of 50, 20, and 10 ,ug/ml, respectively. For B.
subtilis sporulation, Schaeffer medium was used (41).
Cloning of a cwlM homolog (cwlC) of B. subtilis. PstIdigested fragments of B. subtilis 168S chromosomal DNA
were separated by agarose gel electrophoresis, and 5- to 8-kb
fragments were recovered from the agarose gels with Geneclean Kit II (Bio 101). Ligation of the DNA fragments into
the dephosphorylated PstI site of pUC119 was followed by
transformation. Ampicillin-resistant (Apr) transformants
were subjected to colony hybridization analysis (40) with the
Corresponding author.
6260
VOL. 175, 1993
B. SUBTILIS SPORULATION-SPECIFIC CELL WALL HYDROLASE
6261
TABLE 1. B. subtilis strains used in this study
Strain
Genotype
168S
AC327
AC334
1S38
1S60
1S86
327CL1
327CL2
387CL2
607CL2
867CL2
334CL2
ANC1
trpC2 strA smo-1
purB his-1 smo-1
purB flaDl (sin)
trpC2 spoIIIC94
leuB8 tal-1 spoIIG41
trpC2 spoILA1
purB his-1 smo-1 spoVJ::pUDCL1
purB his-1 smo-i cwlC::pUDCL2
his-I smo-1 spoIIIC94 cwlC::pUDCL2
purB smo-1 spoIIG41 cwlC::pUDCL2
purB smo-I spoILU1 cwlC::pUDCL2
purB smo-I flaDi(sin) cwlC::pUDCL2
purB his-I smo-I AcwlC::cat
Source or reference
42
42
26
BGSCa
BGSC
BGSC
This study
This study
1S38 +327CL2b
lS60->327CL2
lS86--327CL2
AC334--327CL2
This study
a BGSC, Bacillus Genetic Stock Center, The Ohio State University.
b Arrows indicate construction by transformation.
32P-labeled 0.6-kb EcoRI-HindIII fragment of pLA41D1S1R
(27) containing a truncated cwlM gene as a probe. From
among 200 colonies, a positive clone harboring pCC65P
(plasmid pUC119 containing a 6.5-kb PstI fragment) was
isolated. A 0.7-kb HindlIl fragment from pCC65P was
subcloned into the HindIII site of pUC119, the resultant
plasmid being designated pCC07H (Fig. 1A). A 2.4-kb BglIIEcoRV fragment from the B. subtilis chromosome, designated pCC24BE (Fig. 1), was cloned into the BamHI-HincII
site of pUC119 with the 32P-labeled 0.7-kb HindIII fragment
of pCC07H as a probe.
DNA sequencing. Nucleotide sequencing was performed
by the dideoxy chain termination method with a modified T7
polymerase (Sequenase; Toyobo). Electrophoresis was performed on 8% (wt/vol) polyacrylamide-8 M urea gels. The
sequences of both strands were determined for the 1.1-kb
HindIII-EcoRV fragment of pCC24BE and partially for the
flanking region (spoVJ), as indicated in Fig. 1A.
B. subtUis transformation. Conventional transformation of
B. subtilis was performed according to the procedure of
Anagnostopoulos and Spizizen (1).
Introduction of an in-frame cwlC-acZ fusion gene into the
B. subtilis chromosome by Campbell-like recombination. The
1.6-kb NcoI-EcoRI fragment containing the 5' region of cwlC
and almost the entire region of spoVJ was isolated from
pCC24BE, blunt-ended with mung bean nuclease, and then
inserted into the unique SmaI site of pMC1871 (Pharmacia)
containing the ,-galactosidase gene (lacZ) (Fig. 1B). The
orientation of the insert in one of the resultant plasmids,
pMCCZ, containing an in-frame cwlC-lacZ fusion gene was
determined from the distance between the asymmetric HindIII site of the insert and the SalI site of the vector and also
confirmed by sequencing of its connection site (Fig. 1B). An
in-frame spoVJ-lacZ fusion gene was generated through the
inverse insertion of this fragment, the plasmid being designated pMCJZ. A 4.6-kb SalI fragment containing the cwlClacZ fusion gene of pMCCZ was inserted into the Sall site of
pUD1 (42), and the resultant plasmid was designated
pUDCL2 (Fig. 1B). pUDCL2 was then integrated into the B.
subtilis AC327 chromosome by means of conventional transformation. The chloramphenicol resistant (Cm') transformant was designated B. subtilis 327CL2. A plasmid,
pUDCL1, was constructed by inserting the spoVJ-lacZ
translational fusion gene (a 4.6-kb Sall fragment from pMCJZ)
into the Sail site of pUD1 and was also integrated into the B.
subtilis chromosome. The resultant transformant was desig-
nated B. subtlis 327CL1. Campbell-like integration of these
plasmids into the B. subtilis chromosome was confirmed by
Southern hybridization analysis. Sporulation mutants derived
from B. subtils 327CL2 were constructed by conventional
transformation and confirmed by the lack of brown pigmentation (Table 1).
A 3.3-kb HindIII-SalI fragment containing the cwlC-lacZ
translational fusion gene from pMCCZ was inserted into the
HindIII-Sall site of pHY300PLK, the resultant plasmid
being designated pHY2HS. On the other hand, the 4.6-kb
SalI fragment containing the cwlC-lacZ from pMCCZ was
inserted into the SalI site of pHY300PLK. The orientation of
the insert was determined by checking the distance between
the asymmetric HindIII sites of the insert and the vector
pHY300PLK. Subsequently, we designated the plasmid
pHY2S, in which the orientation of the insert was the same
as that in pHY2HS.
1-Galactosidase assay. The P-galactosidase assay was performed basically as described by Shimotsu and Henner (43).
B. subtilis cells (1 ml) were centrifuged for 3 min in a
microfuge and then stored at -80°C. The frozen cells were
suspended in 1 ml of Z buffer (34) containing 300 ,ug of
lysozyme per ml and 0.1% (vol/vol) Triton X-100 and then
incubated at 30°C for 10 min. The extract was assayed for
,B-galactosidase activity. One unit of ,B-galactosidase activity
is defined as the amount of enzyme necessary to increase
A420 of 2-nitrophenol released from 2-nitrophenyl-P-D-galactopyranoside (ONPG) by 0.001 in 1 min (34).
RNA analysis. RNA preparation and primer extension
analysis were performed as described previously (26). The
24-mer oligonucleotide primers, Al (5' TAAAGCGA1TJlG
CAGGGTTAACGT 3') and A2 (5' CFl- GTACGATTAT
CATTCAACTGA 3'), were complementary to 75 to 98 bp
downstream and 44 to 67 bp upstream of the putative
translational start point of cwlC, respectively (Fig. 2). The
primers were 5' labeled with [_y-32P]ATP (3,000 Ci/mmol;
Amersham) and T4 polynucleotide kinase (Takara) according to the manufacturers' instructions. Northern (RNA) blot
analysis of RNAs fractionated by electrophoresis in agaroseformaldehyde gels was performed as described by Sambrook
et al. (40).
Construction of an in vitro-derived deletion mutant. For
construction of pBECM containing an insertionally inactivated cwlC gene, pCC24BE was digested with NcoI, bluntended with the Klenow fragment, and then ligated with a
1.1-kb blunt-ended EcoRI-BamHI fragment containing cat
J. BAC7ERIOL.
KURODA ET AL.
6262
H
V H
I
I
I I
H N
Bg
A
I4 <
I
IP-
Spores were purified as described previously (10, 24). Crude
spore peptidoglycan was prepared basically as described by
Warth and Strominger (46). After inactivating spore-bound
autolysins by autoclaving (121°C, 30 min), the spores were
disrupted with glass beads (diameter, 0.1 mm) in a BeadBeater (Biospec, Bartlesville, Okla.). Spore integuments
were sedimented at 20,000 x g for 10 min, washed with 0.2
M sodium phosphate buffer (pH 7.0) and then with deionized
water, and digested with trypsin (0.5 mg/ml). The crude
spore peptidoglycan was sedimented and suspended in a 4%
(wt/vol) sodium dodecyl sulfate (SDS) solution and then
boiled for 10 min. After several washes with 1 M NaCl and
then deionized water, the cell wall preparation was stored at
I-
1 kb
-200C.
B
N/S
H
Sa
pUDCL2
E
4kb
a
8.
1 Kb
FIG. 1. Restriction map of a cwlM homologous gene (cwlC) of B.
subtilis (A) and construction of pCC24BE derivatives (B). (A) Thick
arrows indicate the coding regions of the respective genes with their
transcriptional direction. Stem-loop structures and thin arrows
indicate putative terminators and the sequencing strategy, respectively. The spoVJ gene and its putative terminator are depicted on
the basis of the nucleotide sequence data obtained by Foulger and
Errington (13). (B) Closed arcs, B. subtilis chromosomal DNA; open
arcs, pUC119 or pUC19 DNA; thin arcs, pMC1871 DNA; hatched
arcs, fragment containing the chloramphenicol acetyltransferase
gene (cat, cm). Abbreviations: Bg, BglII; H, HindIII; N, NcoI; V,
EcoRV; Ba, BamHI; E, EcoRI; Hi, HincII; P, PstI; Sm, SmaI; Sa,
Preparation of autolysin-containing fractions. B. subtilis
AC327 and ANC1 were cultured on Schaeffer medium for 13
h and then sedimented by centrifugation (5,000 x g, 5 min,
40C). Proteins in the culture supernatant (1 ml) were precipitated with trichloroacetic acid (final concentration, 2%) and
then centrifuged (12,000 x g, 5 min). After being washed
with 70% ethanol, the pellet was dried, resuspended in 40 ,ul
of SDS-polyacrylamide gel electrophoresis (PAGE) sample
buffer (28), and then boiled for 5 min. A SDS-cell extract was
prepared as follows. The cell pellet from 1 ml of culture was
directly resuspended in 40 ,ul of the SDS-PAGE sample
buffer (28) and then boiled for 5 min. After centrifugation to
remove insoluble materials, the supernatant was used as the
SDS-cell extract. A protoplast cell extract was prepared as
follows. The cell pellet from 40 ml of culture was washed
with SMM buffer (42) and then resuspended in 5 ml of SMM
buffer containing 1.5 mg of lysozyme (Sigma). After 15 min
of incubation at 370C, the protoplasts were sedimented by
centrifugation (5,000 x g, 5 min, 4C), washed with 5 ml of
SMM buffer, and then suspended in 1.6 ml of the SDS-PAGE
sample buffer (28). The extraction was performed as described above. Cell membrane and cytoplasmic fractions
were prepared as follows. The protoplast pellet was suspended in 0.8 ml of Z buffer (34) and then disrupted by
ultrasonication (Tomy UR-1SOP; 150 W, 4°C, 20 s, twice).
After the removal of spores and undisrupted cells by centrifugation (12,000 x g, 5 min, 4°C, two times), cell membranes were sedimented by centrifugation (200,000 x g, 4°C,
20 min). The supernatant was used as the cytoplasmic
fraction. Electrophoresis of gels containing the cell wall
preparation and renaturation of enzymes were performed as
described previously (21).
Other methods. Assays for heat and lysozyme resistance
of spores were performed as described by Nicholson and
Setlow (35). Spore germination was monitored at A580 as
described previously (24).
Nucleotide sequence accession number. The DDBJ, EMBL,
and GenBank accession number for the cwlC sequence is
D14666.
SalIl.
RESULTS
(Fig. 1). Then, pBECM was linearized with PstI and used to
transform B. subtilis AC327, and Cmr transformants were
selected. To examine the predicted recombination, the chromosomal DNA of the transformant was extracted and then
subjected to Southern hybridization analysis (40).
Preparation of B. subtiis vegetative cell walls and spore
peptidoglycan and Micrococcus cell walls. Walls from B.
subtilis vegetative cells and Micrococcus luteus ATCC 4698
were prepared essentially as described previously (10, 23).
Molecular cloning of a cwlMf homolog of B. subtilis 168S.
Previously, we have reported the cloning of the cell wall
hydrolase gene, cwlM, from B. licheniformis (27). To determine whether the cwlM homolog is present in the B. subtilis
chromosome, Southern hybridization analysis was per-
formed with the 32P-labeled 0.6-kb EcoRI-HindIII fragment
of pLA41D1S1R (27) as a probe. This fragment contains a
truncated cwlM gene whose product retains lytic activity
(27). A 6.5-kb PstI fragment of the B. subtilis chromosomal
DNA hybridized with the probe under the condition with a
VOL. 175, 1993
B. SUBTILIS SPORULATION-SPECIFIC CELL WALL HYDROLASE
6263
1 AAGCTTAAAACCAGCAGGAATCAGTGCAGGTTAACCGAACCCATAGTACATACAAACATATGGCGTATGCACAGATTTCATGTCGGACCGTATCTGTTGT
HindI II
101 CGCTTCATGTTGAATTGTGCGCTTTCCCGAGAAATAATTTCTGGATGTGAAAGGGTTATTTCCTCATTTTTCAGTTGAATGATAATCGTACAAGCAGAAG
A2
tYIfM V K I F I D P G H G G S D P G A T G N G
201 CCGTGTTTTTTCATATCCTGTAATGAGGTGATGAAAAATGGTTAAAATTTTTATTGATCCTGGCCATGGCGGGTCTGATCCAGGCGCAACAGGTAATGGC
SD
NcoI
Q E K T L T L Q I A L A L R T I L T N E Y E G V S L L L S R T S D
301 CTTCAGGAGAAAACGTTAACCCTGCAAATCGCTTTAGCCTTACGTACGATATTAACTAATGAATATGAAGGCGTTTCTCTGCTGCTGAGCCGGACAAGCG
Al
L
Q Y V S L N D R T N A A N N W G A D F F L S I H V N S G G G T G F
401 ACCAATATGTCAGCTTAAACGACCGGACAAATGCCGCAAATAACTGGGGAGCAGATTTCTTTTTGTCCATTCACGTTAATTCCGGGGGAGGCACAGGTTT
E S Y I Y P D V G A P T T T Y Q S T I H S E V I Q A V D F A D R G
501 TGAAAGCTATATTTATCCAGATGTAGGAGCCCCGACGACGACTTATCAATCGACAATTCACTCTGAAGTGATACAAGCTGTCGACTTTGCCGATCGCGGC
K
K
T
A
N
F
H
V
L
R
E
S
A
M
P
A
L
L
T
E
N
G
F
I
D
T
V
S
D
A
N
K
L
K
601 AAAAAAACAGCGAACTTCCACGTCCTAAGGGAGTCGGCAATGCCTGCCCTCTTGACCGAGAACGGCTTCATTGATACCGTTTCCGATGCAAATAAGCTGA
T S S F I Q S L A R G H A N G L E Q A F N L K K T S S S G L Y K V
701 AAACGAGCAGTTTTATTCAAAGCTTAGCGAGAGGACATGCAAACGGGCTGGAGCAAGCCTTTAACCTTAAAAAGACTTCCAGCTCAGGGTTATATAAGGT
HindIII
Q I G A F K V K A N A D S L A S N A E A K G F D S I V L L K D G L
801 TCAAATCGGCGCATTTAAAGTCAAAGCGAATGCCGACTCGCTCGCAAGTAATGCCGAAGCCAAAGGTTTTGACTCGATTGTCCTTTTAAAGGACGGATTA
Y K V Q I G A F S S K D N A D T L A A R A K N A G F D A I V I L E S
901 TACAAAGTGCAGATTGGCGCATTTTCATCCAAAGACAATGCAGACACCCTCGCTGCCAGAGCGAAAAATGCCGGCTTTGACGCTATTGTGATCCTAGAAT
*
1001
CATAGCCGAGACGGGGACGAGCGTCTCATAAAAAAACCCGGCTCTCATCGC_AGAAACCGGGTTTTTTTATTCAAGAATATCAACAACAAACTGTGACC
1101 ATGTTTCCAGACGGTTGTAGATATC
EcoRV
FIG. 2. Nucleotide sequence of the B. subtilis cwlC gene. Only the sequence of the nontranscribed DNA strand is shown, from position
+1 (HindIII site) to +1125 (EcoRV). The deduced amino acid sequence of cwlC (nucleotides 238 to 1,002) is given above the nucleotide
sequence. An asterisk indicates a stop codon. A putative ribosome-binding sequence (SD) (nucleotides 225 to 232) and a putative
rho-independent terminator sequence (nucleotides 1,028 to 1,072) are indicated. Downward-pointing arrowheads indicate the positions of the
5' termini of cwlC transcripts determined on primer extension analysis (Fig. SB). The nucleotide sequences complementary to synthetic
oligonucleotides Al and A2 used in the Northern blot and primer extension experiments are underlined. Foulger and Errington (13) have
published the DNA sequence (spoVJ region) which starts from the 75 bp upstream of the HindIII site (position +1).
hybridizing solution consisting of 30% formamide, 5 x Denhardt's reagent, 0.5% SDS, and 100 p,g of salmon sperm
DNA per ml at 37°C for 18 h (20). The level of the
hybridization signal was almost the same as that of the
2.2-kb PstI fragment (cwlMA) of the B. licheniformis chromosomal DNA (20). The 0.7-kb HindIII and 2.4-kb BglIIEcoRV fragments of the B. subtilis chromosomal DNA
hybridized with the probe (20).
We cloned the 5- to 8-kb PstI fragments into the PstI site
of pUC119 and selected the cwlM homolog by means of
colony hybridization under the above condition. A positive
clone harbored a plasmid (pCC65P) containing a 6.5-kb PstI
insert but grew very poorly. Thus, we constructed pCC07H
by subcloning a 0.7-kb HindIII fragment from pCC65P into
the HindIII site of pUC119 (Fig. 1A). Nucleotide sequencing
of the 0.7-kb HindIlI fragment revealed extensive homology
with the 3' terminal region of cwlM (20). Furthermore, we
cloned the 2.4-kb BglII-EcoRV fragment from the B. subtilis
chromosome containing the cwlM homologous gene and its
upstream region into the BamHI-HincII site of pUC119 by
means of colony hybridization with the 32P-labeled 0.7-kb
HindIII fragment of pCC07H as a probe. Two positive
transformants grew normally and harbored the same plasmid
(pCC24BE) containing the entire cwlM homologous gene
(Fig. 1).
Nucleotide sequence of the cwlf homolog of B. subtilis.
Both strands of the 1.1-kb HindIII-EcoRV region and a part
of the 1.2-kb BglII-HindIII region were sequenced (Fig. 1A).
Figure 2 shows the nucleotide sequence of the 1.1-kb HindIII-EcoRV region. An open reading frame encoding 255
amino acids with a molecular mass of 27,146 Da is preceded
by a typical Shine-Dalgamo sequence (GAGGTGAT, AG =
-17.1 kcal (-71.5 kJ)/mol) and followed by a typical p-independent terminator (AG = -28.7 kcal (-120 kJ)/mol). The
deduced amino acid sequence showed an overall identity of
73% with that of cwlM (Fig. 3). Therefore, we regarded this
open reading frame as a cwlM homologous gene and designated it cwlC. In the C-terminal region of CwlC, there are
two intramolecularly repeated sequences (68% identity over
28 amino acids) (Fig. 3). Previously, we suggested that
C-terminal repetition of CwlM may be involved in its substrate specificity (27). The N-terminal half of the CwlC
protein region exhibits sequence similarity (40% identity
over 170 residues) with the C-terminal half (presumed active
domain) of the vegetative major autolysin (CwlB protein) of
B. subtilis (24) (Fig. 3). The N-terminal region of the CwlC
protein shows no typical signal sequence characteristic and
is homologous with those of the CwlM and CwlB proteins.
Therefore, the N-terminal region of OArlC seems to be
essential for its activity. No significant amino acid sequence
6264
J. BACTERIOL.
KURODA ET AL.
Cw1M
1
MVKIFIDPGHGGSDTGASANGLQEKQLTLQTALALRNMLLNEYQNVSVLLSRTSDQTVS
Cw1C
1
MVKIFIDPGHGGSDPGATGNGLQEKTLTLQIALALRTILTNEYEGVSLLLSRTSDQYVS*
Cw1B
319
GETIFIDPGHGDQDSGAIGNGLLEKEVNLDIAKRVNTKLNAS--GALPVLSRSNDTFYS
Cw1M
60
**
*
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****
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**
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*
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*
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LTQRTNAANSWGADYFLSIH--- MNAGGGTGFEDYIYPGVGAPTTT-YRDIMHEEILKV
*
******
*
****
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*****
*******
****
*******
*
*
Cw1C
60
LNDRTNAANNWGADFFLSIH---VNSGGGTGFESYIYPDVGAPTTT-YQSTIHSEVIQA
*
*
* *A*
*
**Y*****
*
*
*L
*
Cw1B
376
LQERVNKAASAQADLFLS IHANANDSSSPNGSETYYDTTYQAANSKRLAEQIQPKLAAN
Cw1M
115
VDFRDRGKKTANFHVLRETAMPALLTENGFVDNTNDAEKLKSSAFIQSIARGHANGLAR
Cw1C
115
VDFADRGKKTANFHVLRESAMPALLTENGFIDTVSDANKLKTSSFIQSLARGHANGLEQ
*
Cw1B
435
LGTRDRGVKTAAFYVIKYSKMPSVLVETAFITNASDASKLKQAVYKDKAAQAIHDGTVS
Cw1M
174
AFNLSK-NAAALYKVQIGAFRTKANADSLAAQAEAKGFDALVIYRDSLYKVQIGAF-SS
*
* ********* **
*******
********
*********
**** *
CW1C
174
AFNLKKTSSSGLYKVQIGAFKVKANADSLASNAEAKGFDSIVLLKDGLYKVQIGAF-SS
Cw1B
494
YYR
SpoIIB
181
Cw1M
231
KENAEALVQQAKNAGFDTFIYQE
Cw1C
232
KDNADTLAARAKNAGFDAIVILES
SpoIIB
228
KEVSQQLGQVLIDSDFEA
***
***********
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***
***
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YAVQAGKFSNEKGAETLTEQLTEKGYSAVSLSKDDGYTYVIAGLASE
*
*
**
*
*
*******
*
* *
FIG. 3. Alignment of the deduced amino acid sequences of the CwlM protein, the CwIC protein, the CwlB protein, and the SpolIB protein.
Asterisks indicate identical amino acids. The repeated amino acid sequences in the CwlC and CwlM proteins are indicated by arrows above
the sequences. The numbers are the positions with respect to the N-terminal amino acids of CwlM, CwlC, CwlB, and SpolIB.
homology was observed between CwlC and CwlA. We
found similarity (30% identity over 64 amino acids) between
the C-terminal regions of the CwlC and SpoIIB proteins (30)
(Fig. 3).
The partial nucleotide sequence (937 bp) determined for
the 1.2-kb BglII-HindIII region (Fig. IA) was consistent with
that of spoVJ (6, 13), except that nucleotide C, which is 93
bp upstream of the translational start codon, was deleted and
the 237th codon, AAA, had changed to AAG without an
amino acid substitution (20). Consequently, we found that
cwlC is located 625 bp upstream of spoVJ in the opposite
direction (Fig. 1A). Recently, Fan et al. (9) showed that
spoVK (1680 on the genetic map) is identical with spoVJ, and
therefore, the map position of cwlC could be assigned as
168°.
Expression of the cwlC gene in the wild type and sporulation-deficient strains. To study the expression of the cwlC
gene, an in-frame cwlC-lacZ fusion gene (Fig. 1B) was
introduced into the B. subtilis chromosome by conventional
transformation. Southern hybridization analysis of the resultant transformant indicated the correct integration of the
gene into the B. subtilis chromosome (20). Figure 4 shows
the time course of expression of the cwlC-lacZ gene. In the
parent strain, 327CL2, the fusion gene was not significantly
expressed during vegetative growth (less than 4 U of 3-galactosidase activity per unit of optical density at 660 nm
(OD6W) of 0.45), but it was induced at 6 to 7 h after the start
of sporulation (t6 to t7) (Fig. 4). Phase-bright spores (by
phase-contrast microscopy) began to appear in the t6 cells.
To study the pattern of dependence of the expression of the
cwlC gene on sporulation developmental genes, we constructed a cwlC-lacZ fusion in sporulation-deficient or
flaD(sin) mutants (Table 1). The greatly reduced P-galactosidase activities of B. subtilis 387CL2, 607CL2, and 867CL2
indicated that the expression of the cwlC gene requires the
300
0
0
>200
co1
0
100
0
i
15
10
Time after the start of sporulation
( hr )
FIG. 4. Time course of the production of the cwIC-lacZ fusion
protein. cwlC-directed ,B-galactosidase activity. was determined at
the indicated times after the start of sporulation in the parent strain,
B.subtilis 327CL2 (-); sporulation mutants 607CL2 (spoIfiG)(A),
867CL2 (spoIL4) (O), and 387CL2 (spoIIIC) (O); and a flaDI(sin)
mutant, 334CL2 (A). The start of sporulation (to) was defined as the
point at which cell growth was no longer exponential. B. subtilis
327CL2 exhibited less than 4 U of 0-galactosidase activity per OD6w0
unit at the growing phase (OD6.0 of 0.45). The spoVJ-directed
,B-galactosidase activity was determined in parent strain 327CL1
(A)-
VOL. 175, 1993
B. SUBTILIS SPORULATION-SPECIFIC CELL WALL HYDROLASE
products of spoIIIC, spoIIG, and spoIL4, respectively.
Thus, it is likely that the expression of cwlC is driven by oai
(the product of a composite of two truncated genes,
spoIVCB and spoIIIC [44]). The requirement for the spoIIG
and spoIL4 products for the cwlC expression can be understood as being an indirect consequence of the dependence of
o~K expression on the stage II regulatory protein(s). The
induction of spoVJ-directed ,B-galactosidase synthesis
(327CL1) preceded that of cwlC (Fig. 4). The induction time
of spoVJwas not in good agreement with the previous report
that the expression of a spoVJ-lacZ fusion became maximum
at t4 (13). This may be due to the differences in the sporulation medium and growth conditions (13). The regulatory
mutation for vegetative autolysins, the flaDI (sin) mutation
(334CL2), did not affect the expression of the cwlC-lacZ
fusion (Fig. 4); in contrast, the flaDl(sin) mutation greatly
reduced the expression of the cwlB-lacZ translational fusion
gene (26).
Transcriptional start point(s) of cwlC. To determine the
cwlC promoter region, we constructed plasmids pHY2S and
pHY2HS, containing about 1.5 and 0.24 kb upstream regions
of cwlC, respectively. B. subtilis AC327 harboring pHY2S or
pHY2HS was cultured on Schaeffer medium, and P-galactosidase activity was determined as a function of time after
sporulation. cwlC-directed 3-galactosidase activity due to
the multicopy plasmid was very similar in B. subtilis AC327
(pHY2S) and AC327 (pHY2HS), i.e., the maximum 3-galactosidase activities were 9,000 U per OD6w at tl5 and 7,800 U
per OD6. at t16, respectively (20). Thus, we considered the
upstream boundaries of cwlC promoter activities to be
located inside the HindIlI site (nucleotide 1 in Fig. 2).
However, the induction time of the fusion gene in both
strains was about 2 h late relative to that of a chromosomal
copy of the cwlC-lacZ fusion gene (20). This may be ascribed
to the fact that the addition of tetracycline to maintain the
multicopy plasmids in cells slightly inhibited the growth rate.
We isolated RNA from B. subtilis AC327 at various times
after sporulation. The RNA was fractionated by electrophoresis in an agarose-formaldehyde gel, blotted onto a
nylon membrane, and then hybridized to oligonucleotide
probes. Probe Al hybridized to a transcript which was
detected at t7.5 and tg (Fig. 5A). The time of appearance of
the cwlC transcript agrees reasonably with that of cwlCdirected 13-galactosidase activity (Fig. 4). No transcript
significantly hybridized to probe A2 (20). These results
indicated that the cwlC transcript starts from a point between Al and A2.
To determine the transcriptional start point(s), we performed extension analysis with primer Al. The Al extension
product was subjected to high-resolution gel electrophoresis
alongside a dideoxy nucleotide sequencing ladder generated
from single-stranded cwlC DNA with Al as a primer (Fig.
SB). The -10 (CATATCCTG) region upstream of the start
points was very similar to the consensus -10 (CATA---TA)
region for r'F-dependent promoters (13, 47, 48) and the
consensus -10 (CATACA-T) region for &1-dependent promoters (13, 36). For primer A2, no apparent extension
product could be obtained (20).
Inactivation of cwlC in the B. subtilis chromosome. We
constructed a plasmid, pBECM, containing an insertional
inactivated cwlC gene by means of a chloramphenicol acetyltransferase gene (Fig. 1B). The linearized pBECM was used
for transformation of B. subtilis AC327. To confirm the
inactivation of cwlC, we performed Southern hybridization
analysis of chromosomal DNA from one of the Cmr transformants, B. subtilis ANC1, with pCC24BE DNA as a
A
12 3
B
4
6265
G A TC 1 2 3 4
.Wc
< 23S
4
16s
A
T
T
A
C
44.-
Tw
C
AA
//
C/
FIG. 5. Northern blot analysis of cwlC mRNA (A) and determination of transcriptional start sites by primer extension analysis (B).
(A) Each lane contains 5 p,g of RNA from B. subtilis AC327 at t4.5
(lane 1), t6 (lane 2), t7 (lane 3), or tg (lane 4). Northern hybridization
was performed with AP-5'-end-labeled primer Al, which is complementary to 75 to 98 bp downstream of the putative translational start
point of cwlC, as a probe (Fig. 2). Hybridizing RNA is indicated by
an arrow. The hybridizing RNA for primer A2 could not be obtained
(20). 23S and 16S indicate the positions of the 23S rRNA (2.9 kb) and
16S rRNA (1.6 kb) in the B. subtilis rmB operon, respectively (14).
(B) RNA was hybridized with 32P-5'-end-labeled primer Al. Primerextended products obtained with reverse transcriptase were subjected to electrophoresis in a 6% (wt/vol) polyacrylamide sequencing gel and then autoradiography. Dideoxy DNA sequencing
reaction mixtures of the cwlC promoter region, with the same
primer, were electrophoresed in parallel (lanes G, A, T, and C).
Lanes 1 to 4 correspond to RNA (20 ,ug) from AC327 at t4.5, t6, t7.5
and tg, respectively. The positions of the two major products are
indicated by arrowheads on the sequence.
probe. On HindIII digestion, the two hybridizing bands at
3.2 and 0.7 (overlapping) kb for B. subtilis AC327 and the
three bands at 3.2, 1.7, and 0.7 kb for B. subtilis ANC1
indicated the predicted double-crossover integration of the
linearized pBECM (20). EcoRI digestion of the chromosomes also supported these results (20).
Cell wall hydrolase profile of the cwlC mutant. Figure 6A
shows the cell wall hydrolase profile of sporulating cells (tg)
of B. subtilis AC327 and ANC1 with the Micrococcus cell
wall preparation as a substrate, which is more preferable for
the CwlC protein than the B. subtilis vegetative cell walls
(20), as well as for the CwlM protein (27). A 31-kDa lytic
band corresponding to the CwlC protein was detected for the
extract from AC327 protoplasts, but not for that from ANC1
(Fig. 6A, lanes 3 and 4). Since there was an undetectable
amount of CwlC protein in the supernatant fraction and a
small amount in the SDS-cell extract fraction (Fig. 6A, lanes
2 and 6), the CwlC protein may be located within a cell until
its lysis. The membrane and cytoplasmic fractions contain
almost the same amount of the CwlC protein (Fig. 6B, lanes
7 and 8). Foster (12) reported that during sporulation, the
band A2 (50 kDa), A3 (34 kDa), A4 (30 kDa), and A5 (23
kDa) materials were lytic for B. subtilis vegetative cell walls.
But we detected only one lytic band (31 kDa) in this
condition, because CwlB (A2) does not lyse Micrococcus
cell walls (8, 27), and the A3 and A5 exhibit relatively weak
activity (12). When we used gels containing B. subtilis spore
peptidoglycan as a substrate, we detected a 31-kDa lytic
band corresponding to the CwlC protein of B. subtilis AC327
6266
J. BACrERIOL.
KURODA ET AL.
A
c
4<97k
|.4 66k
4 97k
4 97k
< 66k
| 43k
4 4 3k
'431k
4 31k
|466k
|< 43k
-. 31k
-422k
|422k
4 22k
FIG. 6. Cell wall hydrolase profile in a cwlC-deficient mutant. Samples (0.5 or 1 ml) were taken from cultures at tg (A and B) or t4o (C),
respectively. Gels contained 0.1% (wt/vol) Micrococcus cell walls (A and B) or 0.2% (wt/vol) B. subtilis spore peptidoglycan (C) as the
substrate. After electrophoresis, the proteins were renatured by treatment with 0.1 M Tris-HCl (pH 8.0) and 0.1 M KCI containing 1% Triton
X-100 (27). Each gel was stained with 0.1% methylene blue in 0.01% KOH before being photographed. Lanes: 1 and 2, culture supematants
at tg; 3 and 4, protoplast extracts; 5 and 6, SDS-cell extracts; 7, membrane fraction; 8, cytoplasmic fraction; 9 and 10, culture supematant at
strain ANCL. The molecular masses of the protein
t4o. Lanes 2, 4, 6, 7, 8, and 10, B. subtilis AC327; lanes 1, 3, 5, and 9, cwlC-deficient
standards (Bio-Rad) are indicated on the right. Rabbit muscle phosphorylase b (97 kDa), bovine serum albumin (66 kDa), hen egg white
ovalbumin (43 kDa), bovine carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (22 kDa) were individually stained with Coomassie
brilliant blue. Spore peptidoglycan hydrolases (31-kDa CwlC and 33-kDa protein) are indicated by arrows.
(Fig. 6C, lane 10). We also found a weak spore peptidoglycan hydrolase (33-kDa protein) (Fig. 6C, lanes 9 and 10).
Characterization of the cwlC mutant. The sporulation and
germination of B. subtilis AC327 and ANC1 were examined.
The sporulation frequencies of AC327 and ANC1 were 77
and 66% at t20, respectively. The heat and lysozyme resistance of spores of ANC1 were also similar to those of AC327
(20). The time course of spore germination monitored atA580
was also similar in both strains (20). The time course of free
spore production (mother-cell lysis), monitored by means of
phase-contrast microscopy, was also similar in both strains
(20). Therefore, the function of the CwlC protein remains
obscure.
DISCUSSION
B. subtilis produces at least four distinct cell wall hydrolases during vegetative growth and, in addition, two during
sporulation (12). We previously cloned cell wall hydrolase
genes, cwUA (23) and cwlB (24), for B. subtilis and cwlM (27)
for B. lichenifonnis. All enzymes encoded by these genes are
amidases (21, 24, 27). We show here that a cwlM homolog of
B. subtilis (cwlC) is located upstream of the spoVJ gene.
Recently, Fan et al. (9) reported the identity of spoVJ to
spoVK, which is located at 1680 on the B. subtilis chromosome. The amino acid sequence of CwlC exhibits 73%
overall identity with that of CwlM (Fig. 3). Thus, CwlC may
be an N-acetylmuramoyl-L-alanine amidase. However, it
might remain the possibility that cwlC encodes a regulator.
There are amino acid sequence repetitions in the C-terminal
region of the CwlC protein (Fig. 3). Such amino acid
repetitions have often been observed in the noncatalytic
domains of several cell wall hydrolases (4, 24, 27, 39). We
have previously reported that the N-terminal to central
region of the CwlM protein is a catalytic domain and that the
C-terminal repetition may be involved in its substrate specificity, i.e., the CwlM protein hydrolyzes the Micrococcus
cell wall preparation more efficiently than those of B. licheniformis and B. subtilis, but the truncated CwlM protein
(lacking the C-terminal repetition) has lost this preference
(27). In the case of CwlB, there are three repetitions in the
N-terminal region which may be involved in the specific and
tight binding to the B. subtilis cell wall (24). On the other
hand, the C-terminal half of CwlB is a presumed catalytic
domain and exhibits 40% identity (over 170 amino acids)
with the CwlC protein (Fig. 3), as well as with the CwlM
protein (27).
The dependence of cwlC-lacZ expression on the spoIIIC
gene indicates that the cwlC gene is transcribed by the orK
form of RNA polymerase (Fig. 4), which is known to
function in the mother cell (18). Recent alignments of oEand oK-dependent promoters revealed consensus for the
-35 (kmATATT, where k is G or T and m is A or C) and -10
(CATACA-T) recognition sequences at a spacing of 14 to 15
bp for the e-dependent promoters (13, 36) and consensus
for the -35 (AC) and -10 (CATA---TA) at a spacing of 16 to
17 bp for the o-dependent promoters (13, 47, 48), respectively. Interestingly, the consensus sequences for these
promoters are rather similar, particularly in the -10 regions.
In the' case of cwlC promoter, the -10 region
(CATATCCTG) is very similar to the consensus sequences
for the &-- and oa'-dependent promoters, but the -35 region
(CAAGCAG) found 14 bp upstream from the -10 region
does not agree with the consensus sequences. In the -35
region the sequences of A- and oK-dependent promoters are
relatively diverse when these promoters require positive
regulators, such as SpoIID protein (13, 18, 19, 36) and GerE
protein (47, 48). The sequence of the -35 region of the cwlC
promoter is very similar to that of cotC (CAAGCCG found
14 bp upstream from the conserved -10 region), of which
the transcription with oK RNA polymerase was stimulated in
vitro by GerE protein (47). However, it is not known
whether the transcription of cwlC requires GerE protein.
The cwlC promoter sequence does not show significant
homology with promoters recognized by 0G or oF, the ca
factors known to function in the forespore (45). The cwlClacZ expression was delayed relative to that of spoVJ-lacZ
(Fig. 4). Foulger and Errington have reported that spoVJ
expression is driven by dual promoters, P1 and P2, which are
under the control of different sigma factors, oE and oa,
respectively (13). Since promoter P2 is relatively weak,
spoVJ expression is mainly directed by the oF form of RNA
polymerase (7, 13). The timing of expression of cwlC-lacZ
supports the above idea that the cwlC gene is transcribed by
the rK form of RNA polymerase.
Previously, Foster described that a 30-kDa lytic protein
dramatically increases just prior to mother cell lysis and after
cortex synthesis (12). From the similarities of the induction
times and molecular masses of this enzyme and the CwlC
protein (31 kDa on SDS-PAGE), the two enzymes seem to
VOL. 175, 1993
B. SUBTILIS SPORULATION-SPECIFIC CELL WALL HYDROLASE
be identical. The insertional inactivation of the chromosomal
cwlC gene, which eliminates the 31-kDa lytic protein, did not
affect mother-cell lysis, spore germination, or some other
spore characteristics. However, some possible functions
may be considered. (i) It may cause some subtle change in
the peptidoglycan structure that is not detectable on testing
of the heat resistance or germination of spores, (ii) its
function may become apparent only when cwlA, cwlB,
and/or the other cell wall hydrolase gene(s) is inactivated, or
(iii) it may play some role in the antimicrobial potentiality of
B. subtilis. To examine the compensatory effect, we are now
attempting to construct a double or triple mutant for lytic
enzymes.
Illing and Errington (17) distinguished three substages of
sporulation stage II: stage III, in which the asymmetric
septation is completed; stage IIij, in which hydrolysis of
peptidoglycan from the center of the septum correlates with
the bulging of the septum into the mother cell; and stage IIiii,
in which hydrolysis of the peptidoglycan extends to the
periphery of the septum. We (22, 25) and Lazarevic et al.
(29) reported that the product of the spoIID gene, which is
required for entry into stage IIiii (17), shows sequence
homology with a modifier protein (CwbA). CwbA stimulates
cell wall lytic amidases, including the CwlM protein, and
therefore, the spoIID product may exhibit stimulatory activity toward some sporulation-specific autolysin(s). Recently,
Margolis et al. (30) found that the spoIIB mutation causes a
severe block at stage IIj when combined with a mutation in
another sporulation gene, spoVG, and interestingly, the
C-terminal region of SpolIB shows low but possibly significant similarity to that of CwlM. We also found that the
C-terminal repeated region of the CwlC protein (amino acids
186 to 249) shows 30% identity (over 64 amino acids) with
that of SpoIlB (amino acids 181 to 245) (Fig. 3). It is
conceivable that SpoIIB might directly affect cell wall metabolism at stage II.
ACKNOWLEDGMENT
We thank N. Sato for technical assistance in the nucleotide
sequencing of cwlC.
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