Partial Characterization of an Enzyme Fraction with Protease Activity Which Converts the Spore Peptidoglycan Hydrolase (SleC) Precursor to an ...
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JOURNAL OF BACTERIOLOGY, June 2001, p. 3742–3751 Vol. 183, No. 12
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.12.3742–3751.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Partial Characterization of an Enzyme Fraction with Protease Activity
Which Converts the Spore Peptidoglycan Hydrolase (SleC)
Precursor to an Active Enzyme during Germination of
Clostridium perfringens S40 Spores and Analysis of
a Gene Cluster Involved in the Activity
SEIKO SHIMAMOTO, RYUICHI MORIYAMA, KAZUHIRO SUGIMOTO,
SHIGERU MIYATA, AND SHIO MAKINO*
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
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Nagoya University, Nagoya, Aichi 464-8601, Japan
Received 15 November 2000/Accepted 28 March 2001
A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early
stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence
and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by
processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The
present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethyl-
ammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization
of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to
668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and
52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained
activity after heat treatment at 55°C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and
Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl2
indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated
that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC,
which are positioned in a tandem array just upstream of the 5ⴕ end of sleC. The amino acid sequences deduced
from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of
homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases.
Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the
exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex
hydrolytic reactions.
Bacterial spore germination, defined as the irreversible loss intact spore peptidoglycan, which likely leads to un-cross-link-
of spore characteristics, is triggered by specific germinants and ing of cortex peptidoglycan; this step is followed by further
proceeds through a set of sequential steps. Spore germination degradation of the polysaccharide moiety of SCLE-modified
is essential to allow spore outgrowth and the formation of a cortex peptidoglycan by CFLE (5, 6, 23, 24, 28, 29). Thus,
new vegetative cell; once triggered, it proceeds in the absence SCLE and CFLE differ from each other in bond specificity and
of germinants and germinant-stimulated metabolism. This fact recognition of the morphology of the substrate. It is most likely
indicates that spore germination is a process controlled by the that the in vivo activity of CFLE is regulated by its requirement
sequential activation of a set of preexisting germination-re- for partially un-cross-linked spore cortex. On the other hand,
lated enzymes but not by protein synthesis (10, 26). SCLE, which acts on intact spores, needs some activation pro-
Among the key enzymes involved in the spore germination cess for the expression of activity. The mechanism of activation
of Bacillus subtilis 168, Bacillus cereus IFO 13597, and Clostrid- is crucial to an understanding of bacterial spore germination.
ium perfringens S40 are a group of cortex-lytic enzymes which SCLE of C. perfringens S40 is a mature form of SleC, which
degrade spore-specific cortex peptidoglycan. In the spores, at is synthesized at an early stage of sporulation as a precursor
least two cortex hydrolases, spore cortex-lytic enzyme (SCLE) consisting of four domains: an N-terminal presequence (113
and cortical fragment-lytic enzyme (CFLE), are suggested to residues), an N-terminal prosequence (35 residues), mature
cooperatively function for cortex degradation. That is, cortex
enzyme (264 residues), and a C-terminal prosequence (25 res-
hydrolysis during germination is initiated by attack of SCLE on
idues) (24, 33, 40). During spore maturation, the N-terminal
presequence and the C-terminal prosequence are sequentially
processed; the resulting inactive proenzyme, with a mass of 35
* Corresponding author. Mailing address: Department of Applied kDa (termed proSCLE) and consisting of the N-terminal pro-
Molecular Biosciences, Graduate School of Bioagricultural Sciences,
sequence and a mature region which exists as a complex with
Nagoya University, Nagoya, Aichi 464-8601, Japan. Phone: 81 (52)
789-4132. Fax: 81 (52) 789-4120. E-mail: makino@nuagr1.agr the cleaved N-terminal prepeptide (termed the prepeptide-
.nagoya-u.ac.jp. proSCLE complex) (33), is deposited on the outside of the
3742VOL. 183, 2001 GERMINATION PROTEASES OF C. PERFRINGENS SPORES 3743
cortex layer in the dormant spore (25). Proteolytic cleavage of 0.18, and the decrease in OD600 was monitored at 32°C to detect SCLE activity
the promature junction of proSCLE in the complex (the link- (20). One unit of activity was defined as a decrease in the OD600 of 0.100 per min.
Preparation of GSP. Purification procedures were carried out at 20°C, unless
age between Val-149 and Val-150 of SleC) during germination noted otherwise, and GSP activity was monitored in each fractionation step as
generates active SCLE with a mass of 31 kDa (24, 33). The described above. Spores were germinated as described previously (23), and GSP
protease involved in the conversion of proSCLE to SCLE, was extracted by incubating germinated spores (4 g of packed weight) in 20 ml of
denoted germination-specific protease (GSP), has been de- 0.25 M KCl–50 mM potassium phosphate (pH 7.0) containing 0.2% 3-[(3-chol-
amidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at 30°C for 2
tected in germinated spores (40), but its enzymatic entity re-
h. After debris were removed by centrifugation (15,000 ⫻ g for 10 min at 4°C),
mains to be established. the extract was heated at 52°C for 30 min to inactivate existing SCLE, and the
A part of the nucleotide sequence of the gene, hereafter supernatant (20 ml) was recovered by centrifugation (15,000 ⫻ g for 10 min at
denoted cspC (C. perfringens serine protease C; see below), 4°C). The supernatant was concentrated threefold by using a UK-10 membrane
which is present just upstream of the 5⬘ end of sleC has been filter (Advantec MFS, Inc., Tokyo, Japan), and 2 ml was applied to a Superose
12 column (2.0 by 31 cm; bead size, 20 to 40 m; Pharmacia) which had been
reported (24). Comparison of the partial deduced amino acid equilibrated with 0.1 M KCl–40 mM potassium phosphate (pH 7.0) containing
sequence of the gene product with those registered in various 0.1% CHAPS. This procedure was carried out three times. Fractions containing
databases suggested that the CspC sequence is homologous to GSP (total, 18 ml), which eluted near the voided volume of the column, were
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that around the active center of serine proteases from Bacillus applied to a hydroxyapatite column (3.0 by 20 cm; fast flow; Wako Pure Chem-
species. Bacterial structural genes are often organized into icals) which had been equilibrated with 40 mM potassium phosphate (pH 7.0)
containing 0.1% CHAPS. GSP did not bind to the column, while most proteins
clusters that include genes coding for proteins whose functions adsorbed to the column.
are related (1, 17). This information raised the possibility that Fractions containing GSP (30 ml) were applied to a TSK gel DEAE-5PW
the cspC gene encodes a protease involved in the activation of high-performance liquid chromatography (HPLC) column (0.75 by 7.5 cm; bead
SleC and prompted us to analyze the cspC gene in parallel with size, 10 m; Tosoh, Tokyo, Japan) preequilibrated with 40 mM potassium phos-
phate (pH 6.0) containing 0.1% CHAPS. Adsorbed materials were eluted with a
attempts to identify GSP. In this paper, we describe the char-
linear gradient of 0 to 0.3 M KCl in 40 mM potassium phosphate (pH 6.0)
acterization of GSP and the cloning of genes which are present containing 0.1% CHAPS. The flow rate was 0.5 ml/min, and fractions were
upstream of the 5⬘ end of sleC. GSP, a serine protease which collected and assayed for GSP activity. The HPLC system used consisted of a
specifically processes the N-terminal prosequence of proSCLE, Jasco 801-SC controller, a Jasco 880-PU pump, a Jasco 875-UV detector (all
was isolated as a fraction consisting of three species of pro- from Japan Spectroscopic Co., Tokyo, Japan), and a Shimadzu C-R3A signal
integrator (Shimadzu Co., Kyoto, Japan).
teins. Several pieces of evidence indicated that these proteins Effect of protease inhibitors and chemicals on GSP activity. All assays were
are products of three tandem genes, cspA, cspB, and cspC, carried out with 40 mM potassium phosphate (pH 7.0), except when we exam-
which are positioned just upstream of the 5⬘ end of sleC and ined the effect of CaCl2 and dipicolinic acid on activity, for which 40 mM
encode subtilisin-like proteases with a triad active center. Tris-HCl (pH 7.0) was used. Purified GSP (1.2 U; 50 l in 0.15 M KCl–40 mM
potassium phosphate [pH 7.0] containing 0.2% CHAPS or 0.15 M KCl–40 mM
However, whether these proteases commit to the activation
Tris-HCl [pH 7.0] containing 0.2% CHAPS) was mixed with 1.25 to 2.50 l of
process for SleC as separate proteases or as a complex is not protease inhibitors and chemicals of appropriate concentrations and the mixtures
known at present. were incubated at 30°C for 1 h. Dimethyl sulfoxide was used to solubilize besta-
tin, E-64, pepstatin A, and PMSF, but the solubilizer included in the assay
MATERIALS AND METHODS medium (maximum, 0.2%) had no effect on activity.
Cloning of the csp genes. Chromosomal DNA from C. perfringens S40 was
Bacterial strains, plasmids, and chemicals. Spores of C. perfringens S40 were digested with EcoRI; fragments in the size range of 6.2 to 8.0 kb were separated
used as the source of proSCLE and GSP. Chromosomal DNA was isolated from by agarose gel electrophoresis, purified with a Geneclean II kit (Bio 101, Inc., La
vegetative cells of the strain. The organism was cultured as described by Miyata Jolla, Calif.), and inserted into pUC118. The ligation mixture was used to trans-
et al. (23). Escherichia coli XL1-Blue (Stratagene Cloning Systems, La Jolla, form E. coli XL1-Blue. The csp genes were cloned from this library with a sleC
Calif.) was used as a host for the screening library. Plasmids pUC118 and probe obtained as a 2.2-kb HindIII fragment from pCP22H (24). The primers
Bluescript II KS(⫹) (Stratagene) were used as cloning vectors. E. coli BL21 used for inverse PCR were 5⬘-TCACAATCTGGAGCTACTCC-3⬘ and 5⬘-TGC
(DE3) (Novagen, Inc., Madison, Wis.) and plasmid pET22b(⫹) (Novagen) were TTGGATTCTTCAAAGAGA-3⬘.
used as a host and as a vector, respectively, for the expression of recombinant Preparation of antiserum against recombinant protein r ⌬1–78CspC. To pre-
protein. E. coli was routinely grown at 37°C in 2⫻ YT medium (1.6% Bacto pare a polyclonal antibody against CspC, a recombinant CspC-His fusion was
Tryptone, 1% Bacto Yeast Extract, 0.5% NaCl [pH 7.0]) with ampicillin added purified. The plasmid used to synthesize this protein was constructed by PCR
to 100 g per ml for plasmid-carrying strains. Transformation was carried out amplification of a part of the cspC gene, encoding Ser-79 to Thr-582, from
according to standard protocols (13). pCP70E using primers containing NdeI and XhoI restriction sites. The NdeI- and
The protease inhibitors used were as follows: Streptomyces subtilisin inhibitor XhoI-digested PCR product was ligated into the NdeI and XhoI restriction sites
(SSI), phenylmethylsulfonyl fluoride (PMSF), and 4-amidinophenylmethanesul- of vector pET22b(⫹) to generate plasmid p⌬1–78CspC, resulting in a sequence
fonyl fluoride (APMSF) from Wako Pure Chemicals (Osaka, Japan); antipain, encoding processed CspC with a C-terminal six-histidine tag. Plasmid p⌬1–
leupeptin, and pepstatin A from Peptide Institute, Inc. (Osaka, Japan); and 78CspC was introduced into E. coli BL21(DE3), and production of His-fused
aprotinin, bestatin, and E-64 from Sigma-Aldrich (Tokyo, Japan). ␣-, - and recombinant protein r⌬1–78CspC was induced with 1 mM isopropyl-1-thio--
-Caseins were purchased from Sigma-Aldrich. Other chemicals were obtained galactopyranoside. The resulting fusion protein obtained from an inclusion body
from Wako Pure Chemicals. was dissolved in 6 M urea and purified using His 䡠 Bind kits (Novagen) as
Preparation of spores and decoated spores. Spores, decoated spores, and the recommended by the supplier. Polyclonal antibody against the purified fusion
spore coat fraction of C. perfringens S40 were prepared by methods described protein was raised in mice as described previously (24).
previously (23, 24). Nucleotide sequencing and analysis. Nucleotide sequencing was performed by
Assay of GSP activity. GSP activity was measured indirectly via the increase in the dideoxynucleotide chain termination method of Sanger et al. (35) using an
SCLE activity after incubation of the prepeptide-proSCLE complex with GSP. ABI PRISM 310 automatic sequencer and an ABI PRISM BigDye Terminator
The complex used as a substrate for GSP was obtained as described by Okamura cycle sequencing kit (Applied Biosystems, Foster City, Calif.). Nucleotide and
et al. (33). A mixture containing 5 l of the prepeptide-proSCLE complex (0.2 amino acid sequence analyses and comparisons with DNA and protein sequences
mg/ml), 134 to 115 l of 40 mM potassium phosphate (pH 7.0), and 1 to 20 l registered in databases (GenBank, EMBL, PIR, and SWISS-PROT) were per-
of GSP fractions in a total volume of 140 l was incubated at 32°C for 3 min, and formed with MacDNASIS software (Hitachi Software Engineering, Tokyo, Ja-
then 10 l of decoated spore suspensions was added. This reaction mixture pan).
contained a large excess of substrates for both GSP and SCLE. The initial optical Other procedures. Protein concentrations were measured according to the
density at 600 nm (OD600) of the mixture in a cell with a 1-mm light path was methods of Bradford (4) and/or Groves et al. (12) using bovine serum albumin3744 SHIMAMOTO ET AL. J. BACTERIOL.
TABLE 1. Purification of GSP activity from C. perfringens basis, the purification of GSP was performed under the con-
germinated sporesa ditions described in Materials and Methods.
Total Total Sp act The steps in the purification procedure and the yields are
Yield Purification
Procedure protein activity (U/mg of
(%) (fold) shown in Table 1. A 668-fold purification was achieved, with a
(mg) (U) protein)
final yield of 10.6% enzyme activity. This final purified fraction
Exudate 69.0 14,394 208.6 100 1 contained three predominant proteins, termed GSP-60, GSP-
Superose 12 18.4 6,282 341.4 43.6 1.6 57, and GSP-52; the proteins had apparent molecular masses
Hydroxyapatite 0.23 3,620 15,739.1 25.2 75.5
DEAE-5PW 0.011 1,533 139,363.6 10.6 668.1
of 60, 57, and 52 kDa, respectively, as estimated by SDS-PAGE
under reducing conditions (Fig. 1A). The same electrophoretic
a
C. perfringens germinated spores (4 g of packed weight) obtained as described pattern was obtained under nonreducing conditions. The ap-
in Materials and Methods were used as the enzyme source for this experiment.
parent nonstoichiometric molar ratios of GSP-60, GSP-57, and
GSP-52 were 1:1:1. The N-terminal amino acid sequences of
the three components were determined to be STSPIEA
as a standard. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
SKVENFHNSPYLKLTGK DVIVXII (31 residues) for GSP-
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(PAGE) was carried out on slab gels using a Laemmli buffer system (19) at a
constant current of 20 mA. Immunoblotting and immunoprecipitation were 60, ESPVEDSKAPVFHRNP (16 residues) for GSP-57, and
performed by use of anti-r⌬1–78CspC antiserum as described previously (24). AYDSNRASXIPSVWNNYNLTGEGILVGFLDTGIDY (35
Analysis of the N-terminal amino acid sequence was carried out with a protein residues) for GSP-52 (X denotes an unidentified residue).
sequencer (Procise cLC model 494; Applied Biosystems) according to the Treatment of the prepeptide-proSCLE complex with the
method of Matsudaira (22).
Nucleotide sequence accession number. The nucleotide sequence data re-
purified GSP fraction generated a 31-kDa polypeptide (Fig.
ported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide 1B), in parallel with the appearance of spore cortex-lytic ac-
sequence databases under accession number AB042154. tivity. Protein sequence analysis of the 31-kDa product pro-
vided an N-terminal sequence of VLPEPXVPEYIV (12 resi-
RESULTS dues), which is identical to that of SCLE.
The purified GSP fraction consisting of three protein com-
Purification of GSP. GSP was extracted from germinated ponents was available in limited quantities (Table 1), which
spores in 0.25 M KCl–50 mM KH2PO4 (pH 7.0) containing hampered further biochemical characterization of its gross
0.2% CHAPS. The presence of CHAPS (⬎0.05%) was needed conformation and compositional stoichiometry. Indeed, the
for the stabilization of enzyme activity. Although most anion amount of GSP in the spore was estimated to be about 1/100
exchangers tested retained GSP even at 1 M KCl over the pH that of the prepeptide-proSCLE complex, which is the in vivo
range of 6.5 to 8.5, the enzyme could be eluted from a DEAE- substrate of GSP (2 mg/g of wet spore) (unpublished results).
5PW column with 0.15 M KCl at pH 6.0, which is an acidic pH Characteristics of GSP. GSP was active over a pH range of
limit used to retain enzyme activity. The addition of dithio- 5.8 to 8.5 in the presence of 0.1% CHAPS (at least 2 months
threitol (0.2 mM) or EDTA (1 mM) or both in the extraction at 4°C and pH 7.0) and was stable after heat treatment at 55°C
and purification steps had no effect on the activity. On this for 40 min at pH 7.0. However, the enzyme lost its activity
FIG. 1. SDS gel electrophoretic patterns of the purified active enzyme fraction (A) as well as the prepeptide-proSCLE complex and ␣-, -, and
-caseins treated or not treated with GSP (B). (A) An aliquot (1 ml) of the main peak fraction containing GSP activity which eluted from a
DEAE-5PW column was dialyzed against H2O, dried by using of rotary evaporator, dissolved in 40 l of 10 mM Tris-HCl (pH 8.0) containing 1%
SDS and 1% 2-mercaptoethanol, and electrophoresed on a 0.1% SDS–10% polyacrylamide gel. (B) The prepeptide-proSCLE complex (lanes 1
and 2), ␣-casein (lanes 3 and 4), -casein (lanes 5 and 6), and -casein (lanes 7 and 8) (5 to 10 g each) were incubated with (odd-numbered lanes)
or without (even-numbered lanes) GSP (1.5 U) in 40 mM potassium phosphate (pH 7.0) at 30°C for 4 h and electrophoresed on a 0.1% SDS–13.3%
polyacrylamide gel. In lanes 1 and 2, 35- and 18-kDa bands are proSCLE and prepeptide, respectively. The standard proteins run were rabbit
muscle phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin
inhibitor (22 kDa), and lysozyme (14 kDa).VOL. 183, 2001 GERMINATION PROTEASES OF C. PERFRINGENS SPORES 3745
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FIG. 2. Physical map of a csp gene cluster of C. perfringens. The ORFs and direction of transcription are indicated by the arrows. The solid lines
below the ORF arrows represent the inserts of recombinant plasmids pCP22H, from which the sleC probe was isolated, and pCP70E, from which
the nucleotide sequence was derived. Restriction sites are indicated as A (AccI), E (EcoRI), Hc (HincII), Hd (HindIII), Nc (NcoI), Nd (NdeI),
P (PvuII), and S (SacI). FPCR is a fragment of DNA cloned by reverse PCR. Sequence data for the HindIII-HindIII region marked by asterisks
are given in Fig. 3.
(⬎80%) within 72 h at 4°C in the absence of CHAPS. Deter- ing frames (ORFs); 5⬘-truncated cspA, cspB, and cspC and
gents such as Triton X-100, nonaethyleneglycol n-dodecyl 3⬘-truncated sleC. The sequence of the 5⬘ end of the cspA gene
ether, octylglucoside, and deoxycholate, which are often used was determined by sequencing of an amplicon obtained by
to stabilize membrane proteins but dissociate aggregated pro- inverse PCR after HindIII chromosomal DNA digestion and
teins more effectively than CHAPS (30), were ineffective in religation. A 5,989-bp DNA sequence encompassing the com-
preventing the inactivation of GSP. plete cspA, cspB, and cspC genes was determined. Figure 3
GSP was inhibited 68% after incubation at 30°C for 1 h with shows the nucleotide sequence of nucleotides 1 to 4,648 and
1 mM PMSF and 20% with 0.1 mM PMSF but not with E-64 the corresponding predicted amino acid sequence. The se-
(0.1 mM), pepstatin A (0.1 mM), bestatin (0.1 mM), and quence of nucleotides 4,643 to 5,989 determined in this study
EDTA (5 mM), suggesting that GSP belongs to a family of agreed completely with the sequence reported by Miyata et al.
serine proteases. However, GSP resisted a variety of known (24).
serine protease inhibitors, such as antipain (0.1 mM), APMSF An inverted repeat sequence (nucleotides 31 to 85) was
(0.1 mM), leupeptin (0.1 mM), aprotinin (10 M), and SSI (1 found upstream of cspA. Possible initiation codons are nucle-
M). In order to determine the substrate preference of GSP, otides 171 to 173 (GTG) for cspA, nucleotides 1,923 to 1,925
the hydrolytic activity of GSP was investigated with ␣-casein, (ATG) for cspB, and nucleotides 3,623 to 3,625 (ATG) for
-casein, and -casein as substrates. None of the proteins cspC. The stop codons for cspA, cspB, and cspC occurred at
tested was hydrolyzed by GSP at detectable levels (Fig. 1B). nucleotides 1,905 to 1,907 (TAA), nucleotides 3,618 to 3,620
These results suggest that the enzyme has a strict substrate (TAG), and nucleotides 5,369 to 5,371 (TAG), respectively.
specificity. These findings suggest that cspA encodes a 578-residue protein
Both L-alanine and D-alanine (5 mM each, a germinant and (molecular weight, 64,427), cspB encodes a 565-residue protein
a competitive inhibitor of the normal germination process, (molecular weight, 62,215), and cspC encodes a 582-residue
respectively) (10) had no effect on GSP-mediated hydrolysis of protein (molecular weight, 64,392). A potential stem-loop ter-
the prepeptide-proSCLE complex. This activity was also not minator structure for the mRNA was found downstream of the
affected by the addition of dipicolinic acid (5 mM) or Ca2⫹ (2 stop codon for cspC (24) but not for cspA or cspB.
mM) or both, which are released from the spore core at an A database search revealed that the products of the csp
early stage of germination (16). The enzyme was inhibited 73 genes belong to the superfamily of subtilisin-like serine pro-
and 39% by incubation with 1 mM HgCl2 and 1 mM p-chlo- teases, as demonstrated by the high degree of conservation in
romercuribenzenesulfonate, respectively. The addition of di- the catalytic domain, which includes the three active-site resi-
thiothreitol (10 mM, incubation at 30°C for 1 h) restored dues (Asp, His, and Ser) and the oxyanion binding region
nearly 100% of the GSP activity which had been once lost with (Asn), which stabilizes the hydrolytic reaction intermediate
either inhibitor. (Fig. 4A). Dendrogram analysis suggested that Csp proteins
Nucleotide and deduced amino acid sequences of cspA, cspB, are closely related to each other (Fig. 4B).
and cspC. An EcoRI genomic library from C. perfringens S40 GSP-57, GSP-52, and GSP-60 are processed forms of CspA,
was constructed in pUC118 and transformed into E. coli XL1- CspB, and CspC, respectively. The 16 amino acid residues
Blue. This library was screened with an sleC probe, and a starting from Glu-78 of CspA, the 35 residues starting from
positive clone carrying a 6.9-kb EcoRI fragment was isolated Ala-97 of CspB, and the 31 residues starting from Ser-79 of
(pCP70E in Fig. 2). A 6.4-kb EcoRI-AccI region of this frag- CspC corresponded to the N-terminal sequences of GSP-57,
ment was sequenced, revealing the presence of four open read- GSP-52, and GSP-60, respectively. The results unequivocallyDownloaded from http://jb.asm.org/ on January 14, 2021 by guest
FIG. 3. Nucleotide sequence and deduced amino acid sequence of a 4,648-bp HindIII-HindIII fragment containing cspA, cspB, and partial cspC
genes. The deduced amino acid sequences of cspA (nucleotides 171 to 1,907), cspB (nucleotides 1,923 to 3,620), and part of cspC (nucleotides 3,623
to 4,648) are given below the nucleotide sequences (See reference 24 for the 3⬘-end sequence of cspC and the whole sequence of sleC). The
numbers for the nucleotides and amino acids are shown on the right. An inverted repeat is indicated by arrows. Putative ribosome binding sites
(rbs) with moderate complementarity to the 3⬘ end of the 16S rRNA of C. perfringens (11) are underlined. The asterisks indicate termination
codons. The amino acid sequences of CspA, CspB, and CspC which agreed with the N-terminal sequences of GSP-57, GSP-52, and GSP-60,
respectively, are underlined in bold.
3746VOL. 183, 2001 GERMINATION PROTEASES OF C. PERFRINGENS SPORES 3747
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FIG. 3—Continued.
indicate that GSP-52, GSP-57, and GSP-60 are processed for GSP-52, 55,458 for GSP-57, and 55,585 for GSP-60. Per-
forms of CspA, CspB, and CspC, respectively. The data suggest cent identities among the putative mature protease sequences
that the Csp proteases are produced in a form possessing are 34% between GSP-52 and GSP-57, 54% between GSP-57
propeptides, with the promature junctions in CspA, CspB, and and GSP-60, and 39% between GSP-52 and GSP-60.
CspC being similar to each other (Fig. 3). Thus, the predicted Recombinant protein r⌬1–78CspC, which was obtained in an
molecular weights of the processed GSP proteins are 51,442 insoluble form, did not show GSP activity. Purified r⌬1–78CspC3748 SHIMAMOTO ET AL. J. BACTERIOL.
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FIG. 4. Sequence alignment (A) and dendrogram analysis (B) of Csp proteins and several subtilisin-like serine proteases. (A) Only the
sequences around three canonical residues (asterisks) and an oxyanion binding site (number sign) of the proteases are aligned. Identical residues
in more than five proteins are indicated by black boxes. Bold underlining indicates the N terminus of mature or putative mature enzymes. Amino
acid residue numbers given in parentheses indicate the first and last residues of amino acid alignments represented. The proteases are as follows:
Apy, hyperthermostable protease from Aquifex pyrophilus (7); Aqua, mesothermophile alkaline protease from Thermus aquaticus YT-1 (18); Carls,
subtilisin Carlsberg from Bacillus licheniformis (15); ISP, intracellular alkaline protease from Thermoactinomyces sp. strain HS682 (39); PrtP, cell
envelope-located protease from Lactobacillus paracasei (14); and Pyro, hyperthermostable protease pyrolysin from Pyrococcus furiosus (41). (B)
Dendrogram of Csp proteins and proteases listed above, in which branch lengths are in inverse proportion to the degree of sequence similarity.
The tree was constructed using the programs ClustalX and NJPLOT (38).
was shown by SDS-PAGE to be almost identical in size to logically distinct from GSP-52 and GSP-57. The anti-r⌬1–
GSP-60, and antiserum raised against the recombinant protein 78CspC antiserum failed to immunoprecipitate GSP activity.
recognized GSP-60 but not GSP-52 or GSP-57 (Fig. 5, lane 1). Location of GSP-60 in dormant spores. The coat fraction
Thus, despite the high sequence similarity among GSP proteins was separated from decoated spores as described previously
(especially between GSP-57 and GSP-60), GSP-60 is immuno- (24). Anti-r⌬1–78CspC antiserum was used to detect GSP-60 inVOL. 183, 2001 GERMINATION PROTEASES OF C. PERFRINGENS SPORES 3749
should be noted that hydrophobic compounds such as alcohols,
fatty acids, and surfactants in such concentrations are known to
exert an inhibitory effect on spore germination (31, 42, 43). In
this regard, the results obtained by using protease inhibitors in
vivo, without consideration of possible inhibitory effects on
germination due to the hydrophobic nature itself, should be
viewed with some caution, since neither the relevant proteases
nor their genes have been characterized. Moreover, it was
reported that the inactive proform of a germination-specific
cortex-lytic enzyme from spores of B. megaterium KM is pro-
cessed to release the active enzyme (9), but the processing
enzyme has not been identified.
GSP activity eluted from a DEAE-5PW column as a single
peak which consisted of three protein components, GSP-52,
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FIG. 5. Immunological detection of CspC-related proteins in dor- GSP-57, and GSP-60. There was no disulfide linkage among
mant spores. The spore coat fraction was separated from decoated these proteins. We have detected active GSP as a large mo-
spores as described in Materials and Methods. The decoated spores (1 lecular fraction, as suggested by its elution near the void vol-
g of wet weight) were disrupted with a bead beater in a 20-ml tube ume in Superose 12 column chromatography. A comparison of
containing 10 ml H2O and 10 g of glass beads (diameter, 0.1 mm). The
the N-terminal amino acid sequences of the isolated proteins
supernatant obtained by centrifugation (15,000 ⫻ g for 5 min at 4°C)
was used as an extract of disrupted decoated spores. GSP obtained by with the deduced amino acid sequences of Csp proteins un-
DEAE-5PW column chromatography, the extract obtained from ger- equivocally demonstrated that GSP-57, GSP-52, and GSP-60
minated spores by incubation in CHAPS solution, the spore coat are hydrolyzed forms of CspA, CspB, and CspC, respectively.
fraction, and the extract of disrupted decoated spores were subjected Anti-r⌬1–78CspC antiserum recognized only GSP-60. It is
to 0.1% SDS–10% PAGE followed by immunoblot analysis with anti-
r⌬1–78CspC antiserum. Lanes 1, GSP obtained by DEAE-5PW column known that genes for enzymes with related activities are often
chromatography (⬃5 g of protein); 2 and 4, spore coat fraction (⬃100 organized into clusters in bacteria; indeed, tandem cspA, cspB,
g of protein for lane 2 and ⬃70 g of protein for lane 4); 3, extract and cspC genes were aligned just upstream of the 5⬘ end of the
from germinated spores (⬃100 g of protein); 5, extract of disrupted sleC gene. The failure of immunoprecipitation of active GSP
decoated spores (⬃100 g of protein). Samples for lanes 1 and 2 were
by anti-r⌬1–78CspC antiserum may imply that epitopes are
electrophoresed on a separate gel different from that used for lanes 3
to 5. The standard proteins were the same as those used for Fig. 1. masked in the multiprotein complex. The percentage of
charged amino acids (Asp, Glu, Arg, and Lys) in each GSP
protein is over 20%. It has been shown that salt bridges be-
the spore fractions. As shown in Fig. 5, the antiserum cross- tween charged amino acids are a major factor stabilizing pro-
reacted only with a 60-kDa polypeptide in the extract from tein structure (7). It is likely, therefore, that intermolecular
germinated spores incubated in CHAPS solution (lane 3) as electrostatic interactions contribute to the formation of the
well as purified GSP (lane 1). These findings unequivocally active multiprotein enzyme with a relatively high thermal sta-
show that the antiserum specifically recognizes GSP-60. bility. At present, however, whether all three of the proteins in
GSP-60 was detected in the coat fraction from dormant spores the active enzyme fraction or only one of the proteins is in-
(Fig. 5, lanes 2 and 4) but not in the extract from decoated volved in the process of activation of proSCLE is unknown. In
spores (lane 5), suggesting a peripheral location of GSP-60 in addition to the functional characteristics of each GSP protein,
dormant spores. GSP activity was not detected in the coat knowledge of the gross conformation and compositional stoi-
fraction under reduced alkaline pH, nor was it regained by the chiometry of the active GSP molecule obtained here is needed
addition of CHAPS after dialysis against neutral buffer. to understand the mechanism of GSP function.
Isolated GSP failed to digest different caseins. Furthermore,
DISCUSSION the enzyme attacked only the Val-Val linkage of the promature
junction of proSCLE (residues 149 and 150 of SleC) and not
The present results provide direct evidence demonstrating neighbor Val-Val bonds (residues 155 and 156 and residues
the involvement of GSP in the process of activation of SleC 161 and 162 of SleC), which lie within a prolyl cluster region
during the germination of C. perfringens spores. It has been between Pro-131 and Pro-167 that contains 10 proline residues
confirmed that the germination protease of B. subtilis and (24). GSP is not responsible for processing of the N-terminal
Bacillus megaterium is involved in degrading small acid-soluble prepeptide and the C-terminal propeptide of SleC during
proteins during spore germination (32, 34). However, germi- sporulation (33). It appears that the processing of the extended
nation protease is not responsible for a germination-triggering sequences of SleC during sporulation and germination of C.
reaction, in the sense that the enzyme hydrolyzes the so-called perfringens spores is tightly regulated by proteases which have
storage proteins of spores. Although the involvement of tryp- a rigorous substrate specificity and are specific to the time of
sin-like proteolytic activity in the triggering reaction has been development of the spores.
suggested by the arrest of germination of B. cereus and B. Extraction of active GSP from germinated spores was en-
subtilis spores in the presence of trypsin inhibitors (2, 3), sev- hanced by the addition of CHAPS, a nondenaturing detergent
eral of the inhibitors used are rather hydrophobic and rela- (30). Immunochemical studies using anti-r⌬1–78CspC anti-
tively high concentrations of the inhibitors (on the order of serum revealed that GSP-60 is liberated from dormant spores
millimolar or more) are needed to interrupt germination. It by treatment of the spores with 0.1 M H3BO3 (pH 10.0) con-3750 SHIMAMOTO ET AL. J. BACTERIOL.
taining 1% 2-mercaptoethanol. This treatment also detaches ing in C. acetobutylicum. Unlike the genome organization of
from spores proSCLE and CFLE, which have been shown to both C. perfringens and C. acetobutylicum, the C. difficile sleC
be located on the exterior of the cortex layer (25). These homolog is found at a distance from two csp homologs. Al-
observations suggest the localization of GSP to a hydrophobic though these genome sequences are incomplete, it seems that
and peripheral region of dormant spores, such as the outer SleC- and Csp-like proteins are conserved among the clos-
membrane, which lies underneath the spore coat. Close local- tridia.
ization of cortex degradation-related enzymes is pertinent to
the pursuit of a cascade of cortex hydrolytic reactions during ACKNOWLEDGMENTS
germination. We acknowledge the technical assistance of Y. Tagata.
The results suggested that Csp proteins belong to a family of This work was supported in part by grants in aid for scientific
subtilisin-like proteases, based on DNA sequence analysis. research (10660081 and 11660085) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
This family of proteases is characterized by an N-terminal
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