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Journal of Bacteriology, August 1998, p. 4303-4308, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning and DNA Sequencing of the Genes Encoding
Clostridium josui Scaffolding Protein CipA and Cellulase
CelD and Identification of Their Gene Products as Major Components of
the Cellulosome
Motohide
Kakiuchi,1
Ayako
Isui,1
Katsuhisa
Suzuki,1
Tsuchiyoshi
Fujino,2
Emi
Fujino,1
Tetsuya
Kimura,1
Shuichi
Karita,3
Kazuo
Sakka,1,* and
Kunio
Ohmiya1
Faculty of
Bioresources1 and
Center for Molecular
Biology and Genetics,3 Mie University, Tsu
514, and
Nagoya Seiraku Co., Ltd., Nagoya
468,2 Japan
Received 23 February 1998/Accepted 16 June 1998
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ABSTRACT |
The Clostridium josui cipA and celD genes,
encoding a scaffolding-like protein (CipA) and a putative cellulase
(CelD), respectively, have been cloned and sequenced. CipA, with an
estimated molecular weight of 120,227, consists of an N-terminal signal
peptide, a cellulose-binding domain of family III, and six successive
cohesin domains. The molecular architecture of C. josui
CipA is similar to those of the scaffolding proteins reported so far,
such as Clostridium thermocellum CipA, Clostridium
cellulovorans CbpA, and Clostridium cellulolyticum
CipC, but C. josui CipA is considerably smaller than the
other scaffolding proteins. CelD consists of an N-terminal signal
peptide, a family 48 catalytic domain of glycosyl hydrolase, and a
dockerin domain. N-terminal amino acid sequence analysis of the
C. josui cellulosomal proteins indicates that both CipA and
CelD are major components of the cellulosome.
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TEXT |
Multienzyme complexes having high
activity against crystalline cellulose, known as a cellulosome, have
been identified and characterized in cellulolytic clostridia such as
Clostridium cellulolyticum (5), Clostridium
cellulovorans (8), and Clostridium
thermocellum (3, 4, 18) and in anaerobic cellulolytic
fungi such as Neocallimastix patriciarum and
Piromyces sp. (29). A common feature of the
clostridial cellulosomes is that they consist of a large number of
catalytic components arranged around noncatalytic scaffolding proteins.
The scaffolding proteins have been identified and termed CipA (or
scaffoldin) and CipB in C. thermocellum (2, 11,
22), CbpA in C. cellulovorans (27), and
CipC in C. cellulolyticum (20). These proteins
consist of multiple noncatalytic domains but do not contain any
catalytic domains (Fig. 1); e.g.,
C. thermocellum CipA comprises a cellulose-binding domain
(CBD) classified in family III, a docking domain, termed dockerin, and
nine cohesin domains (11). Each cohesin domain is a
subunit-binding domain which interacts with a dockerin domain of each
catalytic component. Therefore, cohesin domains are responsible for
integrating catalytic components into the cellulosome (15, 26,
34). C. cellulovorans CbpA is similar to CipA in that
both of them contain a family III CBD and nine cohesin domains,
although the former contains additional repeated domains of unknown
function, termed hydrophilic domains, and does not contain a dockerin
domain (27). The strong cellulolytic activity of the
clostridial cellulosome systems may be ascribed to the function of the
CBD of the scaffolding proteins and/or to the ordered structure of the
cellulosomes, because the activity of the dissociated catalytic
components was shown to be only 25 to 30% of the activity of the
intact cellulosome from C. thermocellum (17).
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FIG. 1.
Schematic diagrams of the scaffolding proteins
responsible for cellulosome assembly. The amino acid sequences of
C. cellulolyticum CipC, C. cellulovorans CbpA,
and C. thermocellum CipA and CipB were derived from their
gene sequences. The complete nucleic acid sequences of C. cellulolyticum cipC and C. thermocellum cipB have not
been reported.
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Clostridium josui is a moderately thermophilic cellulolytic
bacterium (28). Previously, we cloned a cluster of cellulase genes (9), the celB gene encoding endoglucanase
CelB (formerly EG-2), classified into family 8 of glycosyl hydrolases
(12), and two incomplete open reading frames (ORFs), ORF1
and ORF2 upstream and downstream of celB, respectively (Fig.
2). The presence of the dockerin domain
sequences in the amino acid sequences deduced from celB and
ORF1 strongly suggests that C. josui also secretes the
cellulosome into the culture medium, although the C. josui cellulosome has not yet been characterized. The organization of this
gene cluster, ORF1-celB-ORF2, is similar to that of the
cellulase gene cluster, celF-celC-celG, from C. cellulolyticum (5, 9) although the physiological
characteristics of C. josui (28) isolated from
compost in Thailand are significantly different from those of C. cellulolyticum (21) in several points, especially in
optimum growth temperature, i.e., 45°C for the former but 32 to
35°C for the latter. The deduced amino acid sequences of the gene
products of ORF1, celB, and ORF2 exhibit extremely high
sequence identities
89.9, 92.3, and 96.1%with those of CelF, CelC,
and CelG, respectively, from C. cellulolyticum. In C. cellulolyticum, the cipC gene, encoding a scaffolding
protein, was identified upstream of the celF gene and a part
of this gene was cloned by an inverse PCR technique (20).
These findings suggested that the gene encoding a scaffolding protein
of C. josui exists in the region upstream of ORF1.
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FIG. 2.
Restriction maps of pMK-1 and pMK-2 (A) and the gene
cluster of cipA, celD, celB, and
celE (B). Thin lines correspond to vector plasmid DNA.
Arrows indicate DNA fragments used as probes for Southern and Northern
hybridizations.
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In this study, we report cloning and DNA sequencing of the
cipA gene encoding the scaffolding protein and the
celD gene, formerly ORF1, of C. josui. We also
describe the existence of the CelD and CipA proteins as major
components of the C. josui cellulosome.
Cloning and DNA sequencing of the celD and
cipA genes.
Plasmid pUCJ2E is a pUC118 derivative that
contains a 3.8-kb EcoRI fragment (9) carrying the
celB gene along with the two incomplete ORFs of C. josui (Fig. 2). Southern hybridization analysis with the
EcoRI-BamHI fragment of pUCJ2E as a probe
indicated that NheI digestion of C. josui
chromosomal DNA gave a 5.8-kb fragment which was associated with the
probe (data not shown). Therefore, we cloned this fragment into the
XbaI site of pBluescript II KS+ (Stratagene) upon screening
by colony hybridization with the same probe. The recombinant plasmid
obtained was named pMK-1 (Fig. 2). DNA sequencing of the genomic DNA
insert of pMK-1 revealed that the ORF of cipA extended
beyond the 5' end of this DNA fragment, although the full-length
celD gene and a part of cipA encoding the
C-terminal region of CipA were contained in this region. Southern hybridization was carried out again, with the 2.0-kb
NheI-PstI fragment of pMK-1 as a probe, to
determine the restriction sites upstream of the 5.8-kb fragment cloned
in pMK-1. As a result, we expected that a 6.0-kb EcoRI
fragment would contain the full-length cipA gene, and we
isolated this DNA fragment (Fig. 2).
The nucleotide sequences of pMK-1 and pMK-2 were determined with a
Licor (Lincoln, Nebr.) model 4000L automated DNA sequencer
with
appropriate dye primers and a series of the nested deletion
subclones.
The nucleotide sequence reported was determined at
least once in each
direction. Sequence data were analyzed with
GENETYX computer software
(Software Development Co., Ltd., Tokyo,
Japan). Homology searches in
GenBank were carried out with a BLAST
program. Figure
3 shows the complete nucleotide sequence
containing
the full-length
cipA and
celD
structural genes along with their
flanking regions. The
C. josui
cipA gene is the third scaffolding
protein gene whose complete
nucleotide sequence has been determined.
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FIG. 3.
Nucleotide and deduced amino acid sequences of
cipA and celD. The putative promoter and
Shine-Dalgarno (SD) sequences are underlined. The amino acid stretches
marked with a broken line were identified in the cellulosomal proteins
by automated gas-phase sequencing. A palindrome is indicated by arrows
facing each other. The duplicated sequences in the dockerin domain are
boxed.  , boundary between a CBD and a hydrophilic domain;  ,
boundary between a hydrophilic domain and a cohesin domain or between
two contiguous cohesin domains.
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The ORF of
cipA consists of 3,486 nucleotides encoding a
scaffolding-like protein, CipA, of 1,162 amino acids with a predicted
molecular weight of 120,227. The correctness of the position of
the
initiation codon was supported by the N-terminal amino acid
sequence of
one of the major components of the
C. josui cellulosome
(see
below). The putative initiation codon ATG was preceded at
a spacing of
7 bp by a potential ribosome-binding sequence (AGGAGG)
(
14). Possible promoter sequences, TTCAGT
for a
35 region and
AAAAAT for a
10 region with 17 bp between them, were observed;
these are homologous with the consensus
promoter sequences for
the
70 factor found in
Escherichia coli (TTGACA and TATAAT
with 17-bp
spacing) (
25). Downstream of the
termination codon TAA, we found
a 15-bp palindromic sequence
corresponding to an mRNA hairpin
loop, a possible transcription
terminator, with a
G of
30 kcal/mol
(ca.
126 kJ/mol)
(Freier) followed by four T's but with a spacing
of 2 bp.
The ORF of
celD consists of 2,157 nucleotides encoding a
putative cellulase, CelD, of 719 amino acids, which was classified
into
family 48 of glycosyl hydrolases, with a predicted molecular
weight of
80,287. The position of the initiation codon of
celD was
also located on the basis of N-terminal amino acid sequence
analysis of
major components of the cellulosome. The putative
initiation codon ATG
was preceded at a spacing of 6 bp by a potential
ribosome-binding
sequence (AGGAAGG). A possible promoter for
celD,
TTGAAT for a
35 region and TATGGC for a
10
region with a 17-bp
spacing, was found upstream of
celD. The
10 sequence overlaps
the predicted terminator for
cipA. It
is not clear whether transcription
starting from the promoter for
cipA terminates at this putative
terminator or continues to
celD to form a polycistronic mRNA.
Amino acid sequences of CipA and CelD.
The N terminus of the
deduced amino acid sequence of CipA contains a typical signal peptide
characteristic of bacterial extracellular proteins (32).
Comparison of the amino acid sequence of CipA with entries in the
SWISS-PROT and PIR sequence databases revealed that the mature CipA has
a molecular structure similar to the scaffolding proteins reported so
far, C. cellulovorans CbpA (27), C. thermocellum CipA (11), and C. cellulolyticum CipC (20); i.e., it contains a CBD at
its N terminus, followed by a hydrophilic domain and six type I cohesin
domains (Fig. 1). In general, scaffolding proteins, which comprise a
CBD and a number of cohesin domains, are thought to have mainly two
functions: first, they bind a series of catalytic subunits together to
form a complex through cohesin-dockerin interaction (15, 26, 30,
34); second, they cause the complex to adsorb onto cellulose and,
furthermore, may disorder the crystalline structure of cellulose to
produce an easily hydrolyzable substrate through the function of its
CBD (7). In C. josui CipA, cohesin domains are
located in amino acid 284 to 428, 429 to 576, 577 to 724, 725 to 872, 873 to 1020, and 1021 to 1062. Sequence identities between respective
cohesin domains are 63 to 97%. In addition, CipA contains a single
hydrophilic domain, which is conserved in C. cellulovorans
CbpA (27) and C. cellulolyticum CipC
(20) but not in C. thermocellum CipA. Despite the
overall similarity of the scaffolding proteins, C. josui
CipA is different from C. cellulovorans CbpA and C. thermocellum CipA in that the former is considerably smaller than
the others; i.e., the numbers of amino acid residues constituting
C. josui CipA, C. cellulovorans CbpA
(27), and C. thermocellum CipA (11)
are 1,162, 1,848, and 1,854, respectively. Although the full-length
scaffolding protein gene, cipC, from C. cellulolyticum has not been cloned, DNA fragments encoding the N-
and C-terminal regions of CipC were cloned and sequenced
(20). The deduced amino acid sequence of the N-terminal
region of CipC, comprising a succession of a signal peptide, a CBD, and
two cohesin domains, is highly homologous with the corresponding region
of C. josui CipA; the overall sequence identity between them
is 82.5%. On the other hand, comparison of the C-terminal regions of
CipA and CipC revealed an apparent difference (Fig. 1); i.e., the last
(sixth) cohesin domain is connected to another (fifth) cohesin domain
in C. josui CipA, although in CipC the last cohesin domain
is connected to a hydrophilic domain. Furthermore, N-terminal amino
acid sequence analysis of the C. cellulolyticum cellulosomal
proteins showed that CipC is a protein of 160 kDa (10),
which is larger than the predicted molecular mass (120 kDa) of C. josui CipA. These findings indicate that the molecular
architecture of CipA is different from that of CipC. The difference can
be explained by assuming that C. josui cipA arose from a
common ancestral gene of C. josui cipA and C. cellulolyticum cipC through the deletion of the region encoding its C-terminal moiety, since the N-terminal moieties between these scaffolding protein genes are highly conserved.
Sequence analysis of CelD showed that this enzyme consists of a signal
peptide, a family 48 catalytic domain, and a dockerin
domain at its C
terminus. The overall sequence of CelD was highly
homologous with
C. cellulolyticum CelF (91% sequence identity)
(
23) and moderately homologous with
C. thermocellum CelS (58%
sequence identity) (
31). Each
of them is known to be one of
the major catalytic components in the
cellulosome of
C. cellulolyticum or
C. thermocellum.
C. thermocellum CelS is believed to play an
important role in the
efficient degradation of crystalline cellulose,
since purification of
components from a cellulosome fraction revealed
that the combination of
two proteins, noncatalytic scaffolding
protein S1, now known as CipA,
and exoglucanase S8, known as CelS,
was able to completely solubilize
Avicel but at very low rates
(
33).
C. cellulolyticum CelF, which has recently been defined
as a
processive endocellulase, also showed hydrolytic activity
toward Avicel
by itself (
24). Presumably, enzymes of family
48, including
C. josui CelD, play important roles in clostridial
cellulolytic systems.
The existence of a dockerin domain in CelD suggested that it is a
member of the cellulosomal proteins. Dockerin domains, which
consist of
two duplicated sequences, each of about 22 amino acid
residues, are
conserved in the components of cellulosomes from
C. cellulolyticum,
C. cellulovorans,
C. josui,
and
C. thermocellum.
As shown in Fig.
4, the dockerins of
C. josui
CelB and CelD are
similar to those of the
C. cellulolyticum
cellulases rather than
those of
C. thermocellum enzymes such
as CelS, which has an amino
acid sequence typical of this bacterium;
i.e., although a DAL
or DAI motif is conserved in the dockerins from
C. josui and
C. cellulolyticum, this motif is
replaced by NST in those from
C. thermocellum. Recently,
Pagès et al. (
19) have shown that the
cohesin-dockerin
interaction in the
C. cellulolyticum and
C. thermocellum cellulosomes is a species-specific phenomenon, and
they have predicted
that four amino acid residues, which comprise a
repeated pair
(AL, AI, or ST) located in the DAL, DAI, or NST motif,
are critical
to binding specificity as a recognition code.
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FIG. 4.
Alignment of dockerin domains of CelB and CelD of
C. josui (Cj); CelA, CelC, CelD, CelE, CelF, CelG, CelH, and
CelJ of C. cellulolyticum (Cc); and CelS of C. thermocellum (Ct). Sequences of C. cellulolyticum
proteins are from reference 19. Asterisks indicate
amino acid residues involved in calcium-binding. The NST motifs in
C. thermocellum CelS are double underlined. Residues
suspected of serving as selectivity determinants are indicated by pound
signs (#). Amino acids which are conserved or have similar chemical
properties (I, L, M, V, K, R, S, and T) in at least 7 of 10 C. josui and C. cellulolyticum sequences are printed in
white on black. Numbers refer to amino acid residues at the start of
the respective lines; all sequences are numbered from Met-1 of the
peptide. Complete amino acid sequences of CelE, CelH, and CelJ are not
known (19).
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Isolation of the cellulosome from culture of C. josui
and identification of CipA and CelD as major components of it.
C.
josui was cultivated at 45°C for 7 days in 500 ml of GS medium
(13) containing 0.5% ball-milled cellulose as a carbon source. The cells were removed by centrifugation at 10,000 × g for 10 min. The supernatant was concentrated 50-fold by
ultrafiltration through an XM50 membrane (nominal molecular weight
cutoff of 50,000) (Amicon). A portion (3 ml) of the concentrated
culture supernatant was subjected to HiPrep Sephacryl S-300HR column
(16/60) (Amersham Pharmacia Biotech) chromatography with 50 mM Tris-HCl
buffer (pH 7.5) containing 12 mM CaCl2 and 500 mM NaCl as
an eluent. A fraction containing protein complexes with a high
molecular mass (ca. 700 kDa) was analyzed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) (16). All
protein samples were prepared by heating at 100°C for 3 min in SDS
gel loading buffer to denature the proteins. As shown in Fig.
5, a large number of proteins were visualized by Coomassie brilliant blue staining, and many of them showed endoglucanase activity in the gel containing
carboxymethylcellulose as a substrate, by the activity staining assay
(1). When the proteins in this fraction were incubated with
insoluble cellulose, all of the proteins were bound to the cellulose
(data not shown). These results suggest that an extracellular
multienzyme complex, cellulosome, may be present in the supernatant of
the C. josui culture. After separation of the cellulosomal
proteins by SDS-PAGE, the proteins were blotted onto an Immobilon P
transfer membrane (Millipore) and subjected to automated amino acid
sequencing (model 476A protein sequencer, PE; Applied Biosystems). The
N-terminal amino acid sequences of two proteins with molecular masses
of 115 and 70 kDa were identified as ADTGVISVQF and
AASPVNKVYQERFESMYNKI, respectively, which were found in the
amino acid sequences deduced from the cipA and
celD genes. These results suggest that CipA and CelD may be
components of a C. josui cellulosome and confirm that
C. josui CipA is quite smaller than C. cellulolyticum CipC.
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FIG. 5.
SDS-PAGE analysis of the cellulase complex from culture
fluid of C. josui. Lane 1, Coomassie brilliant blue
staining; lane 2, activity staining with carboxymethylcellulose as a
substrate; lane M, protein molecular mass standards. Sizes are shown on
the left.
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Taxonomic relatedness between C. josui and C. cellulolyticum.
High-level resemblance in the organization of the
cellulase gene clusters of C. josui and C. cellulolyticum allows us to expect close taxonomic relatedness
between them despite the differences in optimum temperature for their
growth, i.e., 45°C for C. josui and 32 to 35°C for
C. cellulolyticum. Therefore, we determined the nucleotide
sequence of the 16s ribosomal DNA (rDNA) (registered with accession no.
AB011057) of C. josui by amplification of rDNA by PCR and
direct sequencing of the PCR products. Since C. cellulolyticum was placed in cluster III of the genus
Clostridium (6), which consists of cellulolytic
species, the 16s rDNA sequences of C. josui and the species
of cluster III were aligned and analyzed by the GENETYX computer
program. As shown in Fig. 6, the
dendrogram places C. josui in cluster III at a position
close to C. papyrosolvens and C. cellulolyticum,
suggesting that the cellulase gene clusters arise from a common
ancestor with some evolutionary modifications.
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FIG. 6.
Dendrogram of 16s rDNA sequences of cluster III
Clostridium species and C. josui constructed by
the GENETYX program. The accession numbers for the rDNA sequences are
as follows: C. papyrosolvens, X71852; C. cellulolyticum, X71847; C. cellobioparum, X71856;
C. termitidis, X71854; C. aldrichii, X71846;
C. thermocellum, L09173; C. stercorarium, L09174;
C. thermolacticum, X72870. Bar, 1% sequence divergence.
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Nucleotide sequence accession number.
The nucleotide sequence
data reported herein appear in the DDBJ, EMBL, and GenBank nucleotide
sequence databases under accession no. AB004845.
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ACKNOWLEDGMENTS |
This research was partly supported by the Bio-Oriented Technology
Research Advancement Institution (BRAIN) of Japan. We thank Yasuaki
Okamoto of the Tateyama Brewery Co., Ltd., for financial support.
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FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Bioresources, Mie University, 1515 Kamihamacho, Tsu 514-8507, Japan.
Phone: (81) 59-231-9621. Fax: (81) 59-231-9684. E-mail:
sakka{at}bio.mie-u.ac.jp.
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Journal of Bacteriology, August 1998, p. 4303-4308, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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