Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein in Clostridium cellulolyticum: Comparison of Various ...
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JOURNAL OF BACTERIOLOGY, Mar. 1999, p. 1801–1810 Vol. 181, No. 6
0021-9193/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Sequence Analysis of Scaffolding Protein CipC and ORFXp,
a New Cohesin-Containing Protein in Clostridium cellulolyticum:
Comparison of Various Cohesin Domains and Subcellular
Localization of ORFXp
SANDRINE PAGÈS,1 ANNE BÉLAÏCH,1* HENRI-PIERRE FIEROBE,1 CHANTAL TARDIF,1,2
CHRISTIAN GAUDIN,1 AND JEAN-PIERRE BÉLAÏCH1,2
Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique,1 and
Université de Provence,2 Marseilles, France
Received 8 September 1998/Accepted 6 January 1999
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The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial
sequence was published earlier (S. Pagès, A. Bélaı̈ch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P.
Bélaı̈ch, J. Bacteriol. 178:2279–2286, 1996; C. Reverbel-Leroy, A. Bélaı̈ch, A. Bernadac, C. Gaudin, J. P.
Bélaı̈ch, and C. Tardif, Microbiology 142:1013–1023, 1996), was completely sequenced. The corresponding
protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain
(HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and
a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains.
In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein,
called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed
that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction.
Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same
dockerins and have similar affinity for CelA (Ka 5 4.8 3 109 M21) whereas the cohesin from ORFXp, although
it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 3
108 M21). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.
Clostridium cellulolyticum, a mesophilic anaerobic bacte- which the enzymatic components interact with CbpA remains
rium, secretes cellulolytic complexes called cellulosomes, unclear or at least would appear to involve a different mech-
which have a molecular mass of about 600 kDa (17, 31). These anism, since dockerinless enzyme, EngD, was found to be part
complexes are composed of at least 13 enzymes called cellu- of the cellulosome (12, 13, 45–47). More recently, the cipA
lases, as well as a large scaffolding protein of about 160 kDa gene, encoding the cellulosome scaffolding protein CipA from
that is devoid of catalytic activity, called CipC (for “cellulo- C. josui (a bacterium related to C. cellulolyticum as demon-
some-integrating protein”) (17). It has been previously shown strated by comparison of 16S rDNA), was sequenced. The
that the assembly of the cellulosome is due to strong interac- deduced amino acid sequence of CipA reveals that this scaf-
tions between the cohesin domains of CipC and the dockerin folding protein contains six cohesin domains and, as in CbpA
domains of the catalytic subunits (35). This organization is from C. cellulovorans, does not contain a type II dockerin
similar to that of the cellulosome produced by C. thermocellum domain, as found in the scaffolding protein CipA from C.
(1, 2, 7, 8, 24), for which it has also been demonstrated that the thermocellum.
cohesin domains of the scaffolding protein (CipA) act as re- The cellulolytic complex from C. papyrosolvens C7 was char-
ceptors for the dockerin domains of the enzymatic components acterized biochemically some years ago (37). Seven celluloso-
(14, 30, 43, 49, 50, 51). Furthermore, the C terminus of CipA mal fractions ranging from 500 to 600 kDa were separated by
contains a slightly divergent dockerin domain (type II), which gel filtration chromatography and analyzed by sodium dodecyl
interacts with a second class of cohesin domains present in at sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The
least three cell surface proteins (SdbA, OlpB, and ORF2p) (15, only subunit present in all the fractions was a 125,000 Mr
18, 26, 27, 29). It is believed that this second kind of cohesin-
glycoprotein with no detectable enzymic activity. The latter
dockerin complex is involved in the attachment of cellulosome
could be cellulosomal scaffolding protein, but unfortunately
to the cell surface whereas the interaction between the type I
the sequence of the gene is not still available.
cohesin and dockerin domains involves only the cellulosome
The partial sequence (59 and 39 extremities) of cipC has
assembly. Another cellulosome-producing clostridium, C. cel-
lulovorans, has also been extensively studied, and although the already been published (35, 40). In the present study, the
scaffolding protein CbpA contains cohesin domains similar to complete sequence of cipC was determined and the amino acid
those of C. thermocellum and C. cellulolyticum, the way in sequence of the corresponding protein was analyzed and com-
pared to those of CipA and CbpA. In addition to the cellulose
binding domain (CBD) and two hydrophilic domains, CipC
contains only eight cohesin domains. The first seven cohesin
* Corresponding author. Mailing address: Bioénergétique et Ingé-
niérie des Protéines, Centre National de la Recherche Scientifique, 31 domains are highly homologous, while the last (cohesin 8),
chemin Joseph Aiguier, BP 71, 13402 Marseilles Cedex 20, France. located at the C terminus, displays a much lower degree of
Phone: (33) 91 16 40 70. Fax: (33) 91 71 33 21. E-mail: abelaich@ibsm homology to the other seven. Furthermore, the sequencing of
.cnrs-mrs.fr. a new gene, ORFX, encoding ORFXp, located in the C. cellu-
18011802 PAGÈS ET AL. J. BACTERIOL.
lolyticum gene cluster, revealed that the protein encoded by the amplification and introduction of NdeI (59) and XhoI (39) sites. The reverse
this gene is composed of three domains, the last one being a primer was also designed to graft five histidines at the C terminus of cohesin X
(the His codons are underlined). The amplified fragment was digested with NdeI
cohesin domain, called cohesin X. and XhoI and cloned into plasmid pET22b(1) linearized with the same restric-
By using surface plasmon resonance (BIAcore) and Western tion endonucleases. The resulting plasmid, pETCX, was checked by sequencing
blot procedures, the recognition patterns of cohesin domains 8 as described above and used to transform E. coli BL21(DE3).
and X were determined and compared to that of cohesin do- Purification of recombinant proteins. miniCipC8 and miniCipCX were puri-
fied as described by Pagès et al. (35).
main 1. The subcellular localization of ORFXp was also de- Cells harboring plasmid pETCX were grown in 1 liter of Luria-Bertani me-
termined. On the basis of these data, new hypotheses on the dium at 37°C to an optical density of 1.5 at 600 nm. IPTG at a final concentration
assembly of the C. cellulolyticum cellulosome and the role of of 0.5 mM was then added to the culture, which was continued for 3 h. The cells
ORFXp during this step are discussed. were then harvested by centrifugation (10 min at 5,000 3 g), and resuspended in
40 ml of 0.1 M NaCl–30 mM Tris-HCl (pH 8.0). The cells were broken by passing
the suspension in a French press. DNase I (5 mg/ml) was added to the crude
MATERIALS AND METHODS extract, and this solution was incubated at 4°C for 30 min. The solution was then
Bacterial strains, plasmids, and growth conditions. Escherichia coli loaded onto 5 ml of Ni-nitrilotriacetic acid resin (Qiagen) equilibrated in the
BL21(DE3) was used as the host for pET22b(1) (Novagen) derivative expres- same buffer. The resin was washed with 30 mM imidazole–30 mM Tris-HCl (pH
sion vectors (pETCip1, pETCip8, and pETCipX) (34). E. coli was grown at 37°C 8), and cohesin X was eluted with a linear gradient from 30 to 250 mM imidazole
in Luria-Bertani medium supplemented with ampicillin (100 mg/ml) when re- (two elutions with 200 ml each). Analysis by SDS-PAGE of the cohesin X-con-
quired. C. cellulolyticum ATCC 35319 was grown anaerobically at 32°C on basal taining fractions indicated that no further purification was required. The frac-
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medium supplemented with either cellobiose (2 g/liter) (Sigma) or MN 300 tions were pooled (30 ml) and dialyzed overnight at 4°C against 5 liters of 20 mM
cellulose (5 g/liter) (Serva) as the carbon and energy source (20). Tris-HCl (pH 8). The sample was used as stock solution.
DNA manipulation. Chromosomal DNA was obtained from C. cellulolyticum Antibody preparation. Polyclonal antibodies against miniCipC1 and miniCipX
as described by Quiviger et al. (38). Large-scale and small-scale plasmid purifi- were raised in rabbits by subcutaneous injection of the pure proteins. Antisera
cations were performed by the alkali lysis method (33) with the Qiagen kit. were stored at 220°C with 0.3% NaN3. Antisera raised against miniCipC1 and
Digestion was performed as specified by the manufacturer. miniCipX were preadsorbed with E. coli BL21DE3[pET22b(1)] extract. The
DNA sequencing of cipC. The 59 and 39 ends of cipC have already been antiserum raised against miniCipX was further preabsorbed with pure recombi-
sequenced (35, 40). The internal fragment of cipC (3,462 bp) was amplified nant protein called miniCipC0 (CBD-HD1) (34). The antiserum raised against
by PCR with the two synthesized oligonucleotides ciph1 (59 GTA-GGA- miniCipC1 recognizes the polypeptides miniCipC1 (CBD-HD1-C1) and miniCipC0
GGA-ACT-CTT-GCT-TA 39) and ciph2 (59 TCA-AAA-GAT-GCA-GTT-GAA- (CBD-HD1), the CBD, and the native protein CipC from C. cellulolyticum. This
GGA-GT 39). These two primers were designed to bind DNA regions encoding antibody preparation was called Ab a CipC. The antiserum raised against
the two hydrophilic domains. The PCR fragment was cloned into pUC18, and the miniCipX was preabsorbed with miniCipC0 to avoid cross-reactions with the
extremities of the insert were sequenced with the M13 forward and M13 reverse CBD or the hydrophilic domain. This antibody preparation was called Ab a CX.
primers. This fragment was subsequently digested with HindIII, and the resulting Fractionation of C. cellulolyticum cultures. The fractionation method was de-
fragments were cloned into pUC18 and sequenced. rived from the procedure described by Lemaire et al. (29). The preliminary steps
Expression and purification of recombinant proteins. Cohesin domain 1, of the fractionation were different depending upon the growth substrate. For
present in the polypeptide miniCipC1 (CBD-HD1-C1) (35), was replaced by cellobiose, 1 liter of C. cellulolyticum culture, grown to an optical density at 450
cohesin domain 8. The pCip12A plasmid (34) containing the DNA fragment nm of 1.2, was centrifuged. The culture supernatant was concentrated on a
encoding miniCipC1 and cloned into the p-Mos-Blue-T-vector was digested with Millipore polysulfone membrane (10-kDa cutoff) to 10 ml. It was called the F0
SmaI and SalI, and the smaller fragment, encoding miniCipC1 (containing a fraction. Cells were washed with buffer A (50 mM phosphate, 150 mM NaCl [pH
NdeI site upstream of the coding sequence and a SalI site downstream a codon 7.5]), resuspended in 10 ml of the same buffer, and broken in a French press. This
stop), was cloned into the pUC18 vector. The resulting plasmid, pUCC12, was sample was called fraction F1. The F1 fraction was centrifuged at 1,000 3 g for
digested with EcoRV and SalI to remove the part of the DNA fragment encoding 10 min. Then the pellet, containing the intact cells, was discarded, and the
cohesin domain 1. Two synthetic primers, cip 810 (59 GGG-AAT-TCC-ATA- supernatant was centrifuged for 30 min at 46,000 3 g. The supernatant was called
TGT-CGC-GAC-CAG-TTC-TGA-C 39) and cip 811 (59 ACG-CGT-CGA-CTT- fraction F2. This fraction contains the cytoplasmic soluble proteins. The pellet
AAT-TAA-GTT-TTG-CAC-TTC-C 39), having partial homology to the 59 and 39 was resuspended in 10 ml of buffer B (50 mM phosphate, 150 mM NaCl, 1% SDS
DNA regions, respectively, of the DNA fragment encoding cohesin domain 8 [pH 7.5]). This sample was called fraction F3. This fraction contains the mem-
were synthesized. cip 810 created a NruI site upstream of the coding sequence, brane proteins and the proteins associated with the cell surface. Fraction F3 was
and cip 811 introduced a SalI site and a stop codon downstream of the coding heated at 100°C in a water bath for 15 min and centrifuged for 30 min at 46,000 3
sequence. The amplified fragment (encoding cohesin domain 8) was digested g. This treatment removed proteins that were noncovalently associated with the
with NruI and SalI and cloned into pUCC12 digested with EcoRV and SalI. The cell surface. The supernatant was called fraction F4. The pellet was resuspended
resulting plasmid, pUCC9, was digested with NdeI and SalI, and the DNA in 10 ml of buffer B (fraction F5). After being heated at 100°C for 15 min,
fragment encoding a polypeptide called miniCipC8 (CBD-HD1-C8) was cloned fraction F5 was centrifuged for 30 min at 46,000 3 g. The supernatant was called
into NdeI-SalI-linearized pET22b(1). The resulting plasmid, pETCip8, was ver- fraction F6, and the pellet resuspended in 10 ml of buffer B was called fraction
ified by DNA sequencing (Genome Express Society) with a Perkin-Elmer 373 F7.
fluorescence sequencing apparatus (Applied Biosystems dye terminator meth- When C. cellulolyticum was grown on cellulose (4 days), the cells (from 1 liter
od). of culture) were collected by filtration through a 3-mm-pore-size filter (glass
As described above, cohesin domain 1 present in the polypeptide miniCipC1 microfiber filter GF/D; Whatman). The culture was filtered and washed on the
was replaced by the cohesin domain present in the protein ORFXp. Two syn- filter twice with 50 ml of 50 mM phosphate buffer (pH 7.5) to remove the cells
thetic primers, cip 298 (59 GGG-TTT-AAA-ACT-CCG-GGC-GGA-GAG-G 39) from the cellulose. The eluted fraction containing the cells and the culture
and cip 767 (59 C-ACC-GTC-GAC-TTA-TTT-AAC-TGT-TAT-CTC-ACC 39), medium was centrifuged. The supernatant constituted fraction F0 as described
having partial homology to the 59 and 39 DNA regions, respectively, of the DNA above, and the pellet was called fraction F1. The same procedure was used to
fragment encoding cohesin domain X were synthesized. cip 298 created a DraI obtain fractions F2 through F7. The cellulosome (fraction Fc) was eluted from
site upstream of the coding sequence, whereas cip 767 created a SalI site and a the cellulose with water as described by Gal et al. (17).
stop codon downstream of the coding sequence. After amplification, the DNA SDS-PAGE. SDS-PAGE was performed by the procedure developed by
fragment (encoding cohesin domain X) was digested with DraI and SalI and Laemmli (23) with precast 4 to 20% polyacrylamide (Novex) gels.
cloned into pUCC12 digested with EcoRV and SalI. The resulting plasmid, Detection of ORFXp in subcellular fractions of C. cellulolyticum. Immunode-
pUCCX, was digested with NdeI and SalI, and the DNA fragment encoding a tection of ORFXp was performed by Western blotting as previously described by
polypeptide called miniCipX (CBD-HD1-CX) was cloned into pET22b(1). The Gal et al. (17), using the Ab a CX sample.
resulting plasmid, pETCipX, was verified by sequencing as described above. Biotinylation of proteins and biotin-labelled detection. Biotinylation of
pETCip8 and pETCipX were used to transform E. coli BL21(DE3) strains miniCipC8 and miniCipX was performed with biotinyl-N-hydroxysuccinimide
containing inducible T7 polymerase under the control of the lac promoter. The ester as described by Bayer and Wilcheck (6) (biotin-labelling kit; Boehringer
transformed cells were grown at 37°C on Luria-Bertani medium supplemented Mannheim) as specified by the manufacturer.
with ampicillin to an optical density at 600 nm of 2. The expression was triggered Study of the cohesin-dockerin interaction. The interaction of the cellulosomal
by the addition of 400 mM isopropyl-b-D-thiogalactoside (IPTG), and the cells subunits with miniCipC1, miniCipC8, miniCipCX, and cohesin X was examined
were grown (at 37°C) for an additional 3 h. by using the biotin-labelled mini-scaffolding proteins as probes against the dif-
To overproduce cohesin X, the DNA region encoding cohesin domain X was ferent polypeptides present in cellulosome fraction Fc.
amplified by PCR from C. cellulolyticum chromosomic DNA. Forward primer 59 The kinetic parameters of the interaction between the recombinant cellulase
G-GTG-GGT-CAT-ATG-GAT-AAA-ACT-CCG-GGC-GGA-GAG 39 and re- CelA and the recombinant polypeptides described above were determined by
verse primer 59-C-CAG-CTC-GAG-CTA-GTG-GTG-GTG-GTG-GTG-TTT- using the BIAcore procedure. The biotinylated miniCipC1, miniCipC8, miniCipCX,
AAC-TGT-TAT-CTC-ACC-C 39, which have partial homology to the 59 and 39 or recombinant cohesin X was coupled to a streptavidin-dextran layer on the
extremities, respectively, of the DNA region encoding cohesin X, were used for surface of the sensor chip. Biotinylated recombinant proteins were injected forVOL. 181, 1999 SEQUENCES OF CipC AND ORFXp IN C. CELLULOLYTICUM 1803
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FIG. 1. Schematic representation of CipC from C. cellulolyticum, CipA from C. thermocellum, and CbpA from C. cellulovorans. (A) The positions of the two
oligonucleotides used for the amplification of the internal fragment of cipC are indicated by arrows. (B) The signal sequences, CBDs, hydrophilic domains, and cohesin
domains are shown. The percent identity between cohesin domains is indicated in each box. Cohesin domains 1, 3, and 6 were used as the reference in CipC, CipA,
and CbpA, respectively.
120 s, resulting in approximately 750 resonance units of immobilized protein. The open reading frame (ORF) therefore encodes a polypeptide
The flow cell was equilibrated with 10 mM CaCl2–0.005% surfactant P20 (Phar- containing 1,547 amino acids (aa). As previously described by
macia)–50 mM Tris-maleate buffer (pH 6.5) at a flow rate of 25 ml/min. The
ligand (CelA) was diluted in the same buffer and allowed to interact with the Pagès et al. (35), the ORF begins by encoding a peptide signal
sensor surface by a 300-s injection. In all cases, three different concentrations of sequence. The N terminal sequence of the mature protein was
CelA ranging from 2.5 to 25 nM were injected. The resulting sensorgrams were previously determined by Gal et al. (17). The mature protein
evaluated by using the biomolecular interaction analysis evaluation software contains 1,519 aa with a calculated molecular mass of 155,726 Da,
(Pharmacia) to calculate the kinetic constants of the complex. Control experi-
ments were performed by injections of CelA directly onto the streptavidin- in agreement with the value of 160 kDa previously established by
dextran surface and by injection of a truncated form of the cellulase, missing the SDS-PAGE analysis of a cellulosomal fraction (17). A stretch of
dockerin domain on cohesin X or miniCipC1. 700 bp upstream of the initiation codon was sequenced, and no
Glycoprotein detection. The glycosylated protein was detected as specified by ORF was found in this region, thus indicating that cipC is the first
the manufacturer: the glycoprotein detection system was from Amersham, and
the transfer was performed on Ba83 nitrocellulose membrane (Schleicher & gene of a large cluster including celF, celC, celG, and celE (16).
Schuell). Like the other two scaffolding proteins, CipA from C. ther-
Nucleotide sequence accession numbers. The entire nucleotide sequence of mocellum and CbpA from C. cellulovorans, CipC is a multido-
cipC has been submitted to GenBank and has been assigned accession no. main protein (18, 46). The molecular organization of the three
U40345. The nucleotide sequence of ORFX has been submitted to GenBank and
has been assigned accession no. AF081458. scaffolding proteins is compared in Fig. 1B. CipC is composed
of a signal sequence, a type III CBD (subfamily a), two hydro-
philic domains, and eight cohesin domains separated by short
RESULTS linker sequences. Compared to CipA, the most striking feature
Primary-structure analysis of the scaffolding protein CipC. of CipC and CbpA is the lack of type II dockerin domains. In
The cipC coding sequence of 4,742 nt was determined. The C. thermocellum, this domain is involved in cell surface attach-
internal DNA fragment of cipC contains several repeats with a ment of the cellulosome (26, 27).
high degree of similarity (about 98%). To check that this in- The internal degree of identity between cohesin domain 1
ternal fragment obtained by PCR (Fig. 1A) has the appropri- and the other cohesin domains of CipC was determined (Fig.
ate size, Southern blot experiments were performed with an 1B). This domain is 95 to 87% identical to cohesin domains 2
internal EcoRV-XmnI 2,741-nt probe obtained from this PCR to 7. Cohesin domain 8, which possesses only 60% identity to
fragment. The genomic DNA was digested with Asp718-HindIII, cohesin 1, is the most divergent. A similar observation was
Asp718-EcoRV, Asp718-BamHI, and XmnI. In each case, the size made for CipA from C. thermocellum (44) and CbpA from C.
of the fragments detected by the probe corresponded to the cellulovorans (11), as illustrated in the phylogenetic tree (Fig.
theoretical values calculated from the sequence (data not shown). 2). It would appear that in the scaffoldins known to date, there1804 PAGÈS ET AL. J. BACTERIOL.
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FIG. 2. Phylogenetic tree of cohesin domains from CipC, CipA (C. thermocellum), and CbpA. The phylogenetic tree was constructed with the program AllAll
accessible on the Computational Biochemestry Research Group server from E.T.H. Zurich, Switzerland. The length of each branch is proportional to the evolutionary
distance between the nodes. The small black circle indicates the weighted centroid of the tree. The distances are expressed in pam (percent average mutation). For
each scaffolding protein, the cohesin are numbered.
is a large group of highly homologous cohesins as well as at 19). Similar hydrophilic domains have been found in nonscaf-
least one cohesin domain, always located at one extremity of folding proteins; two domains have been identified in Cel5
the protein, with significant sequence differences compared to from Bacillus lautus and in CelZ from C. stercorarium, and one
the internal cohesin domains. In this respect, CbpA from C. has been identified in CelY from the latter organism (10, 21).
cellulovorans (11) is the most remarkable since, as shown in Comparison of the interaction of cohesin domains 1 and 8
Fig. 2, cohesin domain 9 (located at the C terminus) is more with cellulosomal subunits. It has been demonstrated that
divergent compared to the other cohesin domains. The phylo- cohesin domain 1 of CipC is a receptor domain for catalytic
genetic tree, however, also indicates that these “divergent” subunits, and the dissociation constant of the miniCipC1-CelA
cohesin domains resemble the other cohesin domains of the complex has been measured (35). In view of the high identity,
same scaffolding more closely than they resemble any cohesin one might suppose that cohesins 2 to 7 recognize the dockerin
domain of another scaffoldin. It is also clear from Fig. 2 that in domains with an affinity similar to that of cohesin domain 1.
terms of similarity, the cohesin domains of CipC are more Since the degree of identity between cohesin domains 1 and 8
homologous to those of CbpA than to those of CipA, even
though sequencing of the gene encoding 16S rRNA (16S
rDNA) indicates that C. cellulolyticum is more closely related
to C. thermocellum than to C. cellulovorans (39). CipC contains
two hydrophilic domains (HD1 and HD2). These two domains
have only 56% similarity, but two short stretches of 18 and 21
aa are highly conserved (83 and 86%, respectively) (Fig. 3).
The role of these two domains remains unknown. The hydro-
philic domains found in CbpA have significant similarities to
HD1 and HD2 (46). A lower degree of similarity between HD1 FIG. 3. Alignment of CipC hydrophilic domains HD1 and HD2. The con-
and HD2 and the hydrophilic domain of CipA was found (4, served amino acids are in gray boxes.VOL. 181, 1999 SEQUENCES OF CipC AND ORFXp IN C. CELLULOLYTICUM 1805
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FIG. 4. Diagram of the recombinant proteins used. Dark gray boxes indicate CBD, light gray boxes indicate hydrophilic domains, and white boxes represent cohesin
domains. Small circles represent the His tag. The underlined residues are the first and last residues of the cohesin domain identified on the basis of the available crystal
structures and sequence comparisons.
drops to 60%, it was of interest to compare the recognition pared to that of cohesin domain 1. As described previously
pattern and the kinetic parameters of the formation of the (17), about 13 bands (94, 89.6, 80.6, 77.9, 72.6, 67.7, 58.9, 54.2,
complex with CelA of these two cohesin domains. 53, 49, 44.5, 43, and 29.5 kDa) yielded a positive signal with
(i) Mini-scaffolding protein constructions. To facilitate both miniCipC1 and no significant difference was observed when
the purification and the comparisons of the binding abilities of miniCipC8 was used as the probe (Fig. 5). Determination
cohesin domains 1 and 8, the cohesin domain 1 in miniCipC1 of the kinetic parameters (association rate constant, kon, and
was replaced by the cohesin domain 8 (Fig. 4A and B). The dissociation rate constant, koff) of the miniCipC1-CelA and
engineered polypeptide was called miniCipC8. This replace- miniCipC8-CelA interaction confirmed that CelA interacts
ment was performed by taking into account the recent deter- with these two domains with the same, very high affinity (Table
mination of the three-dimensional structures of cohesin do- 1), in spite of the sequence differences between the two cohe-
mains 1 (44) and 7 (48) from CipA. These domains form a sin domains. These results suggest that, as proposed by Yaron
nine-stranded b sandwich with a “jelly-roll” topology. In view et al. (51) for the cellulosome of C. thermocellum, incorpora-
of the high sequence homologies among the cohesin domains tion of the cellulosomal subunits into the cellulosome in C.
of C. thermocellum, C. cellulolyticum, and C. cellulovorans, it is cellulolyticum also seems to be a nonselective process, i.e., that
very likely that they have the same overall structure. The oli- any cellulase could interact randomly along the scaffolding
gonucleotides designed for amplification of the DNA fragment protein. A higher Ka value (Ka 5 4.8 3 109 M21) was found for
encoding cohesin domain 8 were therefore chosen as function the miniCipC1-CelA complex in the present study than was
of the known structures. The recombinant cohesin domain 8 proposed elsewhere (1.4 3 108 M21) (35). This is due to the
thus possesses the amino acids involved in the first b strand presence of 10 mM CaCl2 in the running buffer. During the
with respect to a correct folding of this domain (Fig. 4A and first experiments, no calcium was added, and since the running
B). buffer was 50 mM KH2PO4–K2HPO4, the available calcium
(ii) Study of the interaction. The biotinylated miniCipC1 concentration was likely to be very low. This 34-fold improve-
and miniCipC8 were used as a probe for the different cellulo- ment in Ka highlights the importance of calcium in the cohesin-
somal subunits, separated by SDS-PAGE. Formation of the dockerin interactions.
complex was visualized with a streptavidin-peroxidase conju- A new cohesin-harboring protein. In C. thermocellum, two
gate. The recognition pattern of cohesin domain 8 was com- genes encoding proteins involved in the cell surface attach-1806 PAGÈS ET AL. J. BACTERIOL.
FIG. 5. Recognition of SDS-PAGE-separated cellulosomal components by
recombinant cohesin domains 1 and 8 of CipC. SDS-PAGE-separated samples
were blotted onto nitrocellulose and probed with the biotinylated miniscaffolding
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protein desired. Blots were developed with streptavidin-peroxidase conjugate.
Two different concentrations of cellulosomal preparation were used, 3 mg (lanes
1) and 6 mg (lanes 2). (A) SDS-PAGE-separated cellulosomal subunits probed
with miniCipC1. (B) SDS-PAGE-separated cellulosomal subunits probed with
miniCipC8. The presence of the two major cellulases of the C. cellulolyticum
cellulosome, CelE and CelF, previously identified (17), are indicated.
ment of the cellulosome form a cluster with the cipA gene (15).
These genes encode a membrane-associated protein contain-
ing a cohesin domain II able to interact with the type II dock-
erin domain of CipA (15, 26, 27, 29). Analysis of the primary
sequence of CipC indicates that no such domain is present in
CipC. Nevertheless, in the large cluster of gene including cipC,
celF, celG, and celE a new ORF, encoding a protein harboring
a cohesin domain, was discovered. In addition, this new ORF
is located between two cellulase genes (16), which strongly
suggests an involvement at some stage of cellulose degradation
and/or cellulosome assembly.
The initiation codon, ATG, of this new ORF is located 169
FIG. 6. Nucleotide and deduced amino acids sequences of the ORFX gene,
bp downstream of the celE stop codon and is preceded by a encoding the predicted signal sequence (underlined), a putative linker domain,
typical Shine-Dalgarno sequence. The entire sequence of 693 and a cohesin X domain (boldface type). The putative Shine-Dalgarno ribosome
nucleotides codes for a protein made up of 229 aa with a binding site is underlined upstream of the ATG codon.
calculated molecular mass of 23,855 Da. Based on its sequence
analysis, this protein can be divided into three distinct do-
mains. The first domain, of about 25 aa, located at the NH2 (i) Is the corresponding protein produced by the bacterium?
terminus of the protein, is likely to be a typical signal sequence (ii) Where is it located in the bacterium? (iii) Which kind of
of a gram-positive organism. It is followed by a P-T-S rich dockerin domain recognizes the cohesin domain of ORFXp?
region of 58 aa, similar to the linker regions found in many To answer these questions, a new mini-scaffolding protein
cellulases and xylanases, and a putative cohesin domain at the was constructed. Cohesin domain 1 of miniCipC1 was replaced
C terminus of the protein (Fig. 6). The sequence of this last by the cohesin-like domain present in ORFXp while preserving
domain was compared with the sequences of two type I co- a theoretical correct folding of this domain (Fig. 2C). This new
hesins (cohesin domains 1 and 8 from CipC) and with a type II recombinant protein, called miniCipCX, was used to obtain
cohesin present in SdbA from C. thermocellum (27). This co- antibodies (Ab a CX) and to prepare a biotinylated probe for
hesin domain, called cohesin domain X, is 35 and 28% iden- binding experiments.
tical to cohesin domains 1 and 8 from CipC, respectively, and Subcellular localization of ORFXp. The presence of CipX in
has only 15% identity to cohesin domain II from SdbA. The various fractions from a cellulose-grown culture was detected
presence of this new ORF raises at least three basic questions. by Western blotting with Ab a CX, which interacts specifically
with cohesin domain X (Fig. 7A). The fractionation procedure
used is described in Materials and Methods. The protein was
TABLE 1. Recombinant cohesin domains and CelA associations not detected in fractions Fc and F0. ORFXp was detected in
and kinetic parametersa fractions F1 to F4 but was found mainly in fractions F1, F3, and
F4, suggesting that it could be cell associated (Fig. 7B). The
Domain kon (M21 z s21) koff (s21) Ka (M21)
presence of the protein in the cytoplasmic fraction (F2) could
miniCipC1-CelA 1.5 3 106 3.1 3 1024 4.8 3 109 be explained by a release of part of the protein from the
miniCipC8-CelA 1.6 3 106 4.2 3 1024 3.8 3 109 membranes during the French press procedure. Indeed, it has
CohXr-CelA 1.3 3 106 5.0 3 1023 2.6 3 108 been previously reported (29, 42, 43) that some of the cell-
a
Kinetic constants were determined from sensorgrams as described in Mate-
associated proteins discovered in C. thermocellum, i.e., OlpA,
rials and Methods. The apparent equilibrium dissociation constant, Ka, was OlpB, ORF2p, and SdbA, are also present in the soluble frac-
determined from the ratio of the two kinetic constants (kon/koff). tion after cell sonication (26, 29, 42).VOL. 181, 1999 SEQUENCES OF CipC AND ORFXp IN C. CELLULOLYTICUM 1807
hesin domain is able to interact with all the cellulosomal sub-
units containing the dockerin domain (present in the Fc frac-
tion) just like miniCipC1 or miniCipC8. These results clearly
indicate that the cohesin X present in the chimeric protein
miniCipCX was not functional.
By using the BIAcore, the binding parameters for CohXr
with CelA were determined and a 19-fold-lower Ka was found
(Ka 5 2.6 3 108 M21 [Table 1]), compared to that for the
miniCipC1-CelA complex. Interestingly the drop in Ka was
almost exclusively due to the dissociation rate constant (koff),
with the association rate constant (kon) being unchanged, in-
dicating that the dissociation of the CohXr-dockerin complex
is much faster.
FIG. 7. Subcellular localization of ORFXp. (A) Two recombinant proteins,
miniCipC1 (lane 1) and miniCipCX (lane 2), were separated by SDS-PAGE, DISCUSSION
transferred onto nitrocellulose, and probed with antibody raised against cohesin
The cipC gene from C. cellulolyticum has been entirely se-
Downloaded from http://jb.asm.org/ on November 5, 2015 by guest
domain X. The blots were developed with anti immunoglobulin G-peroxidase
conjugate. Incubation (lane 2) with miniCipCX indicates that antibodies (Ab a quenced, and analysis of the deduced amino acid sequence
CX) specifically recognize cohesin domain X in the miniCipCX construction. (B)
The different fractions obtained from the C. cellulolyticum cellulose growth
reveals that CipC is composed of a type IIIa CBD, two hydro-
culture (Fc to F7) were separated by SDS-PAGE and probed with antibody (Ab philic domains, and eight cohesin domains. The properties of
a CX) raised against cohesin domain X (see Materials and Methods for a the CBD are the subject of a previous study (35), and the
discussion of fractionation of C. cellulolyticum cultures). function of the two hydrophilic domains is currently unknown.
Cohesin domains present in CipC (type I cohesin domains) are
highly homologous and, in contrast to CipA from C. thermo-
The estimated molecular mass of ORFXp (33 kDa) is higher cellum, are separated by very short linker regions (4). In spite
than the calculated molecular mass (23,755 Da). This differ- of sequence differences, the two most divergent cohesin do-
ence is probably because ORFXp is highly glycosylated (data mains (cohesin domains 1 and 8) have the same affinity for the
not shown). A similar phenomenon was also observed for the dockerin domain from CelA. In addition, it was previously
cellulosome from C. thermocellum, in which 5 to 7% of the reported that the dockerin domains present in CelF and in
total mass can be attributed to carbohydrates (19). Most of CelA have the same high affinity for cohesin domain 1 (41).
them are formed by O-glycosylation of the Thr residues lo- Taken together, these results suggest that the incorporation of
cated in the linker regions of CipA. Since ORFXp contains a the different cellulases into the cellulosome is not cohesin
P-T-S-rich fragment made up of 58 aa, which accounts for 25% specific. CipC harbors eight cohesin domains, whereas 13 pro-
of the protein, it is probable that this domain can constitute the teins containing a dockerin domain have been detected in the
site of glycosylation, leading to an unexpected migration on cellulosome fraction of C. cellulolyticum. This observation sug-
SDS-PAGE. Despite numerous attempts using SDS-PAGE gests that different cellulosomal particles, with different enzy-
and transfers to polyvinylidene difluoride membranes, it was matic compositions, are produced by C. cellulolyticum, as was
not possible to obtain sufficient amounts of the protein to observed for C. papyrosolvens C7. The fact that CelF and CelE
determine the N-terminal sequence. are much more abundant than the other catalytic subunits
ORFXp was not detected on cellobiose-grown cultures in all could indicate that these two proteins are always integrated in
the fractions tested (F0 to F7). Production of ORFXp can be the complexes and that the heterogeneity of the cellulosomes
induced, however, by addition of 100 mg of cellulose per liter. applies only to the other catalytic subunits. This also highlights
Identification of the partner of ORFXp. To understand the the peculiar role of these two cellulases in the degradation of
role of ORFXp, the binding capacity of the cohesin domain cellulose.
present in ORFXp was studied. First, fraction Fc, subjected to Another interesting feature is the absence, such as in CbpA
SDS-PAGE, was probed with biotinylated miniCipX (Fig. 4C). and CipA from C. josui, of type II dockerin domains in CipC
No binding was detected, suggesting that cohesin domain X (44). This domain, in CipA from C. thermocellum, was found to
does not interact with type I dockerin domains. Similar results be responsible for the attachment of the cellulosomes to the
were obtained with fractions F0 to F7 under cellobiose and cell surface (26, 27). These cellulosomes form cell protuber-
cellulose growth conditions (data not shown), suggesting that ances (25). In C. cellulovorans, however, protuberances similar
cohesin domain X is not able to bind any partner in C. cellu- to that of C. thermocellum were observed on the cell surface of
lolyticum. the bacterium by Bayer and Lamed (3) using scanning electron
Although great care was taken during the construction of microscopy. Recently, the presence of cellulolytic cell surface
miniCipX to maintain the available three-dimensional cohesin protuberances has been confirmed (9), suggesting that the
structures (Fig. 4C), the fusion of cohesin X with the CBD and mechanism of attachment of the cellulosomes in C. cellulo-
the hydrophilic domain may have induced an incorrect folding vorans is different from that in C. thermocellum. Such experi-
of the cohesin domain. Furthermore, this domain is preceded ments have not been carried out with C. cellulolyticum, and it
by a long linker domain in ORFXp whereas cohesin domains is therefore not known if this bacterium possesses similar pro-
8 and 1 are located downstream of an hydrophilic domain. tuberances. If C. cellulolyticum possesses cellulolytic protuber-
Thus, in the chimeric protein miniCipCX, cohesin domain X ances, like the majority of cellulosome-producing organisms, it
has a different environment, which could explain the negative can be assumed that the mechanism of attachment of the
results obtained with miniCipCX. To verify this assumption, a cellulosomes to the cell surface is similar to that of C. cellulo-
new construction was undertaken. Cohesin domain X alone vorans.
was overexpressed with a His tag. Recombinant cohesin do- Cohesin domains 1 to 5 of CipC are highly homologous
main X, called CohXr (Fig. 4D), was studied in terms of (88%) to the five first cohesin domains of CipA from C. josui.
binding abilities. Surprisingly, the results showed that this co- As in C. cellulolyticum, the last cohesin domain of CipA (co-1808 PAGÈS ET AL. J. BACTERIOL.
Downloaded from http://jb.asm.org/ on November 5, 2015 by guest
FIG. 8. Sequence alignment of CipA cohesin domains 2 and 5 (CipA2-ct and CipA5-ct) and OlpA cohesin domain from C. thermocellum (OlpA-ct) (data from
reference 4), and cohesin domains 1, 8, and X from C. cellulolyticum (Co1-cc, Co8-cc, and CoX-cc). For C. thermocellum, residues in boldface type are strictly conserved
among cohesins of the same bacterium and expected to be involved in the interaction with the dockerin domain (group 1). In C. cellulolyticum, the five amino acids
expected to be involved in the interaction with dockerin domain are underlined (group 2). The position of the b strands, based on the structure of cohesin domain 2
of CipA, are numbered and indicated by “b.”
hesin domain 6) is more divergent than the five first (63%) and (8 aa) was found at the NH2 terminus of CelA from Rhodo-
is highly homologous to cohesin domain 8 of CipC (89% ho- thermus marinus, downstream of the putative signal peptide of
mology) (22). the protein. Unfortunately, it was not possible to obtain the
The role of ORFXp is not clear. ORFX is located in the N-terminal sequence of ORFXp, and so it was not possible to
cluster of cel genes, 8,980 bp downstream of cipC. In C. ther- establish whether the putative signal sequence is cleaved. By
mocellum, four genes encoding nonscaffolding cohesin do- analogy to other membrane-bound proteins, however, it is pos-
mains were detected. In all cases, the corresponding proteins, sible that the putative signal sequence is not cleaved. For
SdbA, ORF2p, OlpB, and OlpA (26, 27, 29, 42), contain S- example, such an organization was found in cytochrome c(y) of
layer homology repeats which anchor these proteins to the cell Rhodobacter capsulatus (34). This protein is composed of three
surface. The first three proteins harbor a cohesin type II do- domains: an uncleaved signal-like domain, which anchors the
main, which is the receptor of the dockerin type II domain of protein to the cytoplasmic membrane; a long linker domain (70
CipA and therefore allows the anchoring of the cellulosomes. aa); and the cytochrome domain, which lies in the periplasmic
The gene olpA, encoding a protein harboring a type I cohesin space. One can imagine a similar membrane anchoring in
domain, is located 7,934 bp downstream of cipA at the end of ORFXp; however, only electronic microscopy with labelled
a cluster including the genes encoding OlpB and ORF2p (15). antibodies can provide new insights into the exact localization
OlpA contains also a long PTS domain (55 aa) located between of ORFXp and, in particular, cohesin domain X. One hypoth-
the SLH and the cohesin domain (42). The Ka of the complex esis concerning the role of ORFXp is that it acts as an inter-
OlpA-dockerin domain of CelD was measured (43) and found mediate in the docking of cellulases during cellulosome assem-
to be 6.9 3 106 M21. It has been suggested by Leibovitz (28) bly, such as has been proposed for OlpA. This hypothesis is
that OlpA could anchor the catalytic units on the bacterial supported by the slight difference observed in koff between
surface of C. thermocellum at a temporary stage before the CohXr and cohesin domain 1 (miniCipC1) when interacting
incorporation in the cellulosome. ORFXp does not harbor with CelA. The dissociation of the CohX-CelA complex seems
SLH domains. The very long PTS domain (58 aa) located at to be faster than that of the Coh1-CelA complex, whereas the
the NH2 terminus of the protein has not been observed in association constant kon remains almost unchanged. The bind-
other proteins involved in cellulolysis; only a very small linker ing parameters, however, may prove to be very different inVOL. 181, 1999 SEQUENCES OF CipC AND ORFXp IN C. CELLULOLYTICUM 1809
thèse des oligonuclotides, CNRS, Marseilles, France) for providing the
oligonucleotides used in this study. We are grateful to M. T. Guidici-
Orticoni for help with the use of the BIAcore apparatus.
This research was supported by grants from the Centre National de
la Recherche Scientifique, the Université de Provence, the Région
Provence Alpes Côte d’Azur, and the EEC (BIOTECH contract CT-
97-2303).
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