Tài liệu Báo cáo khoa học: The cellulosomes from Clostridium cellulolyticum Identification of new components and synergies between complexes ppt

The cellulosomes from Clostridium cellulolyticum
Identification of new components and synergies between complexes
Imen Fendri
1
, Chantal Tardif
1,2
, Henri-Pierre Fierobe
1
, Sabrina Lignon
3
, Odile Valette
1
, Sandrine
Page
`
s
1,2
and Ste
´
phanie Perret
1,2
1 Laboratoire de Chimie Bacte
´
rienne, CNRS, UPR9043, IMM, Marseille, France
2 Universite
´
Aix Marseille, France
3 Centre de microse
´
quencage et d’analyse prote
´

omique, IMM, Marseille, France
Biomass from plant cell walls contains large quantities
of structural polysaccharides. Cellulose, the most
abundant polysaccharide, is a linear glucose polymer
forming fibrils with a regular crystalline arrangement
[1–3]. In plant cell walls, cellulose fibrils are sur-
rounded by a complex matrix of polysaccharides such
as hemicellulose and pectin [4], which make plant
cellulose resistant to enzymatic hydrolysis. Some
microorganisms secrete diverse cellulases, hemicellu-
lases (xylanases, mannanases, etc.) and pectinases that
have various and complementary modes of action
(endo, exo and processive) [5]. These plant cell-wall-
degrading enzymes, which include glycoside hydrolases
(GH), polysaccharide lyases and carbohydrate ester-
ases, have been classified into families based on their
sequence homologies (Carbohydrate Active Enzyme
Database; ) [6]. In cellulose-rich
anaerobic biotopes, bacteria such as Ruminococ-
cus flavefaciens [7,8], Bacteroides cellulosolvens [9],
Clostridium cellulolyticum [10], Clostridium thermocel-
lum [11], Clostridium cellulovorans [12] and Clostrid-
ium papyrosolvens [13] secrete multienzyme complexes
called cellulosomes which degrade plant cell walls effi-
ciently. In general, cellulosomes are composed of a
scaffolding protein devoid of enzymatic activity which
binds the complexes to the substrate via its carbo-
hydrate-binding module (CBM). This protein contains
several cohesin modules that serve as anchoring points
Keywords

cellulosome; Clostridium cellulolyticum;
diversity; new components; synergy
Correspondence
S. Perret, Laboratoire de Chimie
Bacte
´
rienne, CNRS, UPR9043, 31 chemin
Joseph Aiguier 13009, Marseille, France
Fax: +33 4 91 71 33 21
Tel: +33 4 91 16 43 40
E-mail:
(Received 18 January 2009, revised 24
March 2009, accepted 27 March 2009)
doi:10.1111/j.1742-4658.2009.07025.x
Cellulosomes produced by Clostridium cellulolyticum grown on cellulose
were purified and separated using anion-exchange chromatography.
SDS ⁄ PAGE analysis of six fractions showed variations in their celluloso-
mal protein composition. Hydrolytic activity on carboxymethyl cellulose,
xylan, crystalline cellulose and hatched straw differed from one fraction to
another. Fraction F1 showed a high level of activity on xylan, whereas
fractions F5 and F6 were most active on crystalline cellulose and carb-
oxymethyl cellulose, respectively. Several cellulosomal components specific
to fractions F1, F5 and F6 were investigated using MS analysis. Several
hemicellulases were identified, including three xylanases in F1, and several
cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein
protease inhibitor were identified in F5 and F6. Synergies were observed
when two or three fractions were combined. A mixture containing fractions
F1, F3 and F6 showed the most divergent cellulosomal composition, the
most synergistic effects and the highest level of activity on straw (the most
heterogeneous substrate tested). These findings show that on complex sub-

strates such as straw, synergies occur between differently composed cellulo-
somes and the degradation efficiency of the cellulosomes is correlated with
their enzyme diversity.
Abbreviations
CBM, carbohydrate-binding module; CipC, cellulosome-integrating protein C; CMC, carboxymethyl cellulose; GH, glycoside hydrolases.
3076 FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS
for the enzymes via a strong interaction with enzyme-
borne dockerin modules.
The cellulosomes produced by C. cellulolyticum
grown on cellulose contain  30 dockerin-containing
proteins [14]. The majority of these proteins are GHs
belonging to families 2, 5, 8, 9, 10, 11, 18, 26, 27, 44, 48,
53, 62, 74 and 95. In addition, 62 ORFs that potentially
encode dockerin-containing proteins have been found in
the genome sequence of C. cellulolyticum (http://www.
ncbi.nlm.nih.gov; GI:220927459), (J. C. Blouzard, per-
sonal communication). Twelve genes were found to be
gathered in a large operon called the cip-cel cluster,
beginning with the gene encoding the scaffolding pro-
tein named cellulosome-integrating protein C (CipC),
followed by genes that code for the major cellulosomal
cellulase Cel48F and nine other dockerin-containing
enzymes [15]. Cellulases encoded by the cip-cel cluster
are essential for the formation of efficient cellulosomes
to degrade crystalline cellulose [16], in particular the
processsive cellulase Cel48F [17].
In C. cellulolyticum, the scaffolding protein contains
eight cohesin modules that potentially bind to 62 dock-
erin-bearing proteins. Previous studies have suggested
that any CipC cohesin can bind to any enzyme docker-

in: Cel5A binds to the most divergent cohesins with
similar affinities [18] and cohesin 1 shows a similar
affinity for Cel5A and Cel48F [19]. In addition, over-
production of a minor cellulosomal enzyme, the man-
nanase Man5K, resulted in mannanase-enriched
cellulosomes [20]. The data strongly suggest that the
composition of C. cellulolyticum cellulosomes is hetero-
geneous and may depend on the relative amounts of
dockerin-containing enzymes available.
The hydrolytic efficiency of cellulosomes has also
been studied in mini-cellulosomes assembled in vitro.
These mini-cellulosomes had a strictly controlled
enzyme composition and contained two or three engi-
neered cellulases [21,22]. Enzyme binding to scaffoldin
was found to enhance the activity of the enzymatic
components, particularly on recalcitrant substrates.
This enhancement was attributed to the physical prox-
imity of the enzymes in the mini-cellulosomes and to
cellulose targeting of the complexes via the CBM of
the mini-scaffoldin [21]. The most active mini-cellulo-
some on microcrystalline cellulose was composed of
the processive cellulase Cel48F combined with endo-
glucanase Cel9G. Adding the C. thermocellum bifunc-
tional esterase ⁄ xylanase Xyn10Z to this cellulase pair
yielded the most active mini-cellulosome on hatched
straw [22]. Compared with naturally occurring cellulo-
somes, however, the most active mini-cellulosomes are
fivefold less active on crystalline cellulose and 3.5-fold
less active on straw. Additional factors present in
naturally occurring cellulosomes may therefore account

for their high efficiency.
Cellulosomes produced by C. papyrosolvens and
C. cellulovorans grown on cellulose have been split into
several peaks using anion-exchange chromatography
[13,23]. The subpopulations had diverse enzymatic
compositions and patterns of activity. However, puta-
tive synergistic activities between several subpopula-
tions were not examined. In this study, we separated
cellulosomes produced by C. cellulolyticum. The acti-
vity and composition of the complexes present in each
fraction were analysed to identify new active compo-
nents and ⁄ or an efficient association of components.
The possible occurrence of synergies between cellulo-
somal fractions which might account for the efficiency
of the cellulosomes was investigated.
Results
Fractionation of cellulosomes
To separate the various cellulosomes of C. cellulolyti-
cum, we first extracted cellulose-bound proteins from
the residual cellulose in a 6-day culture. Cellulosomes
(500–900 kDa) were purified using gel-filtration chro-
matography. The cellulosomal fraction was subse-
quently subjected to anion-exchange chromatography.
The elution profile showed that the cellulosomes were
eluted in a single peak with a long tail (Fig. 1). This
elution profile was systematically obtained with cellulo-
somes originating from several clostridial cultures on
cellulose. Using different NaCl gradients or performing
elution with a pH gradient also yielded a single peak
(data not shown). Cellulosome composition was analy-

sed from the beginning to the end of the large peak;
the peak was arbitrarily divided into six fractions
numbered F1–F6 (Fig. 1) and the protein composition
Fig. 1. Anion-exchange chromatography of the cellulosomal
fraction purified by gel filtration. Three milligrams of protein
were loaded onto the column. F1–F6 are the arbitrarily separated
fractions. The grey line gives the continuous NaCl gradient.
I. Fendri et al. Diversity of cellulosomes and their synergies
FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS 3077
and enzymatic properties of the cellulosomes present
in each fraction were analysed.
Protein analyses of the fractionated cellulosomes
The six fractions, separated as described above, were
first analysed using Native PAGE. In all fractions, a
single major diffuse band was observed, which showed
that the proteins present in all the fractions were in a
‘cellulosome state’ [20] and that anion-exchange chro-
matography did not dissociate the complexes (data not
shown). The subunit composition of these complexes
was therefore analysed further by SDS ⁄ PAGE (Fig. 2).
A control sample (C) was formed by pooling the elu-
tion fractions (F1–F6) corresponding to the entire
peak obtained using anion-exchange chromatography.
In this control sample, the proportions were those of
naturally occurring cellulosomes and the sample was
subjected to the same chromatographic procedures as
each of the fractions analysed separately. Each of the
fractions obtained by anion-exchange chromatography
showed numerous proteins, most of which had molecu-
lar masses in the range 30–160 kDa. As expected, the

scaffolding protein CipC (160 kDa) was detected as a
major protein in all the fractions.
The protein composition of the fractions was found
to differ, particularly F1 and F6 which corresponded
to the extremities of the peak (Fig. 2). In each fraction,
Cel48F and Cel9E were found to be abundant. How-
ever, the distribution patterns of the four cellulases
Cel5A [24], Cel9G [25], Cel8C [26] and Cel9M [27]
were quite different (Fig. 3). Cel5A and Cel5M were
present almost exclusively at the end of the peak (in
fractions F4–F6), whereas Cel8C was detected in only
the first two fractions. Cel9G was present in all the
fractions except the first. A complementary analysis
was then carried out using silver-stained SDS ⁄ PAGE.
The components showing the greatest variation in rela-
tive amounts were numbered 1–14 (Fig. 2). Fraction 1
contained high amounts of proteins 6, 9, 12 and 14,
whereas proteins 1, 2, 3, 4, 5, 7, 8, 10, 11 and 13 were
present in fractions F5 and F6 but absent or barely
detectable in F1–F4. The middle fractions, F3 and F4,
which account for most of the complexes in naturally
occurring proportions, were found to have a fairly sim-
ilar composition, midway between those of F1 and F6.
As expected, the cellulosomes produced during a 6-day
period of growth on cellulose showed considerable
heterogeneity and were partly separated using anion-
exchange chromatography.
Enzymatic properties of the various fractions
The activities of the six fractions and the control
sample were compared on noncrystalline substrates

such as carboxymethyl cellulose (CMC) and xylan,
Fig. 2. Composition of the cellulosome fractions (F1–F6). Five
micrograms of protein were separated on a 10% SDS ⁄ PAGE and
silver stained. C, control sample containing the unfractionated mix-
ture of cellulosomes; F1–F6, fractions separated by anion-exchange
chromatography. Major components CipC, Cel9E and Cel48F are
indicated; bands analysed using MS methods are numbered; the
asterisks indicate a band containing a nonsecreted protein identified
by ion-trap MS ⁄ MS as a ketol-acid reductoisomerase, which is not
a cellulosomal component (data not shown).
Fig. 3. Identification of several components in cellulosomes from
fractions F1–F6. Proteins (5 lg) were separated on 10% SDS ⁄
PAGE, transferred to a polyvinylidene fluoride membrane and
probed with anti-CelA, anti-CelC, anti-CelM and anti-CelG serum.
C, control sample corresponding to the unfractionated mixture
of cellulosomes; F1–F6, fractions separated by anion-exchange
chromatography.
Diversity of cellulosomes and their synergies I. Fendri et al.
3078 FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS
microcrystalline cellulose (Avicel) and hatched straw, a
heterogeneous natural substrate. As shown in Fig. 4,
fractions F1–F6 showed different patterns of activity.
On CMC, the most active fraction was F6, which was
45% more active than F5, and 70–90% more active
than the control sample and fractions F1–F4. On
xylan, rather weak activity was measured with the con-
trol sample and all the fractions, except for F1, which
was found to be approximately fivefold more active
than the others. The cellulosomes present in fractions
F1 and F6 therefore have the most efficient enzymes

for degrading xylan and CMC, respectively.
On straw, and to a lesser extent on Avicel (Fig. 4),
the differences in activity between the fractions were
less pronounced than on CMC and xylan. On straw,
all fractions showed a substantial level of activity that
was never less than half that of the most active frac-
tion, F1. On Avicel, F5 was the most efficient fraction,
and the least active fractions, F1 and F2, showed less
than half the activity of F5. All in all, these results
indicate that the differences in the protein composition
of the cellulosomes are related to different enzymatic
properties.
Identification of new cellulosomal components
MS analysis was performed to identify the specific
components of fractions F1, F5 and F6 (Table 1).
Complete MS data such as the spectra and the corre-
sponding annotation table can be found in Figs S1–
S11 and Table S1. Two of the four components, which
were found to be abundant in F1, were identified as
xylanases belonging to GH family 10 (protein 12),
named Xyn10A [14], and to GH family 11 (protein
14), renamed Xyn11B. In addition a hypothetical
xylanase (protein 9) was detected. The catalytic
domain of this protein showed 29% identity with the
xylanase XynA from Erwinia chrysanthemi (accession
no. AAB53151.1) [28,29]. The latter enzyme contains a
GH catalytic domain that has been reported to be
intermediate between families 5 and 30 [29]. The abun-
dance of proteins 9, 12 and 14 in fraction F1 is consis-
tent with the high xylanase activity seen in this

fraction. However, GH10 (protein 12) is also present
in noticeable quantities in the other five fractions.
All the proteins present in F5 were also present in
F6. Among these, we detected protein 2 (Cel9P) [14]
and proteins 5a (Cel9G) [25] and 5b (Cel9H) [28].
Cel9P and Cel9H show the same modular organization
as the endoglucanase Cel9G (GH9-CBM3c-Doc) char-
acterized previously [25]. Although Cel9P and Cel9H
have not yet been characterized, they are expected to
show enzymatic properties similar to those of Cel9G.
Four enzymes belonging to the GH5 family were also
identified: proteins 7a and 11 correspond to the endo-
cellulases Cel5D [30] and Cel5A [31], respectively, and
protein 8 corresponds to the carboxymethyl cellulase
Cel5N [14], and protein 4, in which the GH5 catalytic
domain shows 33% identity with a mannanase from
Bacillus circulans (accession no. BAA25878.1) [31].
Protein 7b was identified as the mannanase Man26A
[14]. Lastly, protein 13 was identified as an N-terminal
dockerin-borne chagasin domain. Chagasin belongs to
Fig. 4. Enzymatic activities of the cellulosome fractions on various
substrates. Specific activities were measured at 37 °C after 30 min
on 0.8% CMC and xylan, and after 24 h on 0.35% microcrystalline
cellulose Avicel and hatched straw at final protein concentrations of
2, 3, 20 and 6 lgÆmL
)1
, respectively.
I. Fendri et al. Diversity of cellulosomes and their synergies
FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS 3079
a family of cystein protease inhibitors found in lower

eukaryotes and prokaryotes [32].
The specific proteins in F6 either show a GH9-
CBM3c modular organization (protein 1 was renamed
Cel9R and protein 3 was identified as Cel9J) or belong
to the GH18 family (protein 11). Protein 11 is a puta-
tive chitinase in which the catalytic module shows 43%
identity with a chitinase from Bacillus pumilus (acces-
sion no. ABI15082.1) and 39% identity with Chi18A
found in C. thermocellum cellulosomes [33].
Synergistic activities between cellulosomes from
different fractions
The possibility that synergies between various fractions
of cellulosomes might account for the efficiency of the
cellulosome mixture was investigated. It was previously
established that the association of an endo-processive
cellulase active on crystalline cellulose with an endo-
cellulase active on CMC leads to the most efficient
in vitro reconstituted mini-cellulosomes on microcrys-
talline cellulose [19]. First, we studied the hydrolysis of
Avicel, combining the most complementary fractions
F5 and F6, which are most active on Avicel and
CMC, respectively. We further studied the synergies
combining these fractions with F3, which accounts for
most of the cellulosomes. No synergies were measured
with the F3 ⁄ F5 pair (Fig. 5). However, the F5 ⁄ F6 and
F3 ⁄ F6 pairs showed synergies of 1.2 and 1.3, respec-
tively, and a synergy of 1.2 was also obtained with the
combination F3 ⁄ F5 ⁄ F6.
On straw, the activity of the control sample was
higher than that of any of the fractions (Fig. 4). This

indicates that on this natural substrate, this combina-
tion of different fractions results in synergistic activity.
Because straw is a complex substrate composed mostly
of cellulose (40% w ⁄ w) and hemicelluloses (15% w ⁄ w),
we analysed its degradation using the following combi-
nation of fractions showing complementary activities:
the xylanase F1 fraction combined with either the
Table 1. Identification of specific components detected in fractions F1, F5 and F6 using MS analysis. All identifications were based on pep-
tide mass fingerprint analyses using the MALDI-TOF technique, except for the proteins 6, 7a and 7b, and 10 which were identified using the
MS ⁄ MS technique. The modular structure of new proteins was determined by performing PFAM and BLAST analyses. S, signal sequence;
GH, glycoside hydrolase; CBM, carbohydrate binding module, GH and CBM numbers are those of the carbohydrate active enzyme database
classification (); Doc, dockerin domain; Ig, immunoglobulin-like domain of cellulase; X2, Ig-like module of unknown func-
tion; M
r
, theoretical molecular mass of the mature protein. The cleavage site was determined using />Cov, percentage of amino acid coverage in the matched proteins; M
pep
, the number of unique matched peptides; U
pep
, the number of
unmatched peptides in the MALDI-TOF experiments. The function of new proteins is based on the GH family of the catalytic module and
the modular organization of the protein, or on the identity of the catalytic domain with another characterized protein (see text).
Protein GI number
a
Modular structure
M
r
(kDa) M
pep
⁄ U
pep

Cov
(%) Score
Protein name and ⁄ or
description Reference
F1
6 220928204 S-Ig-GH9-doc 66.1 6 15.9 60.2
b
Cellulase Cel9S This study
9 220928101 S-GH5 ⁄ GH30-doc 55.6 12 ⁄ 27 19.0 89.0
c
Putative xylanase This study
12 110588916 S-GH10-doc 44.3 17 ⁄ 37 36.0 135.0
c
Xylanase Xyn10A 9
14 220928199 S-GH11-doc 29.6 5 ⁄ 4 19.0 71.0
c
Xylanase Xyn11B This study
F5 ⁄ F6
2 110588925 S-GH9-CBM3-doc 83.3 9 ⁄ 36 15.0 72.0
c
Cellulase Cel9P 9
4 220927835 S-GH5-CBM32-X2-X2-Doc 78.6 9 ⁄ 25 14.0 77.0
c
Putative mannanase This study
5a 585234 S-GH9-CBM3-doc 76.1 10 ⁄ 48 15.0 5.4 · 10
5d
Cellulase Cel9G 20
5b 12007365 S-GH9-CBM3-doc 78.7 11 ⁄ 47 14.0 5.1 · 10
3d
Cellulase Cel9H 22

7a 121824 S-GH5-doc 63.4 5 9.8 50.2
b
Cellulase Cel5D 25
7b 110588924 S-GH26-doc 61.8 2 10.5 20.2
b
Mannanase Man26A 9
8 220928189 S-GH5-doc 56.6 6 ⁄ 7 10.0 68.0
c
Cellulase Cel5N 9
11 121802 S-GH5-doc 50.7 29 ⁄ 39 45.0 249.0
c
Cellulase Cel5A 19
13 220929230 S-doc-Chagasin 31.0 5 ⁄ 18 16.0 60.0
c
Unknown function This study
F6
1 220929070 S-GH9-CBM3-doc 102.3 12 ⁄ 23 13.0 74.0
c
Cellulase Cel9R This study
3 220928185 S-GH9-CBM3-doc 81.3 23 ⁄ 28 26.0 165.0
c
Cellulase Cel9J 22
10 220928973 S-GH18-doc 51.1 4 9.5 40.2
b
Putative chitinase This study
a
Accession numbers of new components are those of the newly released complete genome (; GI:220927459).
b
Scores obtained using using BioworksBrowser search engine (MS ⁄ MS data).
c

Scores obtained using MASCOT search engine (MALDI-
TOF data).
d
Scores obtained using MS-Fit (MALDI-TOF data). For this latter analysis the top nonhomologous protein shows a score of 94.8.
Diversity of cellulosomes and their synergies I. Fendri et al.
3080 FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS
Avicelase F5 and ⁄ or the carboxymethyl cellulase F6
fractions, with and without fraction F3, which
accounts for most of the cellulosomes. The F1 ⁄ F5 and
F1 ⁄ F6 pairs did not exhibit important synergies (1.1
and 1.16, respectively) and released 30% fewer soluble
sugars than the control sample. However, the combi-
nation of fractions F1 ⁄ F5 ⁄ F6 induced greater synergies
(1.3) and released an amount of soluble sugars similar
to that seen with the control. The highest synergy (1.4)
was measured when fractions F1, F3 and F6 were
combined, which resulted in a larger quantity of
released soluble sugars than with the control sample.
Each individual fraction was therefore less active
than the naturally occurring cellulosome mixture, but
mixing fractions combining the most diverse cellulo-
somes induces important synergies.
Discussion
Cellulosomes from C. papyrosolvens and C. cellulovo-
rans were separated using anion-exchange chromatog-
raphy, giving seven and four elution peaks,
respectively. These different cellulosome subpopula-
tions have distinct protein compositions and patterns
of activity [13,23]. It has been suggested that the pres-
ence of several well-separated peaks in the case of cell-

ulosomes from C. cellulovorans may be partly due to
the existence of various categories of cohesins and
dockerins which determine the composition of the cell-
ulosomes [23,34]. Despite the enzymatic diversity of
the cellulosomes from C. cellulolyticum (there are 62
ORFs encoding dockerin-containing protein versus 8
enzymatic units per cellulosome), anion-exchange chro-
matography gave a single peak followed by a long tail.
This suggests a random assembly of enzymes on the
scaffoldin, leading to a large number of enzyme combi-
nations.
In this study, a GH11 xylanase and a GH5 ⁄ 30 puta-
tive xylanase were identified. In the genome sequence of
C. cellulolyticum, only one gene encoding a cellulosomal
GH11 was found. To date, GH11 modules have been
found in modular bifunctional cellulosomal enzymes,
such as XynA from C. cellulovorans (GH11-Doc-
CE4) [35] and XynA from C. thermocellum strain
ATCC27405 (GH11-CBM6-Doc-CE4) [33], or associ-
ated with a CBM6 module such as in XynB (GH11-
CBM6-Doc) from C. thermocellum strain F1 ⁄ YS [36].
In the C. cellulolyticum enzyme, the GH11 catalytic
module had no such catalytic or CBM partner, which
suggests that the catalytic behaviour of the enzyme may
differ from that of previously described enzymes con-
taining GH11. To date, no GH5 ⁄ GH30 enzymes have
been found in C. cellulovorans cellulosomes, whereas a
bifunctional GH30-a-l-arabinofuranosidase B has been
detected in C. thermocellum cellulosomes [33]. The cata-
lytic domain of C. cellulolyticum GH5⁄ GH30 shows

25% identity with the C. thermocellum GH30 module,
28% identity with the E. chrysanthemi XynA catalytic
module and 24% identity with the B. subtilis XynC
catalytic module. XynC has been characterized as an
endoxylanase cleaving the methylglucuronoxylan chain
in close proximity to a methylglucuronosyl-substituted
xylose residue [37]. The functional role of C. cellulolyti-
cum GH5 ⁄ GH30 enzyme remains to be identified.
Interestingly, a nonenzymatic protein (protein 13)
was detected in substantial quantities in the cellulo-
somes. This dockerin-bearing chagasin (MEROPS
peptidase database identification number I42) is a
putative cystein protease C1A inhibitor (http://merops.
sanger.ac.uk) [38]. A gene encoding a dockerin-con-
Fig. 5. Synergies between cellulosomes. Light grey bars indicate
activity measured for the fraction mixture at total protein concentra-
tions of 20 and 6 lgÆmL
)1
on crystalline cellulose (A) and straw (B),
respectively. White bars indicate the theoretical sum of the activi-
ties of the individual fractions measured independently (at half or
one third of the protein concentration). The dark grey bars indicate
the activity of the control. Synergy values are indicated on the light
grey bars.
I. Fendri et al. Diversity of cellulosomes and their synergies
FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS 3081
taining protein related to cystein protease C1A was
found (GI:220929842) in the genome sequence of
C. cellulolyticum. This cellulosomal chagasin ⁄ cystein
protease system is reminiscent of the serpins ⁄ serine

protease cellulosomal system reported in C. thermocel-
lum [39,40]. A cellulosomal protease inhibitor ⁄ protease
system may, therefore, be more widespread than
expected and have a common and important function
in cellulosome regulation, displacement from the cell
surface, degradation and ⁄ or protection of the cellulo-
somes [40].
All the fractions showed a substantial level of activity
on crystalline cellulose. In previous studies, mini-cellulo-
somes reconstituted in vitro, in which the endocellulase
Cel9G (GH9-CBM3c-Doc) was combined with the
processive enzyme Cel48F, were found to hydrolyse
crystalline cellulose the most efficiently [21,22]. In this
study, all the cellulosome fractions contained Cel48F
and several GH9-CBM3c-Doc (Cel9P, Cel9G, Cel9H,
Cel9J). This enzyme combination may be essential for
efficient degradation of crystalline cellulose by the
cellulosome. The most active fraction on Avicel (F5)
was found to contain a small amount of Cel9J, but large
amounts of Cel9P and Cel9G ⁄ Cel9H. Because the least
active fractions, F3 and F4, contained large amounts of
Cel9G ⁄ Cel9H, but lower amounts of Cel9P, it seems
likely that Cel9P might contribute to the high level of
activity on crystalline cellulose seen in F5. Although F6
contained all the proteins present in F5, it showed lower
levels of activity on Avicel and higher levels on CMC
than F5. This may be because of the presence of addi-
tional proteins such as proteins 1 and 3 (which were
identified as GH9-CBM3c-Doc enzymes) and protein 10
(which was identified as a chitinase), and ⁄ or to varia-

tions in the enzyme ratios. On straw, the activity of the
mini-cellulosomes containing Cel9G ⁄ Cel48F was greatly
enhanced by adding the C. thermocellum bifunctional
xylanase (XynZ) [22]. It is worth noting that all the
naturally occurring cellulosome fractions studied here
contained at least one xylanase (GH10 protein 12) and
showed high levels of activity on straw.
Individual fractions displayed less specific activity on
straw than the control (consisting of a combination of
all fractions in naturally occurring proportions). This
indicated that synergies occur in the naturally occurring
control mixture. The activity of each fraction on straw
probably resulted from synergies between different
cellulosomes. This explains why only low synergies
were observed when two or three fractions were mixed.
However, combinations of these cellulosome fractions
in equal proportions, which differ from the naturally
occurring proportions, resulted in levels of activity
on Avicel and straw higher than those seen with the
control mixture, highlighting cellulosome synergies. On
straw, a mixture combining the most complementary
fractions, i.e. the most active fractions on xylan (F1),
Avicel (F5) and CMC (F6), showed lower levels of
activity and synergy than a mixture consisting of F1,
F3 and F6, which was the most diverse combination of
cellulosomes. This finding strongly suggests that, on
complex substrates, the diversity of the combined cellu-
losomes has a greater impact on the final activity than
do the enzymatic properties of the combined fractions.
In a previous study,  30 dockerin-containing enzymes

were detected by performing proteomic analyses on
cellulosomes produced by C. cellulolyticum on cellulose
[14]. The enzyme diversity they contain and their heter-
ogeneous composition are inherent characteristics of
cellulosomes. Our data suggest that these characteristics
give rise to synergistic effects between diverse com-
plexes, which may account for the great efficiency of
plant cell-wall degradation processes.
Experimental procedures
Bacterial strain and cell culture conditions
C. cellulolyticum ATCC35319 [41] was grown anaerobically
at 32 °C on basal medium [42] supplemented with cellobi-
ose (4 gÆL
)1
; Sigma-Aldrich, St Louis, MO, USA) or
MN300 cellulose (5 gÆL
)1
; Serva, Heidelberg, Germany).
Purification of the cellulose-adsorbed cellulolytic
system from C. cellulolyticum
C. cellulolyticum cultures were inoculated with a cellobiose
culture at D
450
= 0.7, and grown in 800 mL of cellulose-
supplemented basal medium for 6 days. The cell culture
was filtered through a 3-lm pore size GF ⁄ D glass filter
(Whatman, Maidstone, UK). The residual cellulose was
subsequently washed with 50 and 12.5 mm Na
2
HPO

4
/
NaH
2
PO
4
(pH 7.0). The cellulosome-containing fraction
was eluted from the residual cellulose with water, dialysed
and concentrated in 20 mm Tris ⁄ HCl buffer (pH 8.0),
150 mm NaCl and 2 mm CaCl
2
by ultrafiltration.
Chromatography
Liquid chromatography was performed at 4 °C using a fast
protein purification liquid chromatography system (A
¨
kta
Explorer
Ô
; Amersham Biosciences, Uppsala, Sweden).
Gel-filtration chromatography was performed using a
HiLoad 26 ⁄ 60 Superdex 200 column (Amersham Bio-
sciences) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 8.0),
150 mm NaCl and 2 mm CaCl
2
. Fractions of interest were
pooled and dialysed against 20 mm Tris ⁄ HCl (pH 8.0) and
Diversity of cellulosomes and their synergies I. Fendri et al.
3082 FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS
2mm CaCl

2
buffer before loading into a Resource Q column
(6 mL) (Amersham Biosciences) equilibrated with 20 mm
Tris ⁄ HCl (pH 8.0) and 2 mm CaCl
2
buffer. Elution was per-
formed with a linear NaCl gradient of 0–1 m, in the same
buffer. Fractions were concentrated using microconcentators
(30 kDa cut-off; Vivaspin, Vivasciences, Palaiseau, France).
Protein concentration was determined as described by Lowry
et al. [43], using bovine serum albumin as the standard.
Enzyme activity
Avicel microcrystalline cellulose (PH101; Fluka, Buchs,
Switzerland), CMC (medium viscosity; Sigma, St Louis,
MO, USA), oat spelt xylan (Sigma) and hatched straw
(Valagro, Poitiers, France) were used as substrates.
Hatched straw was prepared as described by Fierobe et al.
[22]. Insoluble xylan was washed four times in distilled
water and the concentration of the residual material was
estimated from the dry weight. Enzymatic assays were per-
formed in 20 mm Tris-maleate (pH 6.0) at 37 °C. A suitable
amount of protein (see legend to Figs 5 and 6) was mixed
with the substrate preparation at a final substrate concen-
tration of 0.8% (CMC or xylan) or 0.35% (Avicel or
straw). After incubating for 30 min (CMC and xylan) or
24 h (Avicel and straw), aliquots were analysed to deter-
mine the soluble reducing sugar content using the method
of Park & Johnson [44] with d-glucose as the standard.
SDS
⁄

PAGE and western blot analysis
SDS ⁄ PAGE was performed using Prosieve 50 gel solution
(Lonza, Rockland, ME, USA). Native PAGE was per-
formed with precast 4–15% polyacrylamide gradient gels
using a Phast-System apparatus (Amersham Biosciences).
Gels were either silver stained using the Plus one silver-stain-
ing kit (Amersham Biosciences) or electrotransferred onto
nitrocellulose BA83 membranes (Schleicher & Schuell, Das-
sel, Germany). After saturation, membranes were probed
with polyclonal rabbit antibodies raised against Cel9G,
Cel5A, Cel8C or Cel9M. Antibodies were detected using an
anti-rabbit horseradish peroxidase conjugate (Promega,
Madison, WI, USA) and chemiluminescent substrate kit
(ECL plus; GE Healthcare, Little Chalfont, UK). The same
membrane was stripped and sequentially probed with several
antibodies, in line with the manufacturer’s instructions.
In-gel trypsin digestion of proteins
MS analysis was performed to identify proteins that
differed between the various cellulosome fractions. Proteins
of interest were excised from the silver-stained gel and
prepared on a robotic workstation (freedom EVO 100;
TECAN, Ma
¨
nnedorf, Switzerland). The automated prepa-
ration process included destaining steps (ProteoSliver
TM
;
Sigma), washing, reduction and alkylation, digestion by
trypsin (proteomics grade; Sigma), extraction and drying of
mixed peptides, as described previously [45].

MALDI-TOF MS analyses
Complete experimental procedures of MALDI-TOF MS
analysis are described in Doc. S1. Digested peptides were
treated using MALDI-TOF Voyager DE-RP apparatus
(Applied Biosystems, Foster City, CA, USA) in the positive
reflectron mode. Contaminant peaks were removed prior to
a peptide mass fingerprint search against the nonredundant
NCBI database (20080210), restricted to ‘Other Firmicutes’
(445 464 sequences) using the freely available MASCOT
search engine (). Searches
were performed using a maximum peptide mass tolerance
of 150 p.p.m., one missing cleavage allowed, a fixed modifi-
cation of cysteines by iodoacetamide (carbamidomethyl),
a variable modification of methionines (oxidation) and
N-term glutamine (pyro-glutamine).
Proteins were taken to have been identified only when
they had at least five matching peptides and scores > 60
(P < 0.05). When identification scores < 60 were obtained,
we assessed their reliability using the search engine MS-FIT
v4.27.2Basic (). In the case of
peptides matching multiple members of a protein family,
the proteins selected were those with the largest number of
matching peptides. When several proteins were identified
with equal numbers of matching peptides we checked that
they corresponded to the same gene product and selected
the database entry that was the best annotated.
Ion-trap MS
⁄
MS analyses
Complete experimental procedures of ion-trap MS ⁄ MS anal-

yses are described in Doc. S1. Samples which did not produce
a sufficiently clear signal in the MS analyses were studied
using 2D liquid chromatography in a tandem mass spectro-
meter. Peptides were loaded onto a strong cation-exchange
column and eluted in salt steps with an increasing ammonium
acetate molarity, before being separated in a reversed-phase
PicoFrit
Ô
column (New Objective, Woburn, MA, USA). An
ion trap LCQ-DECA
XP
mass spectrometer (Thermo Finni-
gan, Waltham, MA, USA) was used for the data acquisition.
Maximum coverage identification was carried out using the
big three program included in the data acquisition Xcali-
bur
Ô
Finnigan proteomex 2.0 software program. Protein
identification was performed using the Sequest (v28 rev12)
algorithm in the bioworksbrowser 3.3 software program
(Thermo Electron Corp., Waltham, MA, USA) using both
the nonredundant NCBI database (20071113) (http://www.
ncbi.nlm.nih.gov) and C. cellulolyticum extract containing
6641 entries. The following search parameters were adopted:
two missed cleavage sites allowed, variable methionine
I. Fendri et al. Diversity of cellulosomes and their synergies
FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS 3083
oxidation, cysteine carbamidomethylation and no fixed
modification, and 1.5 and 1.0 Da as the maximum precursor
and fragment tolerance. Positive identification of peptides

was assessed by a cross-correlation number (Xcorr) versus
charge state, as follows: Xcorr > 1.5 for singly charged ions,
Xcorr > 2.0 for doubly charged ions and Xcorr > 2.5 for
triply charged ions, peptide probability was £ 5 · 10
)3
. Pro-
tein identification required maximum coverage or at least
two rank one unique peptides.
Protein sequence analyses
The amino acid sequences of the new proteins were com-
pared with those in the NCBI sequence databases using the
blast program [46]. Protein domain compositions were
analysed using the PFAM database (ger.
ac.uk) [47]. Signal peptide position was determined using
the server [48].
Acknowledgements
Imen Fendri received a doctoral fellowship from the
Tunisian Ministry of Higher Education and Scientific
Research. We are very grateful to Danielle Moinier and
Re
´
gine Lebrun (Centre de microse
´
quencage et d’analyse
prote
´
omique, IMM, Marseille, France) for performing
the MS analysis. Financial support from the Marseille-
Nice Ge
´

nopole and the ANR (contracts PNRB –
HYPAB and ‘non the
´
matique BioH
2
’) is acknowledged.
We thank Jessica Blanc for correcting the English. The
genomic sequence data were provided by the US
Department of Energy’s Joint Genome Institute (http://
www.jgi.doe.gov).
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Supporting information
The following supplementary material is available:
Fig. S1. MS spectrum of protein 9, fraction 1.
Fig. S2. MS spectrum of protein 12, fraction 1.
Fig. S3. MS spectrum of protein 14, fraction 1.
Fig. S4. MS spectrum of protein 2, fraction 5 ⁄ fraction 6.
Fig. S5. MS spectrum of protein 4, fraction 5 ⁄ fraction 6.
Fig. S6. MS spectrum of protein 5, fraction 5 ⁄ fraction 6.
Fig. S7. MS spectrum of protein 8, fraction 5 ⁄ fraction 6.
Fig. S8. MS spectrum of protein 11, fraction 5 ⁄ fraction 6.
Fig. S9. MS spectrum of protein 13, fraction 5 ⁄ fraction 6.
Fig. S10. MS spectrum of protein 1, fraction 6.
Fig. S11. MS spectrum of protein 3, fraction 6.
Table S1. Masses and peptide assignments.
Doc. S1. Complete experimental procedures of mass
spectrometry analyses.
This supplementary material can be found in the
online version of this article.

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Diversity of cellulosomes and their synergies I. Fendri et al.
3086 FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS