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.. As expected, the
Trang 1Identification of new components and synergies between complexes Imen Fendri1, Chantal Tardif1,2, Henri-Pierre Fierobe1, Sabrina Lignon3, Odile Valette1, Sandrine Page`s1,2and Ste´phanie Perret1,2
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; http://www.cazy.org) [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: perret@ifr88.cnrs-mrs.fr
(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 cellulocellulo-somes is correlated with their enzyme diversity
Abbreviations
CBM, carbohydrate-binding module; CipC, cellulosome-integrating protein C; CMC, carboxymethyl cellulose; GH, glycoside hydrolases.
Trang 2for 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 propro-tein 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.
Trang 3and 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 fracelu-tions (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.
Trang 4microcrystalline 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 fracfrac-tion, 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.
Trang 5a 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 fraccombina-tions 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 (http://www.cazy.org); Doc, dockerin domain; Ig, immunoglobulin-like domain of cellulase; X2, Ig-like module of unknown func-tion; Mr, theoretical molecular mass of the mature protein The cleavage site was determined using http://www.cbsdtu.dk/services/SignalP/; Cov, percentage of amino acid coverage in the matched proteins; Mpep, the number of unique matched peptides; Upep, 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
Mr (kDa) Mpep⁄ 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 36.0 135.0c 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 72.0c 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 50.2b 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 45.0 249.0c 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 74.0c 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 (http://www.ncbi.nlm.nih.gov; 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.
Trang 6Avicelase 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 composisubpopula-tions 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 cellulocata-lyticum 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-cumGH5⁄ 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)1on 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.
Trang 7taining 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 32C 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 D450= 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 Na2HPO4/ NaH2PO4 (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 CaCl2by ultrafiltration
Chromatography
Liquid chromatography was performed at 4C using a fast protein purification liquid chromatography system (A¨kta Explorer; Amersham Biosciences, Uppsala, Sweden) Gel-filtration chromatography was performed using a
Bio-sciences) equilibrated with 20 mm Tris⁄ HCl buffer (pH 8.0),
150 mm NaCl and 2 mm CaCl2 Fractions of interest were pooled and dialysed against 20 mm Tris⁄ HCl (pH 8.0) and
Trang 82 mm CaCl2buffer before loading into a Resource Q column
(6 mL) (Amersham Biosciences) equilibrated with 20 mm
Tris⁄ HCl (pH 8.0) and 2 mm CaCl2buffer 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 37C 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 (ProteoSliverTM;
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 (http://www.matrixscience.com) 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 (http://prospector.ucsf.edu) 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 PicoFritcolumn (New Objective, Woburn, MA, USA) An ion trap LCQ-DECAXPmass 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 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
Trang 9oxidation, 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 (http://pfam.sanger
ac.uk) [47] Signal peptide position was determined using
the server http://www.cbs.dtu.dk/services/SignalP [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 BioH2’) 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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