Previous Article | Next Article 
Journal of Bacteriology, November 1999, p. 6720-6729, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Novel Cellulosomal Scaffoldin from
Acetivibrio cellulolyticus That Contains a Family 9 Glycosyl Hydrolase
Shi-You
Ding,1,2
Edward A.
Bayer,1,*
David
Steiner,2
Yuval
Shoham,3 and
Raphael
Lamed2
Department of Biological Chemistry, The
Weizmann Institute of Science, Rehovot,1
Department of Molecular Microbiology and Biotechnology,
Tel-Aviv University, Ramat Aviv,2 and
Department of Food Engineering and Biotechnology,
Technion
Israel Institute of Technology,
Haifa,3 Israel
Received 10 June 1999/Accepted 24 August 1999
 |
ABSTRACT |
A novel cellulosomal scaffoldin gene, termed cipV, was
identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the
protein was based on a combination of properties, including its high
molecular weight, cellulose-binding activity, glycoprotein nature, and
immuno-cross-reactivity with the cellulosomal scaffoldin of
Clostridium thermocellum. The cipV gene is
5,748 bp in length and encodes a 1,915-residue polypeptide with a
calculated molecular weight of 199,496. CipV contains an N-terminal
signal peptide, seven type I cohesin domains, an internal family III
cellulose-binding domain (CBD), and an X2 module of unknown function in
tandem with a type II dockerin domain at the C terminus. Surprisingly,
CipV also possesses at its N terminus a catalytic module that belongs
to the family 9 glycosyl hydrolases. Sequence analysis indicated the
following. (i) The repeating cohesin domains are very similar to each
other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from
other known scaffoldin CBDs by the omission of a 9-residue stretch that
constitutes a characteristic loop previously associated with the
scaffoldins. (iii) The C-terminal type II dockerin domain is only the
second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The
X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in
the scaffoldin. (v) Unlike the other known family 9 catalytic modules
of bacterial origin, the CipV catalytic module is not accompanied by a
flanking helper module, e.g., an adjacent family IIIc CBD or an
immunoglobulin-like domain. Comparative sequence analysis of the CipV
functional modules with those of the previously sequenced scaffoldins
provides new insight into the structural arrangement and phylogeny of
this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C. thermocellum as opposed to the known scaffoldins from the
mesophilic clostridia. The phylogenetic relationship of the different
functional modules appears to indicate that the evolution of the
scaffoldins reflects a collection of independent events and mechanisms
whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.
| |
INTRODUCTION |
The cellulosome is a
multiprotein complex consisting of cellulolytic and
hemicellulolytic enzymes which has been described mainly in anaerobic
clostridia (5, 7, 13, 25). The cellulosomal enzymes are
attached to a large, multimodular, noncatalytic subunit called
scaffoldin. Four scaffoldin genes have been sequenced from the
following clostridial species: Clostridium thermocellum
(cipA) (15), Clostridium cellulovorans
(cbpA) (46), Clostridium
cellulolyticum (cipC) (38), and
Clostridium josui (cipJ) (for clarity, the CipA
scaffoldin from C. josui is renamed CipJ in this
communication) (24). All four contain multiple type I
cohesin domains which integrate type I dockerin-tagged enzymes
into the cellulosome complex. In addition, a family IIIa
cellulose-binding domain (CBD) in the scaffoldin is responsible for the
binding of the complex to its substrate, cellulose (39).
Another class of domain, a unique C-terminal dockerin domain
(categorized as a type II dockerin), has also been identifiedthus
far, only in the scaffoldin of C. thermocellum. The type II
dockerin is involved in anchoring this scaffoldin to the bacterial cell
wall by interacting selectively with a type II cohesin, borne by a
series of cell surface proteins (44). Only three such
anchoring proteins have been described, all in C. thermocellum (7). Finally, X2 modules of unknown function (11a) have been found in all four scaffoldin genes.
Acetivibrio cellulolyticus was first isolated from sewage
sludge and proved to be a highly efficient cellulolytic bacterium (26, 41, 42). The strain was classified in a new genus of cellulolytic, gram-negative, non-spore-forming, anaerobic mesophilic bacteria. Nevertheless, recent 16S ribosomal DNA analysis has suggested
that A. cellulolyticus is closely related to the clostridia (32).
In an earlier work (30), A. cellulolyticus
was found to resemble C. thermocellum in a variety of
cellulosome-related biochemical, immunochemical, and
ultrastructural properties. Notably, the cell surface topology of
A. cellulolyticus exhibited perhaps the most dramatic
display of exocellular protuberance structures yet observed (27). The critical question that remained, however,
was whether such organisms produced cellulosomes.
In recent years, the detection of cellulosome-related "signature
sequences" (such as cohesin or dockerin domains) in a protein has
become a clear indication that a given bacterium produces a cellulosome
(1). We therefore decided to try to supplement the previous
biochemical evidence, obtained for A. cellulolyticus, with genetic information. In doing so, we discovered a 10-kb
EcoRI fragment which contained an intact scaffoldin gene.
The gene, termed cipV, was sequenced in its entirety, and
its modular arrangement and sequence similarities with other known
scaffoldins were analyzed. The structural organization of the CipV
scaffoldin was found to resemble that of CipA from C. thermocellum. Nevertheless, it differs from that and other
scaffoldins thus far described by the inclusion of a sequence
consistent with a family 9 glycosyl hydrolase as an integral part of
its polypeptide chain. The entry of CipV into the family of scaffoldins
warrants phylogenetic treatment of its functional modules
vis-à-vis those of the four other known family members.
| |
MATERIALS AND METHODS |
Organism and growth conditions.
A. cellulolyticus ATCC
33288 was purchased from the German Collection of Microorganisms and
Cell Cultures (Braunschweig, Germany). The cells were grown
anaerobically at 37°C in serum bottles containing an American Type
Culture Collection recommended medium (1207 BC medium for A. cellulolyticus) which included either cellobiose (Sigma Chemical
Co., St. Louis, Mo.) or cellulose (Avicel; E. Merck AG, Darmstadt,
Germany) as a carbon source. The cells were grown to mid-exponential
phase (36 to 48 h), the culture was centrifuged (10,000 × g; 10 min), and both the supernatant fluids and the cells were
stored at 
20°C for further use.
Isolation and identification of candidate scaffoldin band(s) from
A. cellulolyticus.
Cell-free culture fluids of A. cellulolyticus were mixed with a 1% volume of a 10-mg/ml
suspension of amorphous cellulose (29). The suspension was
shaken at low speed at room temperature for 30 min and centrifuged at
10,000 × g for 30 min, and the supernatant fluids were
discarded. The pellet was resuspended in sodium dodecyl sulfate (SDS)
sample buffer (34), and the cellulose-adsorbed proteins were
separated by SDS-6% polyacrylamide gel electrophoresis (PAGE). The
gel was cut into three parts. One was stained with Coomassie brilliant
blue R250, destained, and photographed. The remaining two parts were
transferred electrophoretically onto nitrocellulose sheets, one of
which was stained with rabbit antibodies specific for the cellulosome
from C. thermocellum while the other was stained with
Griffonia simplicifolia GS-I lectin, as described previously
(30). The primary antibody was visualized with a second
(goat anti-rabbit) antibody-peroxidase conjugate, and the glycosylated
bands were visualized by peroxidase-conjugated lectin (both obtained
from Sigma). Prestained, low-range calibrated molecular weight
standards were obtained from Bio-Rad Laboratories (Hercules, Calif.).
The cellulosome from C. thermocellum was prepared by the
affinity digestion procedure (35).
Peptide sequencing.
Candidate protein bands, which were
recognized both by anticellulosomal antibodies and by GS-I lectin, were
extracted from the SDS-PAGE gel. The extracted proteins were subjected
to proteolysis with Lys-C, and the resultant peptides were resolved by
reverse-phase high-performance liquid chromatography and collected
manually (33). The purified peptide peaks were analyzed and
sequenced by Edman degradation (Protein Center, Technion, Haifa, Israel).
PCR cloning.
A. cellulolyticus genomic DNA was
isolated as described by Murray and Thompson (36).
Oligonucleotide primers were generated from the peptide sequence. PCR
was carried out at various annealing temperatures (40 to 60°C) in
order to obtain specific amplified products with the genomic DNA as a
template. The purified PCR products were cloned into the pGEM-T Easy
vector system (Promega, Madison, Wis.) and sequenced. The sequences
were compared with GenBank and known cellulosome-related proteins.
Southern blotting.
Genomic DNA (1 µg) was cleaved with
various restriction enzymes, e.g., BamHI,
HindIII, EcoRI, NcoI,
SacI, and PstI, and the fragments were separated
in 1% agarose gels. Relevant DNA fragments were labeled by the
digoxigenin system (Boehringer Mannheim, Mannheim, Germany), and
Southern blotting was performed according to the manufacturer's instructions.
Construction and screening of genomic library.
A.
cellulolyticus genomic DNA (10 µg) was cleaved by
EcoRI and ligated with EcoRI-predigested and
alkaline phosphatase-treated pUC19. The ligation was transferred into
E. coli XL-1 Blue (Stratagene, La Jolla, Calif.) and plated
onto Luria broth-ampicillin-X-Gal (5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside)-IPTG
(isopropyl--D-thiogalactopyranoside) plates. The
above-described labeled probe was used for screening the genomic
library. Hybridization was carried out according to the manufacturer's
instructions (Boehringer Mannheim). Putative positive clones were
verified by dot hybridization.
Nucleotide sequence accession number.
The DNA sequence for
the cipV gene reported here was deposited in the GenBank
database under accession no. AF155197.
| |
RESULTS |
Identification of scaffoldin and sequencing of relevant peptide
segment.
Cell culture extracts of cellobiose-grown A. cellulolyticus were treated with amorphous cellulose, and the
adsorbed fraction was subjected to SDS-PAGE. The separated proteins
were blotted onto nitrocellulose membranes and examined with
cellulosome-specific antibodies and GS-I lectin from G. simplicifolia. The antibodies were elicited in rabbits with intact
C. thermocellum cells (31). The whole-cell
preparation was adsorbed onto mutant cells and the residual antibody
species proved relatively selective for the cellulosomal scaffoldin
subunit. The Griffonia lectin is selective for
-galactosyl moieties, which appear to be characteristic determinants of cellulolytic bacteria in various species (30). The lectin was previously found to recognize cellulosomal oligosaccharides from
C. thermocellum and Bacteroides
cellulosolvens (16, 17).
With these probes, a 210-kDa protein band was identified by both the
antibodies and the lectin (Fig. 1). It is
noteworthy that this band stained relatively poorly with Coomassie blue
compared to other bands (e.g., the ~180-kDa band). On the basis of
the observed immuno-cross-reactivity and lectin-specific staining pattern, the high-molecular-weight (210,000) band was considered a
primary candidate for the scaffoldin subunit of the A. cellulolyticus cellulosome. This rationale provided a focused
approach for identification of the putative scaffoldin, thereby saving
us time and unproductive efforts on unsuitable prospects.
View larger version (75K):
[in this window]
[in a new window]
|
FIG. 1.
Identification of putative cellulosome-related proteins
in A. cellulolyticus. Ac, SDS-PAGE crude cell-free culture
fluids adsorbed to amorphous cellulose; Ab, Western blot analysis with
antibodies specific for the scaffoldin subunit from C. thermocellum; GSI, blotted protein bands cross-reacting with GS-I
lectin from G. simplicifolia; M, prestained, low-range
molecular mass markers; Ct, C. thermocellum cellulosome
standard. The relative molecular masses of designated bands are
indicated. Note that both the antibody and lectin probes labeled a
relatively faint 210-kDa Coomassie-stained polypeptide band.
|
|
The candidate Coomassie blue-stained protein band was extracted from
the SDS-PAGE gel and subjected to proteolysis with Lys-C protease.
Several peptides were purified and sequenced. The sequences obtained
were compared with those of known cellulosome-related proteins from
other species, and a 17-residue peptide (N'-VEFFNAGTQAQSNSIYP-C') appeared to be remarkably conserved, compared to a known segment of the
family III CBDs.
Amplification of an internal CBD fragment.
A degenerate
forward primer, pAC1 (5'-GTK GAA TTY TTY AAY GCN GG-3'), was
designed from the N terminus (N'-VEFFNA-C') of the above 17-residue
peptide sequence, which was homologous with family IIIa CBDs. A
degenerate reverse primer, pCBD (5'-TGW KYR WAR TTW SWC CAG
TC-3'), was then designed from a particularly conserved region of known
family IIIa CBDs. (Abbreviations for degenerate nucleotides are as
follows: K, G or T; Y, C or T; W, A or T; R, A or G; S, C or G; N, A,
C, G, or T.) With these two primers, a 350-bp fragment (AC3)
was amplified specifically from the A. cellulolyticus
genomic DNA by primers pAC1 and pCBD. Cloning and sequencing showed the deduced polypeptide from AC3 had
significantly high homology (approximate 50% identity) with known
family III CBDs. As expected, the sequence of the AC3 N
terminus was identical with the initial sequenced peptide (i.e.,
N'-GTQAQSNSIYP-C'), which indicated that AC3 was indeed part
of the gene encoding a putative scaffoldin subunit.
Cloning and DNA sequencing of the cipV scaffoldin
gene.
The mass of the putative scaffoldin subunit from the
A. cellulolyticus cellulosome is very similar to that of the
210,000-Da S1 band, which corresponds to the scaffoldin subunit from
C. thermocellum (Fig. 1). Consequently, the gene encoding
the putative scaffoldin from A. cellulolyticus would be
expected to exhibit a size (~6 kb) similar to that of the scaffoldin
gene (cipA) from C. thermocellum.
A. cellulolyticus chromosomal DNA was cleaved with various
restriction enzymes, and Southern blotting analysis, with
AC3 as
a probe, detected a ~10-kb
EcoRI band.
The size of this fragment
was particularly appealing, since, if a
prospective ~6-kb gene
was correctly situated there, it might contain
the entire gene
in a single fragment. To clone the 10-kb fragment, an
A. cellulolyticus genomic library was constructed in pUC19
with fully
EcoRI-digested
chromosomal DNA. The plasmid
library was screened by colony hybridization
with the
AC3
probe, and a colony containing the vector with the
~10-kb
EcoRI insert (pACE) was detected. The authenticity of the
insert was confirmed by Southern blotting of
EcoRI- and
PstI-digested
A. cellulolyticus chromosomal DNA.
By using a combined
PstI and
HindIII digest,
fragments from the insert were subcloned into
pUC19 and then sequenced.
Sequencing analysis indicated that the
10-kb insert contained two open
reading frames (ORFs) apparently
related to cellulosomal proteins. One
ORF represented the entire
gene encoding the scaffoldin subunit of
A. cellulolyticus, termed
CipV. The second ORF
represented an incomplete gene containing
at least two repeating
type II cohesin domains at its N terminus
(unpublished
data).
The complete sequence of the intact
cipV gene is shown in
Fig.
2. The
A. cellulolyticus scaffoldin gene consists of 5,748
nucleotides
encoding a 1,915-residue polypeptide
the largest scaffoldin
described
to date. The deduced polypeptide exhibited a calculated
molecular mass
of 199,496 Da, which is consistent with that (excluding
saccharide
components) of the identified candidate scaffoldin
subunit (Fig.
1).
The authenticity of the reading frame encoding
CipV was also confirmed
by an internal amino acid sequence of
the 17-residue segment located in
the resident CBD which is identical
with that of the peptide originally
identified from the putative
scaffoldin band.
View larger version (140K):
[in this window]
[in a new window]
|
FIG. 2.
Nucleotide and deduced amino acid sequences of the
A. cellulolyticus scaffoldin subunit (CipV). The proposed
Shine-Dalgarno sequence (SD) and the 10 and 35 regions of the
putative promoter are indicated. The signal sequence is shown in
italics, the intermodular linker sequences are underlined, and the
C-terminal dockerin domain is designated by a wavy underline. See Table
2 for the positions of the linkers relative to the adjoining modular
components, i.e., the N-terminal catalytic module, the CBD, the seven
cohesins, and domain X. The overall modular arrangement of CipV is
presented in Fig. 3.
|
|
The start codon (ATG) is preceded 8 bp upstream by a typical
Shine-Dalgarno sequence (GGAGG), homologous to other potential
ribosome-binding sites found in various
C. thermocellum
genes
and completely complementary to 5 bases (boldface) in the 3' end
of the 16S rRNA of
Bacillus subtilis
(3'-UCUUU
CCUCCACUAC-5').
A potential promoter is
located 110 bp from the ATG start codon
with conserved
10
(5'-
TATTAA-3') and
35
(5'-
TTGTTT-3')
regions
(boldface).
The N terminus of CipV commences with a putative 29-residue signal
peptide sequence, with a typical positively charged N-terminal
end, a
hydrophobic core, and a more polar carboxylic end with
alanines at
positions
1 and
3 (
50).
| |
DISCUSSION |
The cellulosome is a massive complex that contains multiple types
of enzymes that work synergistically to degrade crystalline cellulose
and other plant cell wall polysaccharides. The enzymes are
incorporated into the complex by virtue of a noncatalytic scaffoldin
subunit. Continued insight into how such a complex is constructed can
provide a model for other multicomponent protein systems.
Only a few complete scaffoldin sequences are currently available. Each
new sequence contributes new and vital information to our understanding
of the structural organization of cellulosomes and the interaction of
their manifold functional modules with each other and with the substrate.
Previously, biochemical and immunochemical methods were used to detect
cellulosome-like entities in various cellulolytic bacteria. In recent
years, however, conclusive establishment of the presence of
cellulosomes in a given bacterium has involved the identification of cellulosome signature sequences, which typically include cohesin and
dockerin domains (1). In the present study, the entire A. cellulolyticus scaffoldin subunit, CipV, was sequenced.
Comparative sequence analysis of its functional modules
with those of the previously sequenced scaffoldins provides new
insight into the structural arrangement and phylogeny of this
intriguing family of microbial proteins.
The modular organization of CipV.
CipV includes all of the
major domains found so far in cellulosomal scaffoldin proteins, with
the surprising and unprecedented addition of a family 9 glycosyl
hydrolase sequence as an integral part of the deduced polypeptide
chain. Until this work, catalytic modules have been found only in free
enzymes or in nonscaffoldin cellulosomal subunits.
Comparison of the domain organization of the CipV sequence with those
of the other clostridial species (Fig.
3)
reveals the
highest similarity with the
C. thermocellum
scaffoldin (
15).
Both scaffoldins contain an internal CBD,
flanked by multiple
copies of cohesin domains, with a single X2 module
of unknown
function and with a type II dockerin at the C terminus.
Nevertheless,
the precise position of the CBD and the number of
cohesins (seven
versus nine) are clearly different in the two strains.
In contrast,
scaffoldins from
C. cellulovorans,
C. cellulolyticum, and
C. josui exhibit N-terminal CBDs,
with multiple cohesin domains interspersed
with one or more copies of
internal X2 modules.
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 3.
Structural organization of the known scaffoldins from
different cellulosome species. The cohesin domains are designated by
numbers according to their sequential positions relative to the amino
terminus. Also indicated are the CBDs, the X2 domains (X), the type II
dockerins (II), and the CipV GH9. The modular organization is based on
the sequences of the respective scaffoldins, available from GenBank
with the following accession numbers: CipA from C. thermocellum, L08665; CbpA from C. cellulovorans,
M73817; CipC from C. cellulolyticum, U40345; and CipJ
from C. josui, AB004845.
|
|
Despite the intrinsic family 9 catalytic module of the
A. cellulolyticus CipV, it can be catalogued together with that
of
C. thermocellum to compose the class I
scaffoldins, on the basis
of its internal CBD and C-terminal dockerin
domain. These features
distinguish the class from the other currently
known scaffoldins
of class II (Fig.
3).
The signal peptide.
The sequence of the CipV signal peptide is
particularly homologous to that of C. thermocellum and also
has similarity to those of the other known cellulosomal scaffoldins
(Table 1). In fact, the interspecies
homology among the scaffoldin signal peptides is much more pronounced
than that between the genes for the scaffoldins and the other
cellulosomal components even within the same species. The observed
homology among the scaffoldin signal peptides might indicate a
specialized functional role, perhaps related to the assembly of the
cellulosome, such that attachment of the enzymatic subunits to
scaffoldin occurs while the latter is still associated to the cell
surface. In this context, it has been observed early on (4)
that the cellulosomal subunits, and the scaffoldin subunit in
particular, from C. thermocellum do not appear in the free, uncomplexed form.
The catalytic module.
Sequence analysis indicates that the
proposed catalytic module of CipV belongs to family 9 of the glycosyl
hydrolases (GH9). GH9 is a common component of cellulolytic enzymes in
bacteria and plants (11, 11a, 12).
Microbial family 9 cellulases commonly conform to one of four thematic
modular arrangements (
5,
6). The simplest GH9
theme is
typical of many plant cellulases and consists of a solitary
catalytic
module. The others contain different adjoining accessory
or helper
modules. For example, endoglucanase E4 from
Thermomonospora fusca (
43) includes a family IIIc CBD immediately
downstream
from its GH9 catalytic module. A third theme is exemplified
by
endoglucanase CelD from the
C. thermocellum cellulosome
(
23),
which bears an immunoglobulin (Ig)-like domain
upstream of the
catalytic module (
20). A fourth thematic
type also contains
an Ig-like domain in the same position, but in
addition, it includes
an N-terminal family IV CBD. In this respect, the
CipV catalytic
module of the
A. cellulolyticus scaffoldin
subunit is similar
to that of the plant GH9 cellulases in that it
lacks an adjoining
helper
module.
This thematic arrangement of the GH9 cellulases is mirrored in the
sequences of their catalytic modules alone, and the divergent
sequences
are reflected in the phylogenetic relationship of the
parent cellulases
(Fig.
4). Thus, the catalytic modules
from cellulases,
which include a fused family IIIc CBD (group A), all
map within
the same branch. On the other hand, the catalytic modules
that
bear an adjacent Ig-like domain fall into a cluster on the
opposite
side of the tree. Cellulases which have the Ig-like domain
only
(group B1) occupy a small separate branch, and those that also
include a family IV CBD (group B2) develop distally to form a
separate
subcluster. Another large cluster (group C) represents
plant enzymes
that generally lack adjoining helper modules. The
catalytic module of
CipV is distinct from the groups depicted
in Fig.
4, occupying a
position adjacent to the plant enzymes.
View larger version (65K):
[in this window]
[in a new window]
|
FIG. 4.
Phylogenetic analysis of the N-terminal family 9 catalytic module of CipV and its relationship with other family 9 members. The analysis of the following catalytic modules was performed
with GenBee based on the GenBank sequences (accession numbers in
parentheses): solid circle, CipV Acece, CipV scaffoldin from A. cellulolyticus (this communication), and Dictyostelium,
endoglucanase from Dictyostelium discoideum (M33861). Group
A enzymes: CelF Clotm, endoglucanase F from C. thermocellum
(X60545); CelZ Closr, exoglucanase Z from Clostridium
stercorarium (X55299); CelA Calsa, cellulase A from
Caldocellum saccharolyticum (L32742); CelG Cloce,
endoglucanase G from C. cellulolyticum (M87018); CelI Clotm,
endoglucanase I from C. thermocellum (L04735); CelB Celfi,
endoglucanase B from Cellulomonas fimi (M64644); E4 Thefu,
endo- or exoglucanase E4 from T. fusca (M73322). Group B1
enzymes: CelJ Clotm, cellulase J from C. thermocellum
(D83704); CelD Clotm, endoglucanase D from C. thermocellum
(X04584); CelC Butfi, endoglucanase C from Butyrivibrio
fibrisolvens (X55732). Group B2 enzymes: CbhA Clotm,
cellobiohydrolase A from C. thermocellum (X80993); CelA
Psefl, endoglucanase A from Pseudomonas fluorescens
(X12570); CelC Celfi, endoglucanase C from C. fimi (X57858);
CelI Strre, endoglucanase I from Streptomyces reticuli
(X65616); E1 Thefu, endoglucanase E1 from T. fusca (L20094).
Plant enzymes: cellulases from Prunus persica (X96853),
Populus alba (D32166), Citrus sinensis
(AF000135), Persea americana (M17634), Pinus
radiata (X96853), Arabidopsis thaliana (X98543),
Phaseolus vulgaris (M57400), Capsium annuum
(X97189), and Lycopersicon esculentum (U20590).
|
|
The discrete position of the CipV catalytic module in the phylogenetic
tree underscores the fact that it lacks an adjoining
helper module and
represents a new class of scaffoldin-associated
family 9 glycosyl
hydrolase.
Interdomain linker segments.
The sequences of the linkers,
which connect adjacent cellulosomal domains of A. cellulolyticus, are shown in Table
2. With the exception of linkers 6, 8, and 9, the relatively long linker segments of the A. cellulolyticus scaffoldin are rich in prolines and threonines, and
extended stretches of their sequences are remarkably similar. The long
linkers are reminiscent of those of the C. thermocellum
scaffoldin subunit but not those of C. cellulovorans,
C. cellulolyticum, or C. josui (for a comparison, see Table 1 of reference 3). The high incidence of
prolines suggests that the linkers form extended configurations, such
as the plant cell wall extensins (52), that physically
separate the various domains. In addition, proline-rich regions of
proteins have been suggested to cause rapid and nonspecific binding
(51), which in the case of scaffoldins may promote
intermodular and/or intersubunit protein-protein interactions. The
numerous threonines would be suitable glycosylation sites, as
demonstrated for the C. thermocellum scaffoldin
(18).
The first linker sequence that separates the CipV GH9 from cohesin 1 is
indicative of the scaffoldins and dissimilar to those
that usually
flank GH9 modules, notably the characteristic linker
segment that joins
a GH9 module to a family IIIc CBD (
43). The
lack of such a
linker and its replacement by a Pro- Thr-rich linker
underscores the
special nature of the CipV GH9
module.
The cohesin domains.
Multiple alignment of the seven repeating
cohesin domains of CipV reveals sequence homology of between 59 and
92%. The most diversity occurs between CohV-1 and CohV-6. In contrast,
the sequences of the first three N-terminal cohesins are very similar
(about 90% identity).
Multiple-sequence alignment among the type I cohesins showed a close
interspecies relationship, which would suggest that all
of these
cohesin domains would assume the same general structural
fold as that
of the recently determined cohesin structures (
45,
47). The
cohesin sequences can be compared by phylogenetic analysis
(
8). An unrooted phylogenetic tree of the known type I
cohesins
(Fig.
5) indicates that the
cellulosomal cohesins from each species
generally form a tight cluster.
Interestingly, the tree places
the CipV cohesins on a separate branch
between those of the other
mesophilic bacteria (i.e.,
C. cellulovorans,
C. cellulolyticum,
and
C. josui), while the cellulosomal cohesins of thermophilic
C. thermocellum occupy an opposing branch on the tree. The two
known
noncellulosomal type I cohesins (OlpA from
C. thermocellum and OrfX from
C. cellulolyticum) maintain discrete positions
on
the tree which radiate away from the other cohesin clusters. It
should be mentioned that, when analyzed together, the type I and
type
II cohesins form two distinct clusters on opposite sides
of the
resultant phylogenetic tree (
2).
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 5.
Phylogenetic relationship of the CipV cohesin domains
with other type I cohesins. The sequences of the scaffoldin-borne
cohesins were obtained from the GenBank accession numbers shown in the
legend to Fig. 3. The nonscaffoldin, type I cohesins were OlpA from
C. thermocellum (X67506) and OrfX from C. cellulolyticum (AF081458). The darkly shaded oval represents the
weighted centroid of the tree.
|
|
The CBD.
The internal CipV CBD belongs to the family III CBDs,
classified according to sequence alignment (48). The
CBDs of this family are separated into two functionally different
types. One type (comprising the family IIIa and IIIb CBDs) binds
strongly to crystalline cellulose, and another (the aforementioned
family IIIc CBDs fused to a family 9 glycosyl hydrolase) fails to bind crystalline cellulose but reportedly serves in a helper role in the
hydrolysis of a single cellulose chain (5, 43).
The CBDs of families IIIa and IIIb were previously proposed to be
distinguished by the nature of the parent protein, the family
IIIa CBDs
being component parts of cellulosomal scaffoldin subunits
and the
family IIIb CBDs being the targeting agent for free noncellulosomal
enzymes (
3). In this context, the family IIIa CBDs contain
a
9-residue segment called the scaffoldin loop which includes
a proposed
cellulose-binding tyrosine residue (Y67, numbered according
to the
system of Tormo et al. [
49]). This loop, together with
the distinctive tyrosine, is missing in all of the family IIIb
CBDs. In
addition, another putative binding residue (position
57) is different
in the two subfamilies: a histidine appears in
this position in the
family IIIa CBDs, and in those of family
IIIb the residue is a
tryptophan.
The phylogenetic relationship of the family III CBDs follows the
general pattern of their functions (Fig.
6). Thus, all of
the family IIIc CBDs
form a distinct cluster on one side of the
tree. On the opposite side
of the weighted centroid are scattered
the family IIIb CBDs. The family
IIIa CBDs from the clostridial
scaffoldins occupy a single branch,
which emanates from an intermediate
position among the family IIIb
CBDs.
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 6.
Phylogenetic analysis of the CipV CBD and its
relationship to other scaffoldin and nonscaffoldin family III CBDs.
Scaffoldin CBDs are shown as squares, and enzyme-borne CBDs are shown
as circles. The sequences were obtained from the respective GenBank
accession numbers (in parentheses): CelY Closr, endoglucanase Y from
Clostridium stercorarium (Z69359); Man/Cel Calsa, mannanase
or cellulase from Caldocellum saccharolyticum (L01257);
EGase Bacsu, endoglucanase from B. subtilis (M16185); CelN
Erwca, cellulase N from Erwinia carotovora subsp.
atroseptica (L39788); CelA Bacla, cellulase A from
Bacillus lautus (M76588). The sources of the sequences of
other family III CBDs are shown in the legends to Fig. 3 and 4. The
darkly shaded oval represents the weighted centroid of the tree.
|
|
According to the phylogenetic tree, the CipV CBD clearly belongs to the
family IIIb CBDs. It forms a separate subbranch with
several other CBDs
of this family, derived from clostridial cellulases.
In addition, the
CipV CBD lacks the scaffoldin loop (and its intrinsic
tyrosine residue)
and has a tryptophan rather than a histidine
at position 57. These
observations immediately suggest that the
scaffoldin loop is not
strictly definitive of the scaffoldins,
and the relationship among the
family IIIa and IIIb CBDs is not
necessarily a precise function of its
parent protein (i.e., a
free noncellulosomal enzyme versus a
cellulosomal scaffoldin subunit).
In any case, further attempts at
categorizing this particular
family of CBDs should await a larger
collection of relevant
sequences.
The X2 module.
The function of this particular type of module
(currently designated the X2 module by Coutinho and Henrissat
[11a]) remains unknown. Originally, the X2 module was
alternatively referred to as the hydrophilic domain in C. cellulovorans (as opposed to the hydrophobic cohesins
[46]) and as domain X in C. thermocellum (28), and the latter distinction is relevant to the current discussion. Interestingly, the phylogenetic relationship of the homologous domains (Fig. 7) appears to
reflect the overall modular organization of the parent scaffoldin
subunit (Fig. 3). Thus, the sequences of the domains X from the group I
scaffoldins (i.e., A. cellulolyticus CipV and C. thermocellum CipA) form a small cluster on the phylogenetic tree
that maps on the opposite pole of the weighted centroid from the
hydrophilic domains of the group II scaffoldins.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 7.
Phylogenetic distribution of X2 modules. The
scaffoldin-derived modules are shown as squares, the enzyme-derived
modules are shown as circles, and cell wall-associated proteins (Sdr)
from S. aureus (GenBank accession no. AJ005646) are shown as
triangles. The numbers indicate repeated modules, according to their
sequential arrangement with respect to the N terminus of the given
protein. The accession numbers of the other proteins are provided in
the legends to Fig. 3, 4, and 6.
|
|
It is interesting to note that related X2 domains occur in a few free
cellulases as well, which either occupy divergent branches
or cluster
together with the group II hydrophilic domains. Another
set of newly
described cell wall-associated proteins from
Staphylococcus aureus (
21,
22) has also been shown to contain repeated
domains
(called B motifs), which appear to be closely related to the
domains
X in the group I
scaffoldins.
Until we know the precise function(s) of the X2 module, it will be hard
to assess the biological and/or structural consequences
of the observed
sequence-based
clustering.
Type II dockerin domain.
Like that of the CipA scaffoldin from
C. thermocellum, the C terminus of CipV exhibits a type II
dockerin domain, which presumably interacts with a type II cohesin of a
putative anchoring protein. This is only the second such dockerin
domain to be discovered. Moreover, the type II cohesins of the
incomplete ORF that appears immediately downstream from the scaffoldin
gene signify a potential anchoring protein in A. cellulolyticus, suggestive of the anchoring protein that occurs in
a similar position on the C. thermocellum genome
(14). Both of the known type II dockerins appear in tandem with a domain X, and this modular dyad may represent a functional theme
that implies interaction with a type II cohesin and consequent anchoring to the cell surface.
The
A. cellulolyticus CipV dockerin sequence is organized in
a seven-division arrangement, as described previously for both
the type I and type II dockerins (
37). The dockerins
include
a 22-residue duplicated sequence that contains a 12-residue
calcium-binding
loop (Fig.
8)
(
10). The designated calcium-binding residues
usually
involve conserved aspartic acids, asparagines, and sometimes
hydroxyamino acids (serines or threonines) (
9),
and such residues
indeed appear at appropriate positions within
the type II CipV
dockerin. However, the most striking feature of this
dockerin
is a 4-residue insert in the first duplicated calcium-binding
motif. Digressions from the canonical EF-hand calcium-binding
motif are
infrequent but have been observed in some type I dockerins
as well as
in the only other known type II dockerin from
C. thermocellum,
in which one of the usual calcium-binding residues
is replaced
by a valine. Such replacements or inserts could
result in reduced
binding affinity for calcium, or
alternative components (e.g.,
backbone atoms) may compensate
for the replaced side chain (
19).
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 8.
Deduced amino acid sequence alignment of the C-terminal
type II dockerin domain from CipV with that of C. thermocellum and their relationship to selected type I dockerins
from various cellulosomal enzyme subunits. The GenBank accession
numbers for CelA (family 5 glycosyl hydrolase from C. cellulolyticum) and CelS (family 48 glycosyl hydrolase from
C. thermocellum) are M93096 and L06942, respectively.
|
|
Molecular evolution of cellulosome-related proteins.
Comparison of the structural arrangement of the CipV scaffoldin subunit
with the phylogenetic relationship among the different modular types is
revealing. Although ancestral information is lacking from the unrooted
phylogenetic trees, the branching points indicate the relationship
between the neighboring intra- or interspecies homologues.
The phylogeny of the various cellulosomal components from the
cellulosome-producing bacteria does not necessarily reflect
the
phylogenetic relationships of the bacteria themselves (
40).
Moreover, the phylogenetic relationships among the individual
types of
functional modules of the
A. cellulolyticus scaffoldin
are,
in some instances, quite different. For example, the CipV
cohesins are
more similar to other mesophilic cohesins than to
those of
C. thermocellum, whereas the X2 module, the type II dockerin,
and the
linkers are clearly more similar to those of
C. thermocellum.
Furthermore, the CipV CBD is unlike those of the
other known scaffoldins
and is classified in family IIIb on the basis
of sequence
homology.
The phylogenetic clustering of the cohesins of the
A. cellulolyticus scaffoldin indicates that their evolutionary
acquisition
may have involved initial lateral gene transfer of a single
cohesin,
followed by domain insertion, multiplication and/or shuffling,
and then divergence by conventional mutagenesis (i.e., accumulation
of
point mutations leading to compositional assimilation). At
some point
in the process, the genetic material that encoded the
cohesin(s) joined
that of the other types of functional modules
and linkers to form the
scaffoldin gene. It is not clear when
speciation of
A. cellulolyticus occurred, with respect to the
development of the
cellulosomal genes. In this context, it is
interesting to note that
A. cellulolyticus has been proposed to
be a member of the
greater clostridial assemblage on the basis
of 16S ribosomal DNA
sequences (
32).
Taken together, the phylogenetic information appears to indicate that
the evolution of the scaffoldins reflects a collection
of independent
events and mechanisms. The individual functional
modules and other
constituents appear to have been obtained from
different microbial
sources, incorporated into the scaffoldin
gene, and modified to meet
the needs of the overall system. Future
sequence data from additional
scaffoldins and other cellulosome-related
components will undoubtedly
refine our views of the phylogenetics
of cellulosome
structure.
The most extraordinary finding, however, was the discovery that the
family 9 glycosyl hydrolase sequence is an integral part
of CipV.
This may indicate the central importance of such an enzyme
to cellulosome action and may portend a trend in as-yet-undescribed
scaffoldins from other
microorganisms.
| |
ACKNOWLEDGMENTS |
This work was supported by a contract from the European
Commission (Biotechnology Programme; BIO4-97-2303) and by grants from the Israel Science Foundation (administered by the Israel Academy of
Sciences and Humanities, Jerusalem, Israel). Additional support was
provided by the Otto Meyerhof Center for Biotechnology, established by
the Minerva Foundation, Munich, Germany.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Chemistry, The Weizmann Institute of Science, Rehovot,
Israel. Phone: (972)-8-934-2373. Fax: (972)-8-946-8256. E-mail:
bfbayer{at}weizmann.weizmann.ac.il.
| |
REFERENCES |
| 1.
|
Bayer, E. A.,
H. Chanzy,
R. Lamed, and Y. Shoham.
1998.
Cellulose, cellulases and cellulosomes.
Curr. Opin. Struct. Biol.
8:548-557[Medline].
|
| 2.
| Bayer, E. A., S.-Y. Ding, A. Mechaly, Y. Shoham, and R. Lamed. Emerging phylogenetics of cellulosome
structure. In H. Gilbert (ed.), Advances in carbohydrate
bioengineering, in press. The Royal Society of Chemistry, London,
United Kingdom.
|
| 3.
|
Bayer, E. A.,
E. Morag,
R. Lamed,
S. Yaron, and Y. Shoham.
1998.
Cellulosome structure: four-pronged attack using biochemistry, molecular biology, crystallography and bioinformatics, p. 39-67.
In
M. Claeyssens, W. Nerinckx, and K. Piens (ed.), Carbohydrases from Trichoderma reesei and other microorganisms. The Royal Society of Chemistry, London, United Kingdom
|
| 4.
|
Bayer, E. A.,
E. Setter, and R. Lamed.
1985.
Organization and distribution of the cellulosome in Clostridium thermocellum.
J. Bacteriol.
163:552-559[Abstract/Free Full Text].
|
| 5.
|
Bayer, E. A.,
L. J. W. Shimon,
R. Lamed, and Y. Shoham.
1998.
Cellulosomes: structure and ultrastructure.
J. Struct. Biol.
124:221-234[Medline].
|
| 6.
| Bayer, E. A., Y. Shoham, and R. Lamed. The
cellulosome: an exocellular organelle for degrading plant cell wall
polysaccharides. In R. J. Doyle (ed.),
Glycomicrobiology, in press. Plenum, New York, N.Y.
|
| 7.
|
Béguin, P., and M. Lemaire.
1996.
The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation.
Crit. Rev. Biochem. Mol. Biol.
31:201-236[Medline].
|
| 8.
|
Belaich, J.-P.,
C. Tardif,
A. Belaich, and C. Gaudin.
1997.
The cellulolytic system of Clostridium cellulolyticum.
J. Biotechnol.
57:3-14[Medline].
|
| 9.
|
Bränden, C. I., and J. Tooze.
1991.
Introduction to protein structure, p. 22.
Garland Publishers, Inc., New York, N.Y
|
| 10.
|
Chauvaux, S.,
P. Béguin,
J.-P. Aubert,
K. M. Bhat,
L. A. Gow,
T. M. Wood, and A. Bairoch.
1990.
Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.
Biochem. J.
265:261-265[Medline].
|
| 11.
| Coutinho, P. M., and B. Henrissat. 27 August
1999, revision date. CAZy website, Carbohydrate-active enzymes server.
[Online.] http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html. [22
September 1999, last date accessed.]
|
| 11a.
| Coutinho, P. M., and B. Henrissat. 9 September
1999, revision date. CAZyModO website, Carbohydrate-active enzymes
server. [Online.] http://afmb.cnrs-mrs.fr/~pedro/DB/db.html.
[22 September 1999, last date accessed.]
|
| 12.
|
Coutinho, P. M., and B. Henrissat.
1999.
The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach, p. 15-23.
In
K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita, and T. Kimura (ed.), Genetics, biochemistry and ecology of cellulose degradation. Uni Publishers Co., Ltd., Tokyo, Japan
|
| 13.
|
Felix, C. R., and L. G. Ljungdahl.
1993.
The cellulosomethe exocellular organelle of Clostridium.
Annu. Rev. Microbiol.
47:791-819[Medline].
|
| 14.
|
Fujino, T.,
P. Béguin, and J.-P. Aubert.
1993.
Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface.
J. Bacteriol.
175:1891-1899[Abstract/Free Full Text].
|
| 15.
|
Gerngross, U. T.,
M. P. M. Romaniec,
T. Kobayashi,
N. S. Huskisson, and A. L. Demain.
1993.
Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology.
Mol. Microbiol.
8:325-334[Medline].
|
| 16.
|
Gerwig, G.,
P. de Waard,
J. P. Kamerling,
J. F. G. Vliegenthart,
E. Morgenstern,
R. Lamed, and E. A. Bayer.
1989.
Novel O-linked carbohydrate chains in the cellulase complex (cellulosome) of Clostridium thermocellum.
J. Biol. Chem.
264:1027-1035[Abstract/Free Full Text].
|
| 17.
|
Gerwig, G.,
J. P. Kamerling,
J. F. G. Vliegenthart,
E. Morag (Morgenstern),
R. Lamed, and E. A. Bayer.
1992.
Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.
Eur. J. Biochem.
205:799-808[Medline].
|
| 18.
|
Gerwig, G.,
J. P. Kamerling,
J. F. G. Vliegenthart,
E. Morag,
R. Lamed, and E. A. Bayer.
1993.
The nature of the carbohydrate-peptide linkage region in glycoproteins from the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens.
J. Biol. Chem.
268:26956-26960[Abstract/Free Full Text].
|
| 19.
|
Hohenester, H.,
P. Maurer,
C. Hohenadl,
R. Timpl,
J. N. Jansonius, and J. Engel.
1996.
Structure of a novel extracellular Ca2+-binding module in BM-40.
Nat. Struct. Biol.
3:67-73[Medline].
|
| 20.
|
Joliff, G.,
P. Béguin, and J.-P. Aubert.
1986.
Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum.
Nucleic Acids Res.
14:8605-8613[Abstract/Free Full Text].
|
| 21.
|
Josefsson, E.,
K. W. McCrea,
D. Ni Eidhin,
D. O'Connell,
J. Cox,
M. Hook, and T. J. Foster.
1998.
Three new members of the serine-aspartate repeat protein multigene family of Staphylococcus aureus.
Microbiology
144:3387-3395[Abstract/Free Full Text].
|
| 22.
|
Josefsson, E.,
D. O'Connell,
T. J. Foster,
I. Durussel, and J. Cox.
1998.
The binding of calcium to the B-repeat segment of SdrD, a cell surface protein of Staphylococcus aureus.
J. Biol. Chem.
273:31145-31152[Abstract/Free Full Text].
|
| 23.
|
Juy, M.,
A. G. Amit,
P. M. Alzari,
R. J. Poljak,
M. Claeyssens,
P. Béguin, and J.-P. Aubert.
1992.
Crystal structure of a thermostable bacterial cellulose-degrading enzyme.
Nature
357:39-41.
|
| 24.
|
Kakiuchi, M.,
A. Isui,
K. Suzuki,
T. Fujino,
E. Fujino,
T. Kimura,
S. Karita,
K. Sakka, and K. Ohmiya.
1998.
Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome.
J. Bacteriol.
180:4303-4308[Abstract/Free Full Text].
|
| 25.
|
Karita, S.,
K. Sakka, and K. Ohmiya.
1997.
Cellulosomes, cellulase complexes, of anaerobic microbes: their structure models and functions, p. 47-57.
In
R. Onodera, H. Itabashi, K. Ushida, H. Yano, and Y. Sasaki (ed.), Rumen microbes and digestive physiology in ruminants. Japan Scientific Society Press, Tokyo, Japan
|
| 26.
|
Khan, A. W.
1980.
Cellulolytic enzyme system of Acetivibrio cellulolyticus, a newly isolated anaerobe.
J. Gen. Microbiol.
121:499-502.
|
| 27.
|
Lamed, E.,
J. Naimark,
E. Morgenstern, and E. A. Bayer.
1987.
Scanning electron microscopic delineation of bacterial surface topology using cationized ferritin.
J. Microbiol. Methods
7:233-240.
|
| 28.
|
Lamed, R., and E. A. Bayer.
1993.
The cellulosome concepta decade later!, p. 1-12.
In
K. Shimada, S. Hoshino, K. Ohmiya, K. Sakka, Y. Kobayashi, and S. Karita (ed.), Genetics, biochemistry and ecology of lignocellulose degradation. Uni Publishers Co., Ltd., Tokyo, Japan
|
| 29.
|
Lamed, R.,
R. Kenig,
E. Setter, and E. A. Bayer.
1985.
Major characteristics of the cellulolytic system of Clostridium thermocellum coincide with those of the purified cellulosome.
Enzyme Microb. Technol.
7:37-41.
|
| 30.
|
Lamed, R.,
J. Naimark,
E. Morgenstern, and E. A. Bayer.
1987.
Specialized cell surface structures in cellulolytic bacteria.
J. Bacteriol.
169:3792-3800[Abstract/Free Full Text].
|
| 31.
|
Lamed, R.,
E. Setter, and E. A. Bayer.
1983.
Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.
J. Bacteriol.
156:828-836[Abstract/Free Full Text].
|
| 32.
|
Lin, C.,
J. W. Urbance, and D. A. Stahl.
1994.
Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage.
FEMS Microbiol. Lett.
124:151-155[Medline].
|
| 33.
|
Matsudaira, P.
1993.
A practical guide to protein and peptide purification for microsequencing, 2nd ed.
Academic Press, New York, N.Y
|
| 34.
|
Morag, E.,
E. A. Bayer, and R. Lamed.
1990.
Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes.
J. Bacteriol.
172:6098-6105[Abstract/Free Full Text].
|
| 35.
|
Morag, E.,
E. A. Bayer, and R. Lamed.
1992.
Affinity digestion for the near-total recovery of purified cellulosome from Clostridium thermocellum.
Enzyme Microb. Technol.
14:289-292.
|
| 36.
|
Murray, M. G., and W. F. Thompson.
1980.
Rapid isolation of high-molecular-weight plant DNA.
Nucleic Acids Res.
8:4321-4325[Abstract/Free Full Text].
|
| 37.
|
Pagès, S.,
A. Belaich,
J.-P. Belaich,
E. Morag,
R. Lamed,
Y. Shoham, and E. A. Bayer.
1997.
Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain.
Proteins
29:517-527[Medline].
|
| 38.
|
Pagès, S.,
A. Belaich,
H.-P. Fierobe,
C. Tardif,
C. Gaudin, and J.-P. Belaich.
1999.
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.
J. Bacteriol.
181:1801-1810[Abstract/Free Full Text].
|
| 39.
|
Poole, D. M.,
E. Morag,
R. Lamed,
E. A. Bayer,
G. P. Hazlewood, and H. J. Gilbert.
1992.
Identification of the cellulose binding domain of the cellulosome subunit S1 from Clostridium thermocellum.
FEMS Microbiol. Lett.
99:181-186.
|
| 40.
|
Rainey, F. A., and E. Stackebrandt.
1993.
16S rDNA analysis reveals phylogenetic diversity among the polysaccharolytic clostridia.
FEMS Microbiol. Lett.
113:125-128[Medline].
|
| 41.
|
Saddler, J. N., and A. W. Khan.
1980.
Cellulase production by Acetivibrio cellulolyticus.
Can. J. Microbiol.
26:760-765.
|
| 42.
|
Saddler, J. N., and A. W. Khan.
1981.
Cellulolytic enzyme system of Acetivibrio cellulolyticus.
Can. J. Microbiol.
27:288-294[Medline].
|
| 43.
|
Sakon, J.,
D. Irwin,
D. B. Wilson, and P. A. Karplus.
1997.
Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca.
Nat. Struct. Biol.
4:810-818[Medline].
|
| 44.
|
Salamitou, S.,
O. Raynaud,
M. Lemaire,
M. Coughlan,
P. Béguin, and J.-P. Aubert.
1994.
Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA.
J. Bacteriol.
176:2822-2827[Abstract/Free Full Text].
|
| 45.
|
Shimon, L. J. W.,
E. A. Bayer,
E. Morag,
R. Lamed,
S. Yaron,
Y. Shoham, and F. Frolow.
1997.
The crystal structure at 2.15 Å resolution of a cohesin domain of the cellulosome from Clostridium thermocellum.
Structure
5:381-390[Medline].
|
| 46.
|
Shoseyov, O.,
M. Takagi,
M. A. Goldstein, and R. H. Doi.
1992.
Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A.
Proc. Natl. Acad. Sci. USA
89:3483-3487[Abstract/Free Full Text].
|
| 47.
|
Tavares, G. A.,
P. Béguin, and P. M. Alzari.
1997.
The crystal structure of a type I cohesin domain at 1.7 Å resolution.
J. Mol. Biol.
273:701-713[Medline].
|
| 48.
|
Tomme, P.,
R. A. J. Warren,
R. C. Miller,
D. G. Kilburn, and N. R. Gilkes.
1995.
Cellulose-binding domainsclassification and properties, p. 142-161.
In
J. M. Saddler, and M. H. Penner (ed.), Enzymatic degradation of insoluble polysaccharides. American Chemical Society, Washington, D.C.
|
| 49.
|
Tormo, J.,
R. Lamed,
A. J. Chirino,
E. Morag,
E. A. Bayer,
Y. Shoham, and T. A. Steitz.
1996.
Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.
EMBO J.
15:5739-5751[Medline].
|
| 50.
|
Von Heijne, G.
1986.
A new method for predicting signal sequence cleavage sites.
Nucleic Acids Res.
14:4683-4690[Abstract/Free Full Text].
|
| 51.
|
Williamson, M. P.
1994.
The structure and function of proline-rich regions in proteins.
Biochem. J.
297:249-260.
|
| 52.
|
Woessner, J. P., and U. W. Goodenough.
1992.
Zygote and vegetative cell wall proteins in Clamydomonas reinhardtii share a common epitope (SerPro)x.
Plant Sci.
83:65-76.
|
Journal of Bacteriology, November 1999, p. 6720-6729, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Mingardon, F., Chanal, A., Tardif, C., Bayer, E. A., Fierobe, H.-P.
(2007). Exploration of New Geometries in Cellulosome-Like Chimeras. Appl. Environ. Microbiol.
73: 7138-7149
[Abstract]
[Full Text]
-
Rincon, M. T., Cepeljnik, T., Martin, J. C., Lamed, R., Barak, Y., Bayer, E. A., Flint, H. J.
(2005). Unconventional Mode of Attachment of the Ruminococcus flavefaciens Cellulosome to the Cell Surface. J. Bacteriol.
187: 7569-7578
[Abstract]
[Full Text]
-
Demain, A. L., Newcomb, M., Wu, J. H. D.
(2005). Cellulase, Clostridia, and Ethanol. Microbiol. Mol. Biol. Rev.
69: 124-154
[Abstract]
[Full Text]
-
Xu, Q., Barak, Y., Kenig, R., Shoham, Y., Bayer, E. A., Lamed, R.
(2004). A Novel Acetivibrio cellulolyticus Anchoring Scaffoldin That Bears Divergent Cohesins. J. Bacteriol.
186: 5782-5789
[Abstract]
[Full Text]
-
Rincon, M. T., Martin, J. C., Aurilia, V., McCrae, S. I., Rucklidge, G. J., Reid, M. D., Bayer, E. A., Lamed, R., Flint, H. J.
(2004). ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome. J. Bacteriol.
186: 2576-2585
[Abstract]
[Full Text]
-
Xu, Q., Bayer, E. A., Goldman, M., Kenig, R., Shoham, Y., Lamed, R.
(2004). Architecture of the Bacteroides cellulosolvens Cellulosome: Description of a Cell Surface-Anchoring Scaffoldin and a Family 48 Cellulase. J. Bacteriol.
186: 968-977
[Abstract]
[Full Text]
-
Devillard, E., Goodheart, D. B., Karnati, S. K. R., Bayer, E. A., Lamed, R., Miron, J., Nelson, K. E., Morrison, M.
(2004). Ruminococcus albus 8 Mutants Defective in Cellulose Degradation Are Deficient in Two Processive Endocellulases, Cel48A and Cel9B, Both of Which Possess a Novel Modular Architecture. J. Bacteriol.
186: 136-145
[Abstract]
[Full Text]
-
Doi, R. H., Kosugi, A., Murashima, K., Tamaru, Y., Han, S. O.
(2003). Cellulosomes from Mesophilic Bacteria. J. Bacteriol.
185: 5907-5914
[Full Text]
-
Xu, Q., Gao, W., Ding, S.-Y., Kenig, R., Shoham, Y., Bayer, E. A., Lamed, R.
(2003). The Cellulosome System of Acetivibrio cellulolyticus Includes a Novel Type of Adaptor Protein and a Cell Surface Anchoring Protein. J. Bacteriol.
185: 4548-4557
[Abstract]
[Full Text]
-
Rincon, M. T., Ding, S.-Y., McCrae, S. I., Martin, J. C., Aurilia, V., Lamed, R., Shoham, Y., Bayer, E. A., Flint, H. J.
(2003). Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens. J. Bacteriol.
185: 703-713
[Abstract]
[Full Text]
-
Arai, T., Araki, R., Tanaka, A., Karita, S., Kimura, T., Sakka, K., Ohmiya, K.
(2003). Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from Clostridium thermocellum CelJ: Importance of the CBM to Cellulose Hydrolysis. J. Bacteriol.
185: 504-512
[Abstract]
[Full Text]
-
Belaich, A., Parsiegla, G., Gal, L., Villard, C., Haser, R., Belaich, J.-P.
(2002). Cel9M, a New Family 9 Cellulase of the Clostridium cellulolyticum Cellulosome. J. Bacteriol.
184: 1378-1384
[Abstract]
[Full Text]
-
Ding, S.-Y., Rincon, M. T., Lamed, R., Martin, J. C., McCrae, S. I., Aurilia, V., Shoham, Y., Bayer, E. A., Flint, H. J.
(2001). Cellulosomal Scaffoldin-Like Proteins from Ruminococcus flavefaciens. J. Bacteriol.
183: 1945-1953
[Abstract]
[Full Text]
-
Ding, S.-Y., Bayer, E. A., Steiner, D., Shoham, Y., Lamed, R.
(2000). A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins. J. Bacteriol.
182: 4915-4925
[Abstract]
[Full Text]
-
Mechaly, A., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Lamed, R., Shoham, Y., Bayer, E. A.
(2001). Cohesin-Dockerin Interaction in Cellulosome Assembly. A SINGLE HYDROXYL GROUP OF A DOCKERIN DOMAIN DISTINGUISHES BETWEEN NONRECOGNITION AND HIGH AFFINITY RECOGNITION. J. Biol. Chem.
276: 9883-9888
[Abstract]
[Full Text]
Top
Abstract
Introduction
