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Journal of Bacteriology, May 1999, p. 3270-3276, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Three Surface Layer Homology Domains at the N
Terminus of the Clostridium cellulovorans Major Cellulosomal
Subunit EngE
Yutaka
Tamaru and
Roy H.
Doi*
Section of Molecular and Cellular Biology,
University of California, Davis, California 95616
Received 14 December 1998/Accepted 19 March 1999
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ABSTRACT |
The gene engE, coding for endoglucanase E, one of the
three major subunits of the Clostridium cellulovorans
cellulosome, has been isolated and sequenced. engE is
comprised of an open reading frame (ORF) of 3,090 bp and encodes a
protein of 1,030 amino acids with a molecular weight of 111,796. The
amino acid sequence derived from engE revealed a structure
consisting of catalytic and noncatalytic domains. The N-terminal-half
region of EngE consisted of a signal peptide of 31 amino acid residues
and three repeated surface layer homology (SLH) domains, which were
highly conserved and homologous to an S-layer protein from the
gram-negative bacterium Caulobacter crescentus. The
C-terminal-half region, which is necessary for the enzymatic function
of EngE and for binding of EngE to the scaffolding protein CbpA,
consisted of a catalytic domain homologous to that of family 5 of the
glycosyl hydrolases, a domain of unknown function, and a duplicated
sequence (DS or dockerin) at its C terminus. engE is
located downstream of an ORF, ORF1, that is homologous to the
Bacillus subtilis phosphomethylpyrimidine kinase (pmk) gene. The unique presence of three SLH domains and a
DS suggests that EngE is capable of binding both to CbpA to form a
CbpA-EngE cellulosome complex and to the surface layer of C. cellulovorans.
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INTRODUCTION |
Clostridium cellulovorans
is a gram-negative, mesophilic, anaerobic, spore-forming bacterium,
which utilizes not only cellulose but also xylan, pectin, and several
other carbon sources (35). C. cellulovorans
produces an extracellular cellulolytic multienzyme complex, which has
been called the cellulosome (17), has a total molecular
weight of about one million, and is capable of hydrolyzing crystalline cellulose.
Cellulolytic bacteria inhabit natural environments in which cellulose
is present primarily in plant cell walls in association with other
polysaccharides (e.g., xylan, mannan, and other hemicellulose components) (27). We have recently found that the C. cellulovorans enzyme complex can degrade several components of
plant cell walls in addition to cellulose, such as xylan, mannan,
lichenan, and pectin (36).
The cellulosome of C. cellulovorans is comprised of three
major subunits, the scaffolding protein CbpA (4, 32, 34), an
exoglucanase ExgS (20), and a third major endoglucanase
subunit EngE, previously designated P100 (5, 32), with a
molecular mass close to 100 kDa. In addition, the cellulosome contains
several enzyme subunits including endoglucanases EngB (7,
8), EngL, and EngY and a mannanase, ManA (36), each
containing a duplicated sequence (DS or dockerin) consisting of
22-amino-acid repeats.
Our ultimate goal is to elucidate the relationships between the
structure and function and the regulatory systems of a variety of
enzymes involved in the cellulosome system of C. cellulovorans. In this study, we report the properties of
engE and EngE. The derived amino acid sequence of EngE has
revealed the presence of three repeated surface layer homology (SLH)
domains at its N terminus and a duplicated sequence (DS) at its C
terminus. These SLHs also have some homology to the hydrophilic domains
(HLDs) of the scaffolding protein CbpA (36). The interesting
SLH domain structure of EngE suggests that EngE not only binds to the
scaffolding protein CbpA through its DS but that it may also bind to
the cell surface through its SLH domains.
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MATERIALS AND METHODS |
Bacterial strains, cloning vectors, and media.
C.
cellulovorans (ATCC 35296) (35) was used as the source
of chromosomal DNA and for the preparation of cellulosomal proteins. Escherichia coli XL1-Blue MRF' and SOLR strains and 
ZAPII
pBluescript SK(
) and pBluescript II KS(+) plasmids served as the
cloning hosts and vectors (Stratagene, La Jolla, Calif.), respectively. plaques were formed after E. coli cells were transfected
with phages in NZY medium containing 5 mM
isopropyl-
-D-thiogalactopyranoside (IPTG). E. coli was grown at 37°C in Luria-Bertani (LB) medium supplemented
with ampicillin (100 µg/ml) when required. For agar medium, LB was
solidified with 1.5% (wt/vol) agar.
Recombinant DNA techniques.
Chromosomal DNA from C. cellulovorans was isolated as described previously
(33). Plasmids from E. coli were purified with a
Qiagen kit (Qiagen Inc.). Agarose gel electrophoresis, transformation of E. coli, and ligation were done by the methods of
Sambrook et al. (29). Chromosomal DNA was partially digested
with EcoRI and electrophoresed on a 0.7% agarose gel. The
fragments in the range of 4 to 10 kb were excised from the gel and
purified with a GeneClean kit (Bio 101 Inc.). The purified fragments
were ligated into the dephosphorylated EcoRI site of
ZAPII. The ligation mixture was packaged in vitro with GigapackIII
Gold (Stratagene). The C. cellulovorans genomic library in
ZAPII was immunoscreened with an anti-EngE antiserum (diluted 1:500)
previously prepared from C. cellulovorans (22)
and a second antibody, alkaline phosphatase-conjugated goat anti-rabbit
immunoglobulin G (diluted 1:3,000) (Bio-Rad). Immunodetection was
performed with the Western Detection Kit (Bio-Rad) following the
manufacturer's instructions. Positive immunocross-reacting plaques
were isolated, and clones were obtained by in vivo excision and rescued
with ExAssist helper phage (Stratagene). Positive clones were further
screened for endoglucanase activity by overlaying the clones with 0.7%
soft agar containing 0.3% carboxymethyl cellulose (CMC) (low
viscosity; Sigma). Colonies having carboxymethyl cellulase activity
were recognized by the formation of clear halos on a red background
after staining with 0.1% Congo red and destaining with 1 M NaCl
(1).
Nucleotide sequence analysis.
The nucleotide sequences of
both strands were determined by the dideoxy chain termination method
(30). The original EcoRI fragment (pYE1) was
subcloned with restriction enzymes, and a series of nested deletion
mutants from each fragment was constructed by using an
ExoIII-mung bean nuclease kit (Stratagene). Double-stranded DNA templates were sequenced with the Sequenase version 2.0 (U.S. Biochemical Co.). Both strands were sequenced. Homology searches in
GenBank were performed with a BLAST program.
EngE activity and protein assays.
The reaction mixture
consisted of 100 µl of EngE solution, 500 µl of 1% CMC (medium
viscosity; Sigma), and 400 µl of 100 mM sodium acetate buffer (pH
5.0). The mixture was incubated at 50°C for 10 min, and then the
reducing sugar as D-glucose was measured by the
Somogyi-Nelson method (38). One unit of enzyme activity was
defined as the amount of enzyme that liberates 1 µmol of
D-glucose per min under the above conditions. Thin-layer
chromatography (TLC) analysis was performed as described previously
(14). Purified EngE was incubated with cellodextrins
(cellotriose [3G]; cellotetraose [4G]; cellopentaose [5G])
solutions for 16 h at 37°C. The reaction products were separated
on TLC plates (Merck) with a solvent system containing
1-butanol-ethanol-water (5:5:2.5). For detection of the products, the
plate was sprayed with staining reagent (5% H2SO4 in methanol) and baked for 10 min at
100°C.
Protein concentrations were measured by the method of Bradford
(3) with a protein assay kit from Bio-Rad, using bovine serum albumin as a standard.
SDS-PAGE and Western blot analysis.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on
a 7.5% polyacrylamide gel by the method of Laemmli (16).
After electrophoresis, the gel was stained with Coomassie brilliant
blue R. A high-molecular-weight SDS calibration kit (Pharmacia) was
used as the standard.
For Western blot (immunoblot) analysis, the PhastGel system (Pharmacia)
was used. Western blot analysis was performed by using
an anti-EngE
antiserum (diluted 1:2,000). The procedure was done
as described above.
Cellulosomes from
C. cellulovorans were prepared
as
described previously (
22).
Purification of recombinant EngE.
E. coli harboring
pYE2R in pBluescript II KS(+) were cultured to early stationary phase
at 37°C with vigorous shaking. The cells were collected by
centrifugation, and the periplasmic fraction was prepared by the
osmotic shock method (21). The two steps of chromatography
were performed with a fast-protein liquid chromatography system
(Pharmacia). The periplasmic fraction was applied to a Q Sepharose Fast
Flow column (2.6 by 22 cm; Pharmacia) equilibrated with 50 mM Tris-HCl
buffer (pH 7.5). After being washed with 3 bed volumes of the same
buffer, the column was eluted with a linear gradient of NaCl (0 to 1.0 M) at a flow rate of 60 ml/h. The active fractions eluted around 0.6 M
NaCl were collected and concentrated by saturated ammonium sulfate.
After centrifugation, the precipitate obtained was dissolved in a small
volume of 50 mM Tris-HCl buffer (pH 7.5). The concentrated enzyme
solution was applied to a Sephacryl S-200 column (2.6 by 75 cm;
Pharmacia) equilibrated with 50 mM Tris-HCl buffer (pH 7.5). The active
fractions were used as purified enzyme.
Nucleotide sequence accession number.
The nucleotide
sequence data reported in this paper have been submitted to GenBank
under accession no. AF105331.
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RESULTS |
Cloning and nucleotide sequence of the engE gene.
A C. cellulovorans ZAPII library was immunoscreened with
anti-EngE antibody and further screened for endoglucanase activity. From approximately 20,000 transformants, 5 positive clones were isolated and further characterized. All clones contained a common 6.0-kb EcoRI insert. A restriction enzyme map of the cloned
gene is shown in Fig. 1. To determine the
coding region of the enzyme, various subclones were prepared and tested
for the formation of clear halos around the colonies. These results
indicated that the coding region for the engE gene was on
the 4.6-kb EcoRV-EcoRI (pYE2 or pYE2R) fragment.
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FIG. 1.
Restriction enzyme map of the pYE1 insert and the domain
structure of EngE. The transformants harboring the plasmids with
appropriate deletions were transferred to an LB agar plate. After agar
with 0.3% CMC was poured over the transformants, production of
carboxymethyl cellulase (CMCase) was judged by the formation of clear
halos around the colonies (+, visible halo; , no halo). The coding
sequence and the domain structure of EngE are shown at the top of the
figure. Symbols:  , signal peptide;  , SLH domain;  , catalytic
domain;  , unknown domain;  , duplicated sequence (DS). a.a., amino acids.
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Figure
2 shows the complete nucleotide
sequence of the
engE structural gene along with its flanking
regions and a partial
open reading frame, ORF1, upstream of the
engE gene. The
engE gene (nucleotides [nt] 760 to 3849) consists of 3,090 nt encoding
a protein of 1,030 amino acids
with a predicted molecular weight
of 111,796. The putative initiation
codon ATG was preceded by
a spacing of 7 bp by a typical gram-positive
bacterial ribosome-binding
sequence, GGAGG, which was homologous to the
consensus Shine-Dalgarno
sequence. Upstream of the coding region, two
possible promoter
sequences, AGGAAA for the
35 region and
TATAAT for the
10 region
with a 17-bp spacing between
them, were found and showed high
homologies to consensus promoter
sequences for
70 or
A factor, TTGACA
and TATAAT with a 17-bp spacing, found in
E. coli or
Bacillus subtilis (
10). A possible
transcription terminator
that consisted of a 17-bp palindrome,
corresponding to an mRNA
hairpin loop with a
G of
26.3
kcal/mol (ca.
110 kJ/mol), followed
by three T's was found
downstream of the TAA termination codon.
This structure is similar to
the rho-independent terminator factor
of
E. coli
(
28). Upstream of the
engE gene, an incomplete
ORF1
(nt 1 to 508) encoding a peptide with partial homology to the
B. subtilis phosphomethylpyrimidine kinase gene
(
pmk) was found.
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FIG. 2.
Nucleotide and deduced amino acid sequences of
engE and ORF1. The putative promoter and Shine-Dalgarno (SD)
sequences are underlined. A palindrome is indicated by arrows facing
each other. The stop codons are indicated by asterisks. A duplicated
sequence (DS or dockerin) in EngE is shown as white letters on a black
background.
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The codon utilization of the
engE gene shows a bias for A or
T at the wobble position as shown previously for genes from
C. cellulovorans. The percentages of codons terminating in A or T
are
86.0 for EngE, 79.3 for EngB (
8), 80.2 for EngD
(
12),
81.5 for EngF (
31), and 77.5 for ExgS
(
20), thus reflecting
the low G+C content of
C. cellulovorans DNA (
35).
Domain structure of EngE.
The N-terminal amino acid sequence
of EngE exhibited a typical signal peptide and consensus sequence
(Val-X-Ala) (37), where the predicted cleavage site is
located between position 31 (Ala) and position 32 (Ala). Removal of the
signal peptide yields a mature protein of 999 amino acids with a
molecular weight of 108,335.
Analysis of the deduced amino acid sequence of EngE revealed a
multidomain structure with a unique feature, i.e., a triplicated
SLH
domain at the N terminus, followed by a family 5 catalytic
domain, a
domain of unknown function, and a duplicated sequence
(DS or dockerin)
(Fig.
1).
The N-terminal half of EngE (residues 32 to 491) contained three highly
conserved repeats of 163, 126, and 163 amino acids,
with more than 63%
identity (Fig.
3A). In addition, the
three
repeats of EngE showed homology with several SLH domains and most
significantly with the HLDs of CbpA (
34) (Fig.
3B).
Furthermore,
the repeated region was homologous to the S-layer protein,
RsaA
from
Caulobacter crescentus (
2). The region
(residues 63 to
272) of the three repeats in EngE showed 16.4%
identity and 70.5%
similarity with the C-terminal region of RsaA (Fig.
4A), while
the region (residues 304 to
426) had 21.1% identity and 69.5%
similarity with the N-terminal
region of RsaA (Fig.
4B).
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FIG. 3.
(A) Alignment of the N-terminal three repeats found in
EngE. (B) Alignment of the sequences shown in panel A with similar
sequences of HLDs of C. cellulovorans (C.v) CbpA, C. cellulolyticum (C.c) CipC, Clostridium josui (C.j)
CipA, and Clostridium stercorarium (C.s) AviII and EngZ.
Amino acid residues identical or similar to those in the consensus
region are shown as white letters on a black background. Similar amino
acids are as follows: F, I, V, L, and M; R and K; S and T; D and E; N
and Q; and F, Y, and W. Gaps left to improve alignment are indicated by
dashes. The numbers refer to amino acid residues at the start and end
of the respective lines; all sequences are numbered from Met-1 of the
peptide.
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FIG. 4.
Alignment of N-terminal (A) and C-terminal (B) regions
in the triplicated SLH domain of C. cellulovorans (C.v) EngE
with S-layer protein of Caulobacter crescentus (C.c) RsaA
(2). Identical and similar amino acid residues are indicated
by asterisks and dots, respectively. Gaps left to improve alignment are
indicated by dashes. The numbers refer to amino acid residues at the
start of the respective lines; all sequences are numbered from Met-1 of
the peptide.
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The C-terminal half (residues 516 to 839) of EngE showed similarity to
catalytic domains of cellulases which belong to glycosyl
hydrolase
family 5 (
13). In particular, the region had high
sequence
similarity to
C. cellulovorans EngD (44.2% identity)
(
12) and
C. cellulovorans EngB (43.7% identity)
(
8),
Clostridium thermocellum CelE (43.6%
identity) (
11),
Ruminococcus albus EG-I (41.3%
identity) (
25),
Orpinomyces CelB (41.0%
identity)
(
19),
Clostridium longisporum CelA
(40.2% identity) (
24),
and
Clostridium
cellulolyticum CelA (37.0% identity) (
6).
Homology search revealed that a sequence of about 130 amino acids
(residues 840 to 970) downstream of the catalytic domain
did not
resemble any sequences in the protein databases. The function
of this
region therefore remains
unknown.
A duplicated sequence (DS or dockerin) (residues 978 to 1,030) was also
found in the C-terminal region of EngE. The DSs consisting
of
22-amino-acid repeats are well conserved in cellulosomal subunits
from
C. cellulovorans and other
Clostridium species.
Purification and characterization of rEngE.
Recombinant EngE
(rEngE) was purified from the periplasmic fraction of E. coli harboring pYE2R by using the Q Sepharose Fast Flow and
Sephacryl S-200 columns. After purification and SDS-PAGE, rEngE gave a
single band, and the molecular weight of the enzyme was estimated to be
around 100,000 (Fig. 5A). This is in good agreement with the value (108,335), excluding the putative signal peptide, calculated from the deduced amino acid sequence. Western blot
analysis indicated that rEngE immunoreacted with the anti-EngE antibody
prepared from C. cellulovorans cellulosomes and that the
size of the immunoreactive band of EngE was in good agreement with that
of the cellulosomal EngE protein from C. cellulovorans (Fig.
5B). Some cross-reaction of the antibody was evident, perhaps due to
the presence of DS in EngE, but the major band was found at the
migration position of EngE. The optimum pH and temperature for activity
of the purified rEngE were 5.0 and 50°C, respectively. As shown in
Table 1, purified rEngE hydrolyzed CMC
and lichenan but showed little or no activity with acid-swollen
cellulose (ASC), Avicel, laminarin, and xylan. TLC analysis revealed
that the purified EngE cleaved 3G, 4G, and 5G to form cellobiose (2G)
(Table 2) but did not act on cellobiose
(data not shown). rEngE seems to hydrolyze preferentially the
-1,4-cellulosidic linkages situated at position 2 and to have
transglycosylation ability.
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FIG. 5.
Analysis of the purified EngE by SDS-PAGE (A) and
Western blotting (immunoblotting) (B). Lane M, standard markers (myosin
[212 kDa],  2-macroglobulin [170 kDa], -galactosidase [116
kDa], transferrin [76 kDa], and glutamic dehydrogenase [53 kDa]);
lane 1, purified EngE; lane 2, cellulosomal proteins from C. cellulovorans.
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DISCUSSION |
The characterization of EngE has completed the sequence analysis
of the three major subunits of the C. cellulovorans
cellulosome. Since EngE has endoglucanase activity, the three major
subunits include the endoglucanase EngE, the exoglucanase ExgS
(20), and the scaffolding protein CbpA (34). The
combination of these three subunits was shown previously to have
cellulolytic activity on ASC (4) and to comprise the core of
the C. cellulovorans cellulosome (32).
EngE belongs to the family 5 cellulases, which comprise a large and
growing family of cellulases from various microorganisms (13). It is interesting that EngB (8), EngD
(12), EngF (31), and ManA (36) from
C. cellulovorans are also members of family 5, while EngL
and EngY (36) are members of family 9.
In this study, we purified and characterized rEngE and compared some
properties of EngE with those of EngB, EngD, and EngF from C. cellulovorans. EngE had the highest activity with CMC as the
substrate, less activity with lichenan (about 60% of the activity as
shown with CMC), and very little or no activity with xylan, while both
EngB and EngD had much higher activity with lichenan than with CMC and
xylan (7). Furthermore, the hydrolysis pattern of EngE
analyzed by TLC showed that the main products from cellodextrins (3G to
5G) were only cellobiose (2G), while EngB, EngD, and EngF produced
glucose (1G), 2G, and 3G, and EngF did not act on 3G (14).
Thus, the properties of EngE differ from those of EngB, EngD, and EngF
in specific activity, substrates degraded, and the products that are
formed, although all the enzymes belong to family 5.
Electron micrographs have shown that cellulosomes are attached to the
cell surface (17). In C. thermocellum, it has
been proposed that the dockerin II present on CipA interacts with a cell surface protein Olp containing a cohesin II (18) and
that this interaction binds the cellulosome to the cell surface. The C. cellulovorans CbpA (equivalent to CipA), on the other
hand, does not contain a dockerin II but contains four conserved HLDs, which are absent from C. thermocellum CipA. When the
sequences of the HLDs were examined with the BLAST system, it was found that partial homology existed between HLDs and surface layer proteins found on other organisms (Fig. 4). This suggested to us that the HLDs
present in CbpA could play a role in binding the cellulosome to the
cell surface just as the putative dockerin II-cohesin II reaction holds
the cellulosome to the surface of C. thermocellum. This role
of HLDs in CbpA has not been proven to this point but is being actively examined.
It was therefore of great interest when the analysis of the EngE domain
structure revealed the presence of three repeated SLH-like domains at
its N terminus. Furthermore, the triplicated SLH domain of EngE showed
homology with the S-layer protein RsaA from C. crescentus
(2). The C. crescentus S-layer is paracrystalline in nature, exhibiting an array of ring-like subunits (each composed of
six copies of RsaA) arranged on a lattice with p6 symmetry and
interlinked at a threefold rotational axis (2). Although the
association between RsaA and the outer membrane is not completely understood, the protein appears to be anchored to the outer membrane via noncovalent interactions with a specific smooth lipopolysaccharide molecule (2). So far, the only known function of the
C. crescentus S-layer is to protect cells against predation
by a Bdellovibrio-like organism (15). RsaA, as
well as the three repeated regions of EngE, contained no cysteine
residues. In fact, several S-layer proteins have no cysteine residues
(9). Moreover, the N-terminal region of RsaA (residues 1 to
154) was involved in cell surface anchoring, while the C-terminal
region (residues 784 to 907) possessed a secretion signal
(2).
Many microorganisms have been shown to have surface layer proteins
(SLPs) that bind to the peptidoglycan layer (26). Even a
single SLH domain can apparently bind a SLP to the peptidoglycan layer.
One function proposed for these SLPs is that they serve to anchor
extracellular enzymes such as pullulanase to the cell surface
(23).
Since the N and C termini of the SLH domains of EngE exhibited homology
with both the N- and C-terminal regions of RsaA, the SLH domains of
EngE may also possess similar functions, i.e., anchoring the enzyme to
the cell surface and also in some targeting function to the cell surface.
By isolating C. cellulovorans peptidoglycan or cell surface
protein fractions and interacting them with the SLH domains from EngE
and CbpA, we should be able to test the hypothesis that SLH domains are
involved in binding cellulosomes and cellulases to the cell surface
(Fig. 6). A positive result would further
strengthen the hypothesis that the cellulosomes are attached to the
cell surface by interaction of the SLH domains from both CbpA and its constituent enzymes such as EngE. This hypothesis is currently being
investigated.
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FIG. 6.
Hypothetical model for attachment of EngE to the CbpA of
C. cellulovorans and the cell surface. The drawing is not to
scale. Because of its similarity to the S-layer protein and the
presence of a duplicated sequence (DS or dockerin), the three SLH
domains of the N terminus of EngE integrate into the lattice of the
S-layer, while the C terminus of EngE is bound to CbpA through its DS.
Also, CbpA integrates itself into the lattice of the S-layer through
its four SLH domains. CD, catalytic domain.
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ACKNOWLEDGMENTS |
This research was supported in part by grant DE-DDF03-92ER20069
from the U.S. Department of Energy.
Cellobiopentaose was provided by Seikagaku America, Inc.
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FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Molecular and Cellular Biology, University of California, Davis, CA
95616. Phone: (530) 752-3191. Fax: (530) 752-3085. E-mail:
rhdoi{at}ucdavis.edu.
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Journal of Bacteriology, May 1999, p. 3270-3276, Vol. 181, No. 10
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Abstract
Introduction
