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Journal of Bacteriology, October 2000, p. 5906-5910, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Large Gene Cluster for the Clostridium
cellulovorans Cellulosome
Yutaka
Tamaru,1
Shuichi
Karita,2
Atef
Ibrahim,3
Helen
Chan,1 and
Roy H.
Doi1,*
Section of Molecular and Cellular Biology,
University of California, Davis, California
956161; Center for Molecular Biology
and Genetics, Mie University, Tsu, Japan2; and
Biotechnology and Genetic Engineering Institute, Menoufiya
University, Sadat City, Egypt3
Received 6 March 2000/Accepted 14 July 2000
 |
ABSTRACT |
A large gene cluster for the Clostridium cellulovorans
cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39-47, 1998). Gene walking revealed that the
engL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244-247, 2000) was located downstream of the
cbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of
the clustered genes is
cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN and is about
22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene.
This large gene cluster is flanked at the 5' end by a putative
noncellulosomal operon consisting of nifV-orf1-sigX-regA
and at the 3' end by noncellulosomal genes with homology to transposase
(trp) and malate permease (mle). Since gene
clusters for the cellulosome are also found in C. cellulolyticum and C. josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may
arise from a common ancestor with some evolutionary modifications.
| |
TEXT |
Clostridium cellulovorans
(ATCC 35296) (19), an anaerobic, mesophilic, and
spore-forming bacterium, produces extracellular polysaccharolytic
multicomponent complexes called the cellulosome (1, 8),
which has the ability to degrade cellulose, xylan, mannan, and pectin
(19, 21). The C. cellulovorans cellulosome (3) consists of three major subunits, CbpA, P100, and P70, and several minor subunits (10, 16). We have previously
cloned and sequenced several cellulosomal subunits, i.e., the
scaffolding protein CbpA (18), the endoglucanases EngB
(4, 17) and EngE (20), and the exoglucanase ExgS
(9). More recently, we have completely sequenced the
engL gene cluster, which consists of five different open
reading frames (ORFs) containing a cellulosomal ManA-encoding sequence
(21).
In a recent 16S rRNA gene analysis of polysaccharolytic clostridia,
C. cellulovorans was classified in group I of the
phylogenetic tree (13) while most cellulolytic clostridia,
such as C. cellulolyticum, C. josui, C. papyrosolvens, and C. thermocellum, belonged to the same cluster (group III) (7). Although C. cellulovorans was located far from the other cellulolytic
clostridia in the phylogenetic tree, the gene clusters of the C. cellulovorans cellulosome (22) seem similar to those of
C. cellulolyticum (2) and C. josui (6, 7). Since a large gene cluster
in C. cellulolyticum (cipC-celF-celC-celG-celE-ORFX-celH-celJ-celK)
has recently been reported (2), such a gene cluster seems to
be specific for mesophilic clostridia and did not occur in the
thermophilic bacterium C. thermocellum. Furthermore, recent data obtained with C. cellulovorans, C. cellulolyticum, C. josui, and C. acetobutylicum revealed that all of these gene clusters begin with
the scaffoldin gene, followed by a gene encoding a family 48 cellulase
(2). It is of interest to determine the chromosomal
organization of the genes of the cellulosome complex, since it may
provide information concerning the number of genes, the transcriptional
regulation, the coordinate expression, and the evolutionary
relationship of the genes in the complex.
In this paper, we describe the large gene cluster around the
cbpA and exgS genes of C. cellulovorans. We also analyzed the amino acid sequences of the
corresponding proteins and compared them with those of other proteins.
Furthermore, this large gene cluster also codes for a small 25-kDa
protein, hydrophobic protein A (HbpA), that showed homology with
hydrophobic domains (HBDs or type I cohesins) in CbpA (18).
The role of HbpA is still not understood, but it may function in a
manner similar to that reported for OlpA of C. thermocellum
(1) and ORFXp of C. cellulolyticum (11). The occurrence of this small HbpA may be widespread
among mesophilic clostridia that produce cellulosomes.
Cloning and DNA sequencing of the gene cluster.
The major gene
cluster of the cellulosome consists of nine genes, as shown in Fig.
1. We have cloned and sequenced the
cbpA-exgS gene cluster (9) and the
engL gene cluster (pYI-1) harboring five different ORFs,
i.e., engK-hbpA-engL-manA-engM (21). Since it was
expected that the engL gene cluster might be located
downstream of the cbpA-exgS gene cluster, we cloned the
region between exgS and engK by gene walking. As
shown in Fig. 1, the internal fragment between exgS and
engK was amplified by PCR with two synthesized oligonucleotides, YT-12 (5'-CTGATATGAACGGTGATGGAAAAG-3'),
corresponding to exgS, and YT-13
(5'-CCACCAGTTAATGTAGTTGGCA-3'), corresponding to
engK. As a result, a 4.6-kb PCR fragment (pAI-1) was
obtained and cloned into the pCR2.1 vector with a TA cloning kit
(Invitrogen) and then sequenced (Fig. 1). The DNA sequence of the pAI-1
fragment contained the engH and engK genes. No
potential transcription terminator was observed between engH
and engK, while a large potential terminator (14)
was seen after engK. This observation indicated that the
engH and engK genes might be encoded by an
operon. Likewise, since no repeat elements were observed between
cbpA and exgS and between hbpA and
engL, they appear to be encoded as operons; large transcriptional terminators were found between exgS and
engL. There is a potential transcriptional terminator
downstream of manA, indicating that manA is a
monocistronic gene. In fact, ManA production is repressed by cellobiose
(21) while the three major cellulosome subunits are
expressed in the presence of cellobiose (10). Thus, it will
be of extreme interest to study the regulation of expression of these
putative operons. One might expect coordinated expression of the
operons for the enzymatic subunits with the cbpA-exgS
operon.
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FIG. 1.
Restriction enzyme map of a cellulosomal gene cluster.
The genes coding for CbpA, ExgS, EngH, EngK, HbpA, EngL, ManA, EngM,
and EngN are shown at the top. The pin-like marks indicate palindromes.
E, H, and P indicate EcoRI, HindIII, and
PstI restriction sites, respectively.
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|
To obtain the complete
engM gene, Southern hybridization
analysis with a partial
engM fragment of pYI-1 as a probe
was carried
out. Either
HindIII or
PstI
digestion of
C. cellulovorans chromosomal
DNA gave a 3.3- or
4.6-kb fragment which was associated with the
probe (data not shown).
As a result of screening by colony hybridization
with the same probe,
we cloned two kinds of plasmids that were
named pEngM83 (3.3-kb
HindIII fragment) and pEngM53 (4.6-kb
PstI
fragment), respectively (Fig.
1). The DNA sequence of these fragments
contained four ORFs. The first ORF coded for EngM; the second
ORF,
named
engN, encoded only the N-terminal amino acid sequence
of family 9 cellulases. The last two ORFs coded for proteins that
were
homologous to transposase (
trn) and malate permease
(
mle),
respectively (Fig.
1), and these two genes flanked
the cellulosome
gene cluster at the 3' end. On the other hand, the gene
cluster
was flanked at the 5' end by the noncellulosomal gene cluster
nifV-orf1-sigX-regA (S. Karita and R. H. Doi,
unpublished data;
18). There are three cellulosomal
genes that are unlinked to
the major gene cluster and unlinked to each
other, i.e.,
engB (
17),
engE
(
20), and
engY-pelA (
22).
The
engN gene is an anomaly, since the coding sequence,
which has been checked several times in all three reading frames,
indicated that EngN does not have a complete catalytic domain.
Repeated
sequencing experiments indicate strongly that
engN is
a
truncated gene. Furthermore, no duplicated sequence (DS) is
present in
the coding sequence. The cloned
engN gene also does
not
express any endoglucanase activity in
Escherichia coli,
while
the other enzymatic genes are expressed in
E. coli as
active enzymes.
Since
engN is flanked by
engM and
the transposase gene (Y. Tamaru
and R. H. Doi, unpublished data),
there does not appear to have
been some accidental deletion during
cloning.
Amino acid sequences encoded by the gene cluster.
The
cellulosomal subunits of C. cellulovorans are summarized in
Table 1. We have previously characterized
several cellulosomal subunits, i.e., CbpA (18), EngE
(20), ExgS (9), EngB (4, 5), and ManA
(21). Four family 9 cellulases, i.e., EngH, EngK, EngL,
and EngM, have been found in the gene cluster. EngK and EngM
belong to subfamily E1 in family 9, while EngH and EngL belong to
subfamily E2 in family 9. Also, except for EngL, family 9 cellulases in
the gene cluster contain a cellulose-binding domain (CBD). EngH
contains a family IIIc CBD, while EngK and EngM have a family IV
CBD.
The presence of DSs (or dockerins), each sequence consisting of about
22 amino acids, is one of the tell-tale signs of a cellulase
enzyme
belonging to the cellulosome. The cellulosomal gene products
are all
characterized by the presence of a DS, usually at the
C terminus of the
protein, although the DS of ManA is located
at its N terminus (Fig.
2). Although a DAL or DAI motif is
conserved
in the DSs from
C. cellulolyticum and
C. josui and an NST motif
is conserved in those from
C. thermocellum (
7), this motif
of
C. cellulovorans is replaced by NAI. Since the cohesin-dockerin
interaction in
Clostridium species is a
species-specific phenomenon
(
12), the
C. cellulovorans NAI motif may be essential as a recognition
code for
binding specificity. Furthermore, the linkage of the
DS to the
catalytic domain may have a special structure since,
almost invariably,
when these enzyme subunits are expressed in
E. coli, a
protease in
E. coli cleaves off the DS and leaves a
still-active catalytic domain. This suggests strongly that a
protease-accessible
structure is present between the catalytic domain
and DS domains
of
C. cellulovorans cellulosomal enzymes.
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FIG. 2.
Alignment of the DSs of cellulosomal subunits of
C. cellulovorans. Amino acids which are conserved in at
least five of the eight sequences are highlighted. Identical amino acid
residues are highlighted. Pluses indicate amino acid residues involved
in calcium binding. Residues suspected of serving as selectivity
determinants are indicated by pound signs.
|
|
DNA sequence of hbpA and domain structure of HbpA.
Figure 3 shows the complete nucleotide
sequence of the hbpA structural gene along with its flanking
regions. The hbpA gene consists of 720 nucleotides encoding
a protein of 240 amino acids with a predicted molecular weight of
24,930. The putative initiation codon (ATG) is preceded by a spacing of
7 bp and by a typical ribosome-binding sequence, AGGAG, which is
homologous to the consensus Shine-Dalgarno sequence. Downstream of the
TAA translation termination codon, a transcription terminator was not
observed, suggesting that hbpA and engL are in an
operon.
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FIG. 3.
Nucleotide and deduced amino acid sequences of
hbpA and HbpA, respectively. The Shine-Dalgarno (SD) and
signal peptide sequences are underlined. The stop codon is indicated by
an asterisk. The amino acids of the HBD are highlighted.
|
|
The N-terminal amino acid sequence of HbpA exhibits a typical
signal peptide and consensus sequence (Val-X-Ala) (
23),
where
the predicted cleavage site is located between positions 19 (Ala)
and 20 (Gly) (Fig.
3). The N-terminal region of HbpA (residues
20 to 104) contains a surface layer homology (SLH) domain which
shows
homology with S-layer proteins from
Mycoplasma hyorhinis (18.5% identity and 84.5% similarity among 103 amino acids; accession
no.
P29228) and
Plasmodium reichenowi (26.5%
identity; 91.6%
similarity among 83 amino acids; accession no.
Z30339) (Fig.
4A). The SLH
sequences vary among different surface layer proteins
but can be
recognized as SLH domains by a few conserved identical
amino acids
(
15).
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FIG. 4.
(A) Alignment of the N-terminal region of HbpA from
C. cellulovorans (C.v) with the corresponding proteins from
M. hyorhinis (M.h) and P. reichenowi (P.r). (B)
Alignment of the C-terminal region of HbpA with HBDs of CbpA from
C. cellulovorans (C.v). Identical amino acids are
highlighted. Gaps left to improve the 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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|
Also, the N terminus of HbpA has several potential O-glycosylation
sites. Since it does not contain a DS, HbpA most likely
does not bind
to CbpA and is not part of the cellulosome. The
C-terminal region
(residues 105 to 240) shows 32 to 37% identity
with HBDs of CbpA
(
18) (Fig.
4B), while this region has about
the same
identity with type I cohesins of other
Clostridium species
(data not shown). Furthermore, the whole HbpA sequence reveals
29.6%
identity and 86.2% similarity to
C. cellulolyticum ORFXp
(
11) (Fig.
5). The presence of
the N-terminal SLH domain suggests
that HbpA is a cell surface-bound
protein with some function in
cellulosome assembly, as postulated
previously for a similar protein,
ORFXp, from
C. cellulolyticum (
11). It was postulated that the
cohesin
in ORFXp acts as a temporary binding station for cellulosomal
enzymes
that are destined for CipA during the assembly of the
cellulosome
(
11). A significant difference between
C. cellulolyticum ORFXp and
C. cellulovorans HbpA is the
absence of an SLH domain
in ORFXp. The presence of the glycosylation
sites suggests that
HbpA can be glycosylated, while ORFXp is highly
glycosylated (
11).
Thus, the occurrence of this small,
hydrophobic protein may be
widespread among mesophilic clostridia that
produce cellulosomes.
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FIG. 5.
Alignment of C. cellulovorans (C.v) HbpA with
C. cellulolyticum (C.c) ORFXp. The gap left to improve the
alignment is indicated by a dash. Identical and similar amino acid
residues are indicated by asterisks and dots, respectively. The numbers
refer to amino acid residues at the start of the respective lines; all
sequences are numbered from Met-1 of the peptide.
|
|
Nucleotide sequence accession numbers.
The nucleotide sequence
data reported in this paper have been submitted to GenBank under
accession no. U34793 and AF132735.
| |
ACKNOWLEDGMENTS |
This research was supported in part by grant
DE-FG03-92ER20069 from the U.S. Department of Energy.
| |
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, October 2000, p. 5906-5910, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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