From the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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CelR, a protein that regulates transcription of
cellulase genes in Thermomonospora fusca
(Actinomycetaceae) was purified to homogeneity. A
6-kilobase NotI-SacI fragment of T. fusca
DNA containing the celR gene was cloned into
Esherichia coli and sequenced. The celR gene
encodes a 340-residue polypeptide that is highly homologous to members
of the GalR-LacI family of bacterial transcriptional regulators. CelR
specifically binds to a 14-base pair inverted repeat, which has
sequence similarity to the binding sites of other family members. This
site is present in regions upstream of all six cellulase genes in
T. fusca. The binding of CelR to the celE
promoter is inhibited specifically by low concentrations of cellobiose
(0.2-0.5 mM), the major end product of cellulases. The
other sugars tested did not affect binding at equivalent or 50-fold
higher concentrations. The results suggest that CelR may act as a
repressor, and that the mechanism of induction involves a direct
interaction of CelR with cellobiose.
Biodegradation of cellulose by bacteria and fungi is accomplished
by extracellular cellulolytic enzymes encoded by genes that are subject
to transcriptional control. Several regulators of the transcription of
cellulase genes in the cellulolytic filamentous fungus,
Trichoderma reesei, including the activators
ACE I and ACE II and the glucose
repressor, Cre1, have been described (1, 2). However, little is known
about the molecular mechanisms of cellulase regulation in soil bacteria.
The thermophilic actinomycete, Thermomonospora fusca, a
major degrader of cellulose in plant residues, is an extensively
studied cellulolytic bacterium. This species produces six different
extracellular cellulases, designated E1 through E6. Three of the
enzymes (E1, E2, and E5) are endocellulases, two are exocellulases (E3
and E6), and one enzyme possesses both exo- and endocellulolytic
activity (E4). The major product of these enzymes is cellobiose
(3-5).
Biosynthesis of cellulases in T. fusca is regulated by
cellobiose induction and catabolite repression, with any readily
metabolized sugar acting as a repressor (6). The lowest level of
cellulase synthesis (3 nM) was observed with xylose as a
carbon source, and the highest level was found in cultures grown on
microcrystalline cellulose. Endocellulases and exocellulases showed
distinctly different regulation patterns, with exocellulases showing
the highest level of induction (7).
The structural genes for all these enzymes, designated celA
through celE (8-10) and
celF,1 have been
cloned and sequenced. All six cellulase genes in T. fusca
contain the 14-bp inverted repeat sequence TGGGAGCGCTCCCA in their
5'-upstream regions. A regulatory protein that interacts with the
upstream regions of cel genes was detected in T. fusca cultures grown on cellulose. The binding site was identified
as the 14-bp inverted repeat by DNase I footprinting of the
celE gene and by chemical footprinting of the
celB gene (11, 12). The same palindrome was found in the
regulatory regions of cellulase genes from different streptomycetes, in
particular, the cenC gene from Streptomyces
strain KSM9 (13), the celA genes from Streptomyces halstedii (14) and Streptomyces lividans (15), and also
in the cel1 gene from Streptomyces reticuli (16),
where it serves as the operator for a repressor protein (17). The same
sequence was also found in a xylanase gene from Thermomonospora
alba (18). The occurrence of this site in cellulase and xylanase
genes from different species may indicate its special role in
regulating carbohydrate catabolism in streptomycetes and other high GC
Gram-positive bacteria.
Here we report the isolation and properties of CelR, a regulatory
protein from T. fusca that specifically binds to the 14-bp inverted repeat site in cellulase genes, as well as the complete nucleotide sequence of the celR gene.
Bacterial Strains and Plasmids--
T. fusca ER1 is
an extracellular protease-negative strain derived from T. fusca YX (12). Escherichia coli strain DH5 Growth of Organisms--
T. fusca was grown on
Hagerdahl medium (20) supplemented with 0.5% Solka Floc
(microcrystalline cellulose, James River Corp.). E. coli
strains containing recombinant plasmids were grown in Luria broth or
plated on Luria agar plates containing 0.1 mg/ml ampicillin.
Quantitation of DNA Binding Activity--
Plasmid pE5-46 from
E. coli strain D541, which contained the
celE gene from T. fusca (3) was cut with
EcoRI, SalI, and XhoI, and the 3' ends
of the fragments were labeled with [ Purification of the CelR Protein--
T. fusca strain
ER1 was grown in 10 liters of Hagerdahl medium (20) containing 0.5%
Solka Floc and 2 g of yeast extract at 52 °C (pH 7.2 at 40%
oxygen saturation). Cells were harvested after 15 h of
cultivation, sedimented and resuspended in lysis buffer containing 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM
DTT. All purification steps were performed at 4 °C, unless otherwise
noted. Cells were lysed with a French press, and cell debris was
removed by centrifugation at 10,000 × g for 25 min.
Streptomycin sulfate (4%) was added to the supernatant, and the DNA
precipitate was removed after 1 h by centrifugation at 10,000 × g for 25 min. Sodium chloride (0.5 M) was
added, and the supernatant (700 ml) was loaded on a 200-ml
phenyl-Sepharose CL-4B column (Amersham Pharmacia Biotech). The column
was eluted successively with 0.5 M, 20 mM NaCl
in 10 mM Tris-HCl (pH 7.8) containing 1 mM
EDTA, 0.1 mM DTT, the same buffer without NaCl, and with
distilled water. Fractions eluted with distilled water (150 ml) were
applied to a 40-ml heparin-Sepharose CL-4B column (Amersham Pharmacia
Biotech) equilibrated with 10 mM Tris-HCl (pH 7.8), 0.1 M NaCl, 1 mM EDTA, 0.1% Triton X-100, and 0.1 mM DTT (buffer A). The column was eluted with a linear
gradient of NaCl from 0.1 to 1.5 M (400 ml). Appropriate
fractions (120 ml) were pooled, dialyzed against buffer A, and loaded
on a 7-ml DNA-Sepharose column that contained DNA fragments with
multiple copies of the 14-bp inverted repeat (TGGGAGCGCTCCCA) coupled
to Sepharose 4B (Amersham Pharmacia Biotech). DNA affinity
chromatography was performed in buffer A at room temperature. After
washing with 25 ml of buffer A, the column was successively eluted with
0.3, 0.6, and 1.0 M NaCl in buffer A. Electrophoretically
pure (>95%) CelR protein was eluted with 10 ml of warm (50 °C) 1.8 M NaCl, concentrated, and desalted using Centricon-30
concentrators. Protein was determined with the BCA reagent (Pierce)
using bovine serum albumin (Sigma) as a standard.
Molecular Weight Determination--
The molecular weight of CelR
was determined by 12% SDS-PAGE (22) using low and high molecular
weight protein standards (Sigma). Gels were stained with Coomassie
Brilliant Blue R-250 (Sigma). Duplicate gels were cut into 2-mm
sections and washed successively with 50% isopropanol, 10%
isopropanol, and 20 mM Tris-HCl (pH 7.8), 1 mM
EDTA. Gel sections were homogenized in the same buffer, and protein was
extracted overnight at 4 °C. DNA binding activity in gel extracts
was measured as described.
The molecular weight of native CelR was determined by HPLC gel
chromatography. A Waters Protein Pak 200SW glass column (8 mm × 30 cm) was equilibrated and eluted with 10 mM Tris-HCl (pH 8.0), 0.1 M NaCl at 15 ml/h. The column was calibrated with
gel chromatography protein standards (Bio-Rad). Purified CelR (28 µg
in 200 µl) was applied to the column, and fractions of 0.25 ml were
collected and assayed for celE-promoter binding activity.
Effect of Sugars on CelR Protein-DNA Binding--
The effect of
sugars on the CelR-DNA interaction was measured by gel retardation
experiments using purified CelR with the conditions described above.
Sugars (0.1-100 mM) were introduced into the reaction
mixture prior to the addition of CelR (10-90 pg/µl).
Protein Sequencing--
Purified CelR was cleaved with
endo-lys-C, and the products of digestion were separated by HPLC. The
N-terminal sequence of CelR and an internal peptide were determined on
an Applied Biosystems model 492 automated protein sequencer at the
BioResource Center, New York State Center for Advanced Technology
(Cornell University, Ithaca, NY). An internal standard peptide was
included in all sequencing runs in order to independently verify the
performance of the sequencer.
Isolation of Genomic DNA from T. fusca--
Genomic DNA was
isolated from a 125-ml T. fusca culture grown for 24 h
on 0.5% cellobiose. Cells were pelleted by centrifugation (8,000 rpm,
15 min), frozen, thawed, and resuspended in 30 ml of 50 mM
Tris-HCl (pH 8.0), 25 mM EDTA at 4 °C. SDS (1%), and 0.8 ml of diethyl pyrocarbonate were added. Cells were French pressed
at 5,000 p.s.i. directly into 35 ml of phenol:chlorophorm:isoamyl alcohol mixture (25:24:1) on ice. The lysate was mixed and centrifuged (8,000 rpm, 15 min), and the water phase was treated with phenol three
more times. Then the water phase was consecutively extracted with a
chlorophorm:isoamyl alcohol mixture (24:1) and with ether. DNA was
precipitated from the water phase on ice with 0.3 M NaCl and 2.5 volumes of cold ethanol, spooled on a glass rod, washed in cold
ethanol, and redissolved in 1.5 ml of TE buffer (pH 8.0) overnight.
Cloning of the celR Gene--
Degenerate sets of 15-mer
oligonucleotides GC(C/G/T)GT(C/G)ATCAACGGC encoding the N-terminal
region of the CelR protein from Arg25 to Gly29,
and GA(G/A)AACAACCAGAA(G/A), complementary to the strand encoding the
internal part of the protein from Glu73 to
Lys77 were synthesized by the BioResource Center, Cornell
University. Oligonucleotides were labeled with fluorescein by the
3'-tailing reaction with terminal transferase. Total genomic DNA from
T. fusca was completely digested with NotI and
SacI, electrophoresed on a 0.7% agarose gel, Southern
blotted into a Hybond N+ membrane (Millipore), and hybridized with
fluorescein-labeled oligonucleotides. The 3'-oligolabeling and signal
amplification system for the FluorImager (Amersham Pharmacia Biotech)
was used in the hybridization experiments. A 6-kb DNA fragment that
hybridized to both oligonucleotide probes was visualized with a STORM
840 scanner and the ImageQuant program (Molecular Dynamics).
Fragments of ~6 kb were isolated from a
NotI-SacI complete digest of genomic DNA
separated on a 0.7% agarose gel and purified with a QIAquick gel
extraction kit (Qiagen). The DNA was ligated to pBluescript SK+
(Stratagene, La Jolla, CA) digested with the same restriction enzymes.
The ligation mixture was used to transform E. coli strain
DH5 DNA Sequencing--
Double-stranded DNA from pNS1 was used as a
template for sequencing the celR structural gene and its 3'-
and 5'-flanking regions. The sequences of both strands were determined
by the dideoxy chain termination method (23) at the BioResource Center,
Cornell University. The two degenerate 15-mer oligonucleotides were
used to determine the initial nucleotide sequences. Specific primers
for sequencing the celR gene were synthesized by the
BioResource Center.
DNA and Protein Sequence Analysis--
Gapped BLAST and
PSI-BLAST programs (24) were used for searching for sequence homologies
in the GenBank and SwissProt data bases. The Pfam HMM
program3 was used for
creating alignment with the GalR/LacI family. Sequencher (Gene Codes
Corp.) was used for analysis and assembling DNA sequencing data.
Calculations of codon usage, molecular weight, isoelectric point, and
hydrophobicity of the protein product were made with DNASTAR Lasergene software.
CelE Promoter Binding Activity in T. fusca Extracts--
T.
fusca cultures grown on microcrystalline cellulose possessed
considerable celE promoter binding activity (Fig.
1). Preliminary experiments showed that
the maximum level of activity was observed after 12-24 h of
cultivation, and that the levels of binding activity remained high
during an additional 48 h of cultivation. Cells grown on
non-inducing carbon sources had insignificant celE promoter binding activity (data not shown).
Reduced motility of the high molecular weight DNA fragment containing
the E. coli plasmid promoters was also observed (Fig. 1,
lanes 2-4). However, it was independent of the
carbon source used to grow T. fusca. Presumably, the reduced
motility of the high molecular weight fragment was due to the
interaction of RNA polymerase from T. fusca present in the
crude extract with plasmid promoters from pE5-46. No shift was detected
for the 1428-bp DNA fragment, which contained no promoter elements.
Purification of CelR Protein--
The CelR protein was purified
from 10 liters of T. fusca culture by three chromatographic
steps. The process of purification was followed by the gel retardation
assay, Lowry protein measurement (25), and SDS-PAGE (22). A
purification summary is presented in Table
I. Purification yielded highly active
protein of greater that 95% purity (Fig.
2). The resulting CelR bound only to the DNA fragment containing the celE promoter (Fig. 1).
Molecular Weight of CelR Protein--
The molecular mass of pure
CelR protein as determined by SDS-PAGE is 41 ± 1.5 kDa. Samples
of partially purified and pure CelR were separated with SDS-PAGE, and
the gels were cut into 2-mm sections. Protein was eluted from each
section, renatured, and tested for DNA binding activity. In each case
the binding activity was associated with the 41-kDa band (data not
shown). Estimation of the native molecular weight of purified CelR with HPLC gel chromatography showed that the protein aggregated under non-denaturing conditions. About 3% of CelR came off the column as a
dimer (70-85 kDa), a tetramer (145-175 kDa), and an octamer (250-400
kDa). The major part of the protein formed high molecular weight
aggregates that could not be resolved by HPLC. Only traces of CelR
monomer were found.
CelR Binding Affinity--
The dissociation constant was
calculated as the concentration of CelR that caused 50% of the DNA to
form a complex with the protein under the described conditions. The
apparent Kd for the CelR-celE promoter
complex in the absence of cellobiose was 0.5-1 × 10 Effect of Cellobiose on CelR Protein-DNA Binding--
The binding
of CelR to the celE promoter region was inhibited by
cellobiose at concentrations of 0.2-0.5 mM and higher
(Fig. 3). The apparent dissociation
constant for cellobiose was 5 × 10 CelR Gene Cloning and Characterization--
The N terminus and an
internal region of CelR were sequenced. Two degenerate oligonucleotides
prepared on the basis of reverse translation of the amino acid sequence
were used to select E. coli clones. About 200 E. coli transformants from a NotI-SacI library
were screened. Six transformants contained plasmid DNA with a 6-kb
insert that hybridized with the two different oligonucleotide probes.
Plasmid DNA (pNS1) from one of the positive clones was used as a
template for sequencing. The DNA insert contained a three-cistron
operon bglABC coding for two sugar permeases and a
A potential ribosome binding site (GGAA) is located 5 bases upstream of
the start codon. The 5'-regulatory region of celR contains
an inverted repeat that may act as a transcription terminator for the
bglC gene whose termination codon is located 108 nucleotides upstream of the celR start codon. A putative transcription
terminator sequence for the celR gene (a 21-base palindrome)
is located immediately downstream of its structural region.
It is interesting to note that the reverse strand of the
celR gene contains a 1008-nucleotide open reading frame that
begins with GTG start codon (nucleotides 1140-1138) and terminates
with TAG (nucleotides 135-133). A putative ribosome-binding site (AGG) is located 5 nucleotides upstream from the start codon. The
hypothetical 335-amino acid protein product is 23% identical to a
putative ATP/GTP-binding protein from Streptomyces
coelicolor, AL031225.
CelR Sequence Similarities--
The amino acid sequence of CelR
has been scanned against the GenBank and EMBL data bases. CelR shares
significant homology with a number of proteins that belong to the
GalR-LacI family of bacterial transcriptional regulators. It is 49%
identical to a transcriptional regulator from S. coelicolor
(e1309425), 36% identical to a ribose operon repressor RbsR from
E. coli (P25551), 32% identical to a transcriptional
repressor CytR from E. coli (P06964), a transcriptional
regulator DegA from Bacillus subtilis (e1173517), and a
ribose operon repressor RbsR from Haemophilus influenzae
(P44329), 30% identical to a galactose operon repressor GalR (P03024),
a maltose repressor MalI (M60722), and a sucrose operon repressor CscR
(P40715) from E. coli, 29% identical to the lactose
repressor from E. coli (P03023) and to many other members of
the family.
The N-terminal helix-turn-helix motif, located between amino acids 8 and 29, is the most conserved part of the CelR protein. It is 65%
identical and 80% similar to the GalR-LacI consensus (Fig.
6). An unusual feature of CelR is a
cluster of four consecutive arginine residues near the N terminus that
is followed by proline and threonine (amino acids 3-8). This may
represent a phosphorylation site for a cAMP- and
cGMP-dependent protein kinase (26). Location of this site
at the boundary of the DNA-binding motif may indicate its involvement
in regulation of DNA-protein interactions. The amino acid sequence
(residues 205-210, 256-260, and 282-285) of CelR also shares some
homology with elements of the sugar binding sites of the GalR/LacI
proteins (27, 28).
A functionally active DNA-binding protein that interacts
specifically with the 5'-regulatory region of the celE gene
was isolated from T. fusca grown on microcrystalline
cellulose. CelR did not bind to the E. coli promoters in
pE5-46 or to the coding region of the celE gene; it bound
tightly to the celE promoter.
Sequence similarities showed that CelR belongs to the GalR-LacI family
of transcriptional regulators. The GalR-LacI regulators bind to their
target DNA sites as homodimers, and their operator sequences are
inverted repeats. Each monomer of a homodimer interacts with its half
of an operator sequence with an N-terminal DNA-binding domain that
contains a characteristic helix-turn-helix motif (27). The CelR protein
shares a number of common features with other members of this family.
CelR possesses a typical DNA binding domain, based on sequence
comparison, and its binding site is similar to operator sites for the
lactose repressor LacI from E. coli, the amylase repressor CcpA from Bacillus subtilis, and some other members of the
GalR-LacI family (Table II). Unlike other
GalR-LacI proteins that form dimers and tetramers, CelR, because of its
hydrophobicity, shows strong aggregation under non-denaturing
conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(19) was
used for cloning and plasmid isolation. Plasmid pE5-46 has been
described previously (3). Plasmid pNS1 was constructed as described below.
-32P]dCTP or
[
-32P]dATP, using DNA polymerase I Klenow fragment
(21). The following 32P-labeled pE5-46 fragments were
present in the reaction mixture: a high mobility 548-bp fragment
(containing the celE promoter and regulatory region), a low
motility 2656-bp fragment (containing the pE5-46 promoters), and a
1428-bp fragment (containing the coding region of the celE
gene). Binding was carried out in 15-20 µl of 40 mM
Tris-HCl buffer (pH 8.0), 150 mM KCl, 1 mM
MgCl2, 0.1 mM EDTA, 0.1 mM
DTT,2 3 ng of 32P-labeled fragments, T. fusca extract (1-6 µg of protein), 10 µg/ml poly(dG-dC), and
12.5% glycerol. After incubation at 37 °C for 15 min, samples were
electrophoresed through a 1.2% agarose gel in 90 mM Tris
borate buffer (pH 8.3) containing 3 mM EDTA. Gels were
dried at 80 °C under vacuum and radioautographed. The extent of
binding to the celE regulatory region was estimated from
dilutions that yielded 25-75% conversion of the DNA fragment to the
slower mobility DNA-protein complex. One unit of celE
promoter binding activity is the amount of DNA-binding protein that
converts 50% of the DNA fragment to a DNA-protein complex under the
assay conditions.
, and the cells were plated on LB ampicillin plates containing
5-bromo-4-chloro-3-indolyl b-D-galactopyranoside and
isopropyl-1-thio-
-D-galactopyranoside (21).
Transformants were screened by hybridization of E. coli
colonies on Hybond N+ membrane with a fluorescein-labeled probe, as
described. Plasmid DNA was purified from positive transformants with a
plasmid miniprep kit (Qiagen), digested with NotI and
SacI, electrophoresed on 1.2% agarose gel, Southern blotted
into Hybond N+ membrane, and hybridized with both fluorescein-labeled
probes, as described. Plasmid DNA from one of the clones that carried a
6-kb insert which hybridized with both oligonucleotide probes was
called pNS1.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA binding activity of T. fusca
ER1 extract and the purified CelR protein. A gel retardation
assay was used to analyze binding to 32P-labeled DNA
fragments. A, DNA fragment containing the celE
promoter; B, celE promoter DNA-CelR complex;
C, DNA fragment containing celE coding region;
D, DNA fragment containing pE5-46 promoters. Lane
1, no protein; lanes 2-4, crude
T. fusca extract (120, 240, and 480 ng of intracellular
protein); lanes 5-8, purified CelR protein (0.22, 0.44, 0.88, and 1.75 ng).
Purification of the CelR regulatory protein from T. fusca
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Fig. 2.
Electrophoregrams of CelR purification
fractions separated by 12% SDS-PAGE and stained with Coomassie
Brilliant Blue R-250. Lane 1, protein molecular weight
markers; lane 2, crude extract of T. fusca;
lane 3, phenyl-Sepharose; lane 4,
heparin-Sepharose; lane 5, affinity DNA-Sepharose.
9 M.
4 M,
as measured in gel retardation experiments under the above conditions
with 75 pg/µl CelR. The effect of cellobiose on the dissociation
constant of the CelR-celE promoter complex is shown on Fig.
4. A 500-fold increase in cellobiose
concentration (from 0.1 to 50 mM) resulted in a 7-fold
increase in Kd for the CelR-celE promoter
complex. At the same time, equivalent or higher concentrations of other
tested sugars did not affect binding. Cellotriose, sophorose, and
xylobiose showed little inhibition at 50 mM. Other mono-
and disaccharides (glucose, galactose, mannose, xylose, arabinose,
sucrose, lactose, and maltose) had no effect on binding even at 100 mM (data not shown).
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Fig. 3.
Inhibition of CelR protein binding to the
celE promoter by cellobiose. A-D, as
in Fig. 1. Lane 1, no protein; lane
2, 1.2 ng of CelR protein; lanes 3-8,
1.2 ng of CelR protein and cellobiose (0.2, 0.5, 1, 5, 10, or 50 mM).
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Fig. 4.
The effect of cellobiose on the dissociation
constant of CelR-celE promoter complex.
-glucosidase,4 and
celR, coding for the regulatory protein. Both strands were sequenced, and the sequence of celR is shown in Fig.
5. The celR gene has a G+C
content of 68%, which is similar to the 65% G+C content of T. fusca DNA (9). A reading frame from nucleotide 109 to nucleotide
1128 encodes a 340-amino acid moderately hydrophobic protein (125 hydrophobic amino acids). The molecular weight of CelR, inferred from
its amino acid sequence, is 36,863 daltons, lower than its apparent
molecular weight determined by SDS-PAGE. The reading frame that codes
for CelR has a high content of G and C (85.6%) in the third positions
of the codons, typical for cellulase genes from T. fusca
(8-10). The N-terminal (first 37 amino acids) and an internal (amino
acids 60-77) sequence of the protein read from the nucleotide sequence
of the celR gene are identical to the N-terminal sequence of
CelR protein and the sequence of its endo-lys-C cleavage product, as
determined with the protein sequencer.
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Fig. 5.
DNA sequence of the celR
gene and deduced amino acid sequence of CelR protein. The
N-terminal domain of CelR and its endo-lys-C cleavage product
determined by protein sequencing are noted. RBS, ribosome
binding site. Inverted repeats in 5'- and 3'-regions of the gene are
indicated with arrows.
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Fig. 6.
Alignment of the N-terminal sequence of the
CelR protein from T. fusca with the consensus sequence
of 37 bacterial regulatory proteins belonging to the GalR/LacI family
(Pfam HMM program).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
DNA binding half-sites for CelR and other bacterial regulators of the
GalR-LacI family (27)
The 5' regulatory regions of all known cellulase genes in T. fusca contain from one to three copies of this sequence (7). Presumably, all six cel genes in T. fusca are
under transcriptional control of the CelR protein. Apparently,
cellulase regulation in T. fusca follows the general design
of transcriptional control of genes encoding enzymes for carbohydrate
catabolism in other eubacteria. In particular, it resembles the
regulatory system of the amylase and chitinase genes in S. lividans that are controlled by Reg1, a member of the GalR/LacI
family, that acts as a positive regulator of chitinase genes under
inducing conditions, or as a negative regulator of -amylase and
chitinase production in the presence of glucose (29). Similar to
reg1, which is not adjacent to the
-amylase genes in
S. lividans controlled by reg1, celR
is not adjacent to any of the T. fusca cellulase genes.
Cellulase regulation in T. fusca is different from
regulation of cellulase genes in the fungus T. reesei,
although the two evolutionally distant species possess similar
cellulolytic enzyme systems (30). In T. reesei, the
cellulase genes are controlled by the transcriptional activators ACE I
and ACE II and a glucose repressor Cre1 belonging to the family of
Cys2-His2 zinc finger proteins (1, 2).
The vast majority of proteins of the GalR/LacI family bind carbohydrate
or nucleoside effectors (27). Our results show that the DNA binding
activity of CelR is modulated by cellobiose. Results of in
vitro experiments explain the literature data on the induction of
cellulase production in T. fusca cultures by cellobiose (6, 7). The fact that low physiological levels of cellobiose (0.2-0.5 mM) are sufficient for dissociation of the
CelR-celE promoter complex in vitro is evidence
that cellobiose is the true inducer of the cel genes, and
that the mechanism of induction involves a direct interaction of CelR
with cellobiose. In the absence of cellobiose, CelR presumably forms
complexes with the 14-bp inverted repeats next to the cel
genes and blocks their transcription. However, a very low constitutive
level of cellulase synthesis (about 3 nM) was observed even
when cells were grown on non-inducing sugars (7). When cellulose is
present in the environment, cellobiose resulting from its digestion
enters cells and form complexes with CelR, allowing transcription to
proceed. As cellobiose in the cells is exhausted, CelR likely represses
transcription. This simple mechanism may allow quick adaptation of
cells to changing environments, improve the efficiency of the
cel regulon, and help avoid the unnecessary production of
extracellular enzymes. The occurrence of the 14-bp inverted repeat in
the upstream regions of cel genes from different
Streptomyces species (13-16) suggests that this regulatory
mechanism may be also shared by other cellulase genes. One result that
is not explained by this model is the absence of active CelR protein in
cells grown without cellulose or cellobiose. The mechanism of cellulase
regulation in T. fusca under non-inducing conditions
requires further investigation.
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ACKNOWLEDGEMENTS |
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We gratefully thank Diana Irwin, Sheng Zhang, Joseph Calvo and Theodore Thannhauser for advice and discussions, Guifang Lao for preparation of the DNA affinity column, and William Enslow for expert protein sequencing and HPLC gel chromatography.
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FOOTNOTES |
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* This work was supported by Grant DE-FG02-84ER13233 from the Department of Energy Basic Energy Research Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF086819.
Permanent address: Institute of Theoretical and Experimental
Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia.
§ To whom correspondence should be addressed: 458 Biotechnology Bldg., Cornell University, Ithaca, NY 14853. Tel.: 607-255-5706; Fax: 607-255-2428; E-mail: dbw3{at}cornell.edu.
1 D. Irwin, unpublished results.
3 The Pfam HMM program is available via the World Wide Web (http://pfam.wustl.edu/hmmsearch.shtml).
4 N. A. Spiridonov, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DTT, dithiothreitol, PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase pair(s); HPLC, high performance liquid chromatography.
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REFERENCES |
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