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Journal of Bacteriology, April 1999, p. 2290-2292, Vol. 181, No. 7
Department of Oral Biology, The Dental
School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2
4BW, United Kingdom,1 and Centre de
Bioingénierie Gilbert Durand,
Received 6 October 1998/Accepted 20 January 1999
Truncated variants of GTF-I from Streptococcus downei
MFe28 were purified by means of a histidine tag. Sequential deletions showed that the C-terminal domain was not directly involved in the
catalytic process but was required for primer activation. A fully
active catalytic core of only 100 kDa was isolated.
Oral mutans streptococci play a key
role in the induction of human dental caries, virulence being mediated
by extracellular glucose polymers (glucans) synthesized from sucrose by
secreted glucosyltransferases (GTFs) (EC 2.4.1.5) (8). GTFs
catalyze cleavage of sucrose and the transfer of glucosyl residues to a growing glucan chain. It is also possible to observe sucrose hydrolysis (the transfer of glucose to water) or the synthesis of oligosaccharides resulting from the transfer of glucose to an alternative sugar acceptor
(11).
GTFs are of high molecular mass, around 160 kDa. Except for DSR-A of
Leuconostoc mesenteroides B1299 (10), they
possess a signal peptide followed by a highly variable region of about 100 amino acids. They have a highly conserved catalytic core region of
about 900 amino acids (19) followed by a C-terminal
glucan-binding domain covering about 400 amino acids composed of series
of tandem repeats designated A, B, C, and D repeats (14,
15). At least part of the C-terminal domain is required for
glucan synthesis, though there are conflicting reports as to
whether it is involved in sucrose splitting as well (1, 3,
4). The C terminus may influence the structure of the
glucan (12, 20), though such studies have not been
carried out with a GTF that makes an insoluble glucan. GTF-I expressed
by Streptococcus downei produces a water-insoluble glucan
containing Construction of truncated GTF-I forms and expression in
Escherichia coli.
The signal peptide and N-terminal highly
variable region are not required to have fully active GTF (1,
10). We therefore engineered modified gtfI genes
encoding only the conserved core region (GTF-Ic) or the conserved core
region with either full-length (GTF-I1) or truncated C-terminal domains
(GTF-I2, GTF-I3, and GTF-I4) by subcloning PCR-amplified
gtfI fragments using VENT polymerase (Biolabs Inc.)
and primers designed upon the gtfI sequence (3) containing engineered restriction sites into pTrcHisA
(Invitrogen). The resulting plasmids expressed fusion proteins
containing an additional stretch of six histidine residues at the N
terminus (Fig. 1). Overproduction
of these GTF-I variants was achieved by culturing E. coli
XL1-Blue in Luria broth. After induction with
isopropyl- Purification of GTF-I variants.
Previous processes for
purification of GTFs were based upon their glucan binding ability and
necessitated the use of denaturing agents or the addition of dextran
for elution (2, 6, 15). His-tagged GTF-I variants were
purified under native conditions using Ni-nitrilotriacetic acid agarose
resin (Qiagen). Binding was realized with sodium phosphate buffer (pH
7.8) (500 mM NaCl in the presence of 5 mM imidazole) followed by
washing with sodium phosphate buffer (pH 6.0) (500 mM NaCl in the
presence of 20 mM imidazole). Elution was performed with 200 mM
imidazole. Proteins present in the elution peak were dialyzed overnight
at 4°C against 50 mM Tris-HCl buffer (pH 7.5). Overall, 200-ml
cultures yielded about 4 mg of protein purified to a high degree of
purity (Fig. 2B).
Characterization of GTF-I variants.
Reactions were performed
at 37°C in 50 mM Tris-HCl buffer (pH 7.5) containing 100 g of
sucrose liter
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Isolation of an Active Catalytic Core of
Streptococcus downei MFe28 GTF-I
Glucosyltransferase
ABSTRACT
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(1-3) glucosyl linkages (3, 16). Other almost
identical gtfI genes have been isolated from Streptococcus sobrinus, which is taxonomically closely
related to S. downei (1, 17). To extend earlier
observations on the effect of C-terminal truncation on GTF-I activity
(3) and to define what minimum size was required for glucan
synthesis, we have introduced a rapid and efficient method for
purifying recombinant GTF.
-D-thiogalactopyranoside, protein extracts
were obtained by sonication (Fig.
2A). Activity assays carried out using the dinitrosalicylic acid method specific for reducing sugars (9) showed very little difference between GTF-I4 and GTF-I3. Therefore, GTF-I4 was chosen for further study, as were GTF-I1, GTF-I2,
and GTF-Ic.
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FIG. 1.
Schematic representation of protein sequence of GTF-I
and the truncated variants constructed. Molecular sizes of GTF-I,
GTF-I1, GTF-I2, GTF-I3, GTF-I4, and GTF-Ic are indicated. aa, amino
acids. Repeat units (A, B, and C) are also localized in the C-terminal
glucan-binding domain. Numbers in brackets represent the end points of
the different variants in the gtfI sequence (3).
Activity obtained with purified GTF-I1, GTF-I2, GTF-I3, and GTF-Ic is
indicated at right in boldface.
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FIG. 2.
Expression of the GTF-I-derived forms truncated at the C
terminus. Shown is sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis of GTF-I1, GTF-I2, GTF-I3, and GTF-Ic in
E. coli XL1-Blue (A) and after purification (B).
Experimentally determined sizes were 160, 150, 135, and 110 kDa for
GTF-I1, GTF-I2, GTF-I3, and GTF-Ic, respectively, and were in good
agreement with the predicted values.
1. One unit was defined as the amount of
enzyme that catalyzed the formation of 1 µmol of fructose per min. An
activity assay (Fig. 1) showed there is a slight decrease of activity
in variants with truncated C-terminal domains but that it is less
extensive than those reported in earlier studies (1, 3, 4).
GTF-Ic retained almost 70% of the activity obtained with GTF-I1. This difference may be attributable to the fact that previously activity has
only been estimated from crude E. coli extracts with no
allowance for possible variations in expression levels, whereas we used purified enzyme. For GTFs producing (1-6)-rich glucan, a large part
of the C-terminal domain is required (7, 9). In contrast, the present results show that for a GTF producing (1-3)-rich glucan, the core region alone is active.
1 for GTF-I1
to 36.0 ± 3.0 s1 for GTF-Ic). This suggested that
the turnover rate of the reaction but not the ability for substrate
binding was modified by the truncations in the C-terminal domain. This
domain is, however, required for activation by dextran T-10 (Fig.
3). Addition of 0.5 mg of dextran T-10
ml1 to full-size GTF-I1 resulted in an activity increase
of 1.5. This induction level was similar to those obtained with entire GTF-I from S. sobrinus OMZ176 (5) or S. downei MFe28 (13). GTF-I2 was less sensitive, and
the activator effect was abolished with GTF-I3 and GTF-Ic. The results
clearly show that dextran T-10 binds at a site separate from the
sucrose binding site present in the core region.
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FIG. 3.
Effect of dextran T-10 on GTF-I1, GTF-I2, GTF-I3, and
GTF-Ic activities, which were determined at different dextran T-10
concentrations. Activity obtained without dextran T-10 was defined as
100% activity for each enzyme.
-D-glucopyranosyl-D-fructopyranose), which results from the transfer of glucosyl residues onto fructose (18). All the GTF-I variants, even the core molecule GTF-Ic, exhibited the same yields of glucan synthesis, sucrose hydrolysis, and
leucrose (Table 1). Glucan synthesized by
GTF-I variants was analyzed by 13C nuclear magnetic
resonance spectra and recorded with a Bruker AC 300 spectrometer. This
analysis showed that they contained only (1-3) glucosyl linkages,
as reported for intact GTF-I (14), so the presence of the
C-terminal glucan-binding domain of GTF-I does not influence the
structure of the glucan produced.
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ACKNOWLEDGMENTS |
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This work was supported by Wellcome Trust grant 04539 and the European Project BIOTECH CT98-0022.
We are indebted to M. Vignon (CERMAV, Grenoble, France) for performing the nuclear magnetic resonance analyses.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Oral Biology, Dental School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4BW, United Kingdom. Phone: (44) 191 222 8391. Fax: (44) 191 222 6137. E-mail: vincent.monchois{at}ncl.ac.uk.
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Journal of Bacteriology, April 1999, p. 2290-2292, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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