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FEMS MicrobiologyLetters 72 (1990) 299-302
Published by Elsevier
299
FEMSLE04215
Carboxyl-terminal deletion analysis of the Streptococcus mutans
glucosyltransferase-I enzyme
Chiaki K a t o a n d H o w a r d K. K u r a m i t s u
Department of Pediatric Dentistry, University of Texas Health Science Center. San Antonio. TX U.S.A.
Received15 June 1990
Revisionreceived10 July 1990
Accepted 11 July 1990
Key words: Streptococcus mutans; Ghicosyltran~ferase; Deletions
1. S U M M A R Y
Sequential deletion of the carboxyl-terminal
amino acids (including the six direct repeating
units) of the glucosyhransferase-I (GTF-I) enzyme
of Streptococcus mutans revealed differential effects on sucrase and G T F activities. Removal of
all but one repeating unit resulted in a truncated
enzyme with significant sucrase activity but no
detectable G T F activity. These results are compatible with the presence of two functional domains
in the enzyme.
2. I N T R O D U C T I O N
Streptococcus mutans is the principal etiological
agent in the development of human dental caries
[1], One of the important cariogenic properties of
these organisms is their ability to synthesize adherent water-insoluble giucans from dietary
Correspondence to: H.K. Kuramitsu, Department of Pediatric
Dentistry, University of Texas Health Science Center, San
Antonio, TX 78284, U.S.A.
sucrose. This property is dependent upon the concerted action of two groups of glucosyltransferases
(EC 2.4.1.5): GTF-S which synthesize primarily
soluble glucans, and GTF-! which catalyze the
formation of insoluble products [1]. Both biochemical [2] and genetic [3-6] characterization of
these enzymes have been reported.
Nucleotide sequencing of the GTFs produced
by the mutans streptococci [7-9] has revealed the
presence of multiple homologous direct repeating
units consisting of approximately 65 amino acid
residues at the carboxyl-termini of the enzymes.
The isolation of truncated forms of the GTF-I
from Streptococcus downei has suggested that the
carboxyl one-third of the enzyme contains the
sequences required for both enzymatic activity
and ghican binding [7]. In agreement with these
results, a proteolytic fragment of a G T F has been
isolated from this region exhibiting glucan-binding
activity [10]. Therefore, in order to define more
precisely the role of the direct repeating units in
the activity of the GTFs, a deletion analysis of the
GTF-I from S. mutans was carried out. These
results are discussed relative to the h~Jothesis that
multiplt: functional domains are present in these
enzymes.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
300
3. MATERIALS AND METHODS
3.1. Bacterial strains
Plasmid pTSU5 was constructed by ligating the
promoter-containing 1.9 kb PstI-EcoRI fragment
of plasmid pTS20 [7] with the 4.5 kb EcoRI-Pstl
fragment of pSU5 [7] inserted into pUCll9.
Transformation, plasmid DNA preparation and
DNA manipulations were carried out as previously described [31.
3.2. Construction of GTF-I deletion mutants
Plasmid pTSU5 was cleaved with XbaI and
SacI and a series of deletion plasmids were constructed following exonuclease III and mungbean
nuclease digestion as previously described [11].
Nucleotide sequence analysis was carried out by
the dideoxy chain termination procedure [12].
3.3. Preparation and analysis of crude extracts from
deletion derivatives
E. coil HB101 containing the deleted plasmids
was grown at 30°C for 16 h in (2 x ) TY medium,
centrifuged, washed, and extracts prepared following sonic disruption [13], The proteins from each
deletion derivative were analyzed following 7%
SDS-PAGE as recently described [3]. Sucrase and
GTF activities were determined as previously outlined [3].
4. RESULTS AND DISCUSSION
The S. mutans GS-5 GTF-I activity expressed
from the gtfB gene was previously demonstrated
to contain three and one-half direct repeating units
consisting of 65 amino acids at the extreme
carboxyl-terminus of the enzyme [7]. However,
further upstream from the beginning o¢ these repeating units, six repeating units containing 67
amino acids overlapping and including the previously characterized units [71 could be identified
(Fig. 1). In order to determine the function of
these latter repeating units in enzyme activity, the
6.4 kb Pstl fragment of plasmid pTSU5 containing the gtfB gene was sequentially deleted from
the 3' end of the insert. Each deletion plasmid was
ligated, transformed into E. coli HBI01, and the
isolated plasmids analyzed by restriction mapping
and DNA sequencing (Fig. 2).
Extracts of each deletion derivative were prepared and assayed for enzyme expression following SDS-PAGE (Fig. 3). Western blotting (Fig.
3B) indicated that similar quantities of GTF-I
1096
1
WYYFDNNGYM-VTGAQSINGVNYYFLSNGLQLRDAILKNEDGTYAYY-GNDGRRYENGYYQFMSGV
• *******
****
***
**
** .
.
** ** .
.
2
WRHF-NNGEMSV-GLTVIDGQVQYFDEMGYQAKGKFVTTADGKIRYFDKQSGNMYRNRFIENEEGK
3
WLYLGEDGAA-VTGSQTINGQHLYFRANGVQVKGEFVTDHHGRZSYYDGNSGDQIRNRFVRNAQGQ
• *
, * ***
***********************
**************************
4
WFYFDNNGYA-VTGARTINGQLLYFRANGVQVKGEFVTDRYGRISYYDGNSGDQIRNRFVRNAQGQ
5
WFYFDNNGYA-VTGARTINGQHLYFRANGVQVKGEFVTDRHGRISYYDGNSGDQIRNRFVRNAQGQ
• ***~*~*** *******************************************************
6
WFYFDNNGYA-VTGARTZNGQHLYFRANGVQVKGEF%~DRYGRZSYYDANSGERVRIN
.
1475
CS WFY FDNNGYA-VTGARTINGQHLYFRANGVQVKGEFVTDRHGRI
SYYDGNSGDQI RNRFVRNAQGQ
Fig. 1, Amino acid s~uence of the six direct ~ a t i n g units of the G T ~ I en~me, The numbers at the ~ginning and end of the
s~uen~ ~ r ~ the amino~id ~sitions fromthe imtiatormet~onine,The as~ri~s ( ~) indica~the aminoacids~mol~ous with
t~ ~n~nsus s~uen~ (cs).
o
3
i
6 {kb}
i
IOO.
P
H
E'B
HH
B
I
I
It
I
I
I
P
I
-' ,
::::)
gff - C
gtf - B
pTSU 5
pCK28
pCK35
.=
pTS20
pCK36
pCKl|
pCK4|
pCK24
0
Fig. 2. Structures of the deletion derivatives of the GTF-I
enzyme. The restriction map of the 6.4 kb Ps|l fragment is
depicted above the structures of the deletion derivatives. The
..L~
Repeating
units
gtfB
gene. H, Hindlll: P, Pstl: B, BamHl', E, EcoRI.
Fig. 4. Correlation between the number of direct repeating
units and enzyme activities of the GTF-I deletion derivatives.
©-c~, Sucra~ activity; O. . . . . 4 , GTF activity.
protein were expressed by each plasmid but G T F
activity could only be detected on the gels for the
larger p e p | i d e s (Fig. 3C). More sensitive sucrase
and G T F radioactive assays of each derivative
indicated differential effects on the two activities
(Fig. 4). Removal of more than three of the terminal repeat units (containing less than 1288 of the
1475 amino acids of the intact enzyme) resulted in
a marked decrease in b o t h activities with much
greater effects on G T F activity. In addition, the
presence o f at least a portion of one repeat unit
(enzyme with 1139 amino acids) yielded an enzyme with insignificant G T F activity but subs|an-
tial sucrase activity. Removal of all six repeat
units abolished bo:.h activities.
These results confirm an earlier investigation
with the G T F - ! from S. downei [9} indicating that
the carboxyl-terminal repeat units arc important
for enzyme activity. However, in contrast to the
enzyme from S. downei, removal of all but one
repeating unit of the GS-5 enzyme yielded an
enzyme with significant sucrase activity. This difference may reflect functional or structural dissimilarities between the two enzymes [11]. The present
results demonstrating differential effects of a m i n o
acid deletion on sucras¢ and G T F activities are
direct repeating units are depicted at the 3' end of the
A
M
200--
~'
9 71 .146--
~am
66.2--
~
1
2
34
S
5
6
7
8
~=~...
......
1
•
2
........
3
4
~
C
5
6
7
8
I
2
3
4
5
6
7
8
; ~ ~;~ ~
' i;~"',
(KDo)
Fig. 3. SDS-PAGE analysis of the proteins expressed by the deletion derivatives. (A) Cca)massie blue staining. (B) Western blot
analysis with anti,GTF serum. (C) GTF activity staining. Crude extracts from: I, pCK28: 2, pCK35: 3, pTS20; 4, pCK36; 5. pCKI 1;
6, pCK41 ; 7, pCK24; 8, pUC! 19 were analyzed. Molecular mass markers are indicated in the left margin.
302
also compatible with the presence of two functional domains on the enzymes (one involved in
sucrose binding and hydrolysis and the other with
glucan binding and chain extension). This hypothesis is further supported by recent data indicating that the sucrose binding site is present
within the amino-terminal one-third o f the G T F
enzymes (Mooser, personal communication).
ACKNOWLEDGEMENTS
This investigation was supported in part by
Public Health Service G r a n t DE-03258 from the
National Institutes of Health.
REFERENCES
[!1 Loesche, WJ. (1986) Microbiol. Rev. 50, 353-380.
[2] Shimamura, A., Tsumori, H. and Mukasa, H. (1983) FEBS
Lett. 157, 79-84.
[3] Aoki, H., Shiroza, T., Hayakawa, M.. Sato, S. and Kuramitsu, H.K. (1986) Infect. lmmun. 53, 587-594.
[4] Hanada. N. and Kuramitsu, H.K. (1988) Infect. lmmun.
56.1999-2005.
[5] Hanada, N. and Kuramitsu, H.K. (1989) Infect. imman.
57. 2079-2085.
[6] Russell, R.R.B., Gilpin, M.L., Mukasa, H. and Dougan.
G. (1987) Jo Gen. Microbiol. 133, 935-944.
[7] Shiroza, T., Ueda, S. and guramitsu, H.K. (1987) J.
Baeteriol. 169, 4263-4270.
[8] Ueda, S., Shiroza, T. and Kuramitsu, H.K. (1988) Gene
69,101-109,
[9] Ferretti, J.J., Gilpin, M.L. and Russell, R.R.B. (1987) J.
BacterioL 1269, 4271-4278.
[10] Mooser, G. and Wong, C. (1988) Infect, Immun. 56,
880-884.
[11] Russell, R.R.B., Shiroza, T., Kuramitsu, H.K. and Ferreui, J.J. (1988) J. Dent. Res, 67, 543-547,