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Journal of Bacteriology, December 1998, p. 6400-6403, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Role of C-Terminal Domains in Surface Attachment of
the Fructosyltransferase of Streptococcus salivarius
ATCC 25975
Catherine
Rathsam and
Nicholas A.
Jacques*
Institute of Dental Research, Surry Hills,
New South Wales 2010, Australia
Received 18 May 1998/Accepted 16 September 1998
 |
ABSTRACT |
The cell-associated 
-D-fructosyltransferase of
Streptococcus salivarius, which is devoid of the cell wall
anchoring motif, LPXTG, is released on exposure to its substrate,
sucrose. Deletions within the C terminus of the enzyme implicated both
the hydrophobic and the proline-glycine-serine-threonine-rich
wall-associated domain in stabilizing the enzyme on the cell surface.
| |
TEXT |
The
-D-fructosyltransferases (Ftf's) of oral streptococci
and the levansucrases (SacB's) of bacilli form a catalytically distinct family of proteins which polymerize the fructose moiety of
sucrose into extracellular fructans (4, 6, 13, 23). Unlike
the bacilli and mutans streptococci, which secrete their enzymes
directly into the culture fluid (2, 3), the Ftf of
Streptococcus salivarius is initially cell associated
(14). The release of the S. salivarius Ftf from
the surface of the cell occurs upon exposure to its substrate, sucrose
(18). The initial cell surface binding and subsequent
substrate-induced secretion appear to be unique.
It should be noted that since determining the sequence of the Ftf of
S. salivarius (20), we have found that the signal
sequence is cleaved at
TQVKA
DQVTE
to form the mature protein, not at the computer-predicted site,
TLAFLGATQV,
previously published. This suggests that the predicted start site for
translation is at Met-31, not at Met-52. As a result, the numbering of
the amino acids has been modified by subtracting 52 from that
previously published, giving rise to a predicted length of 917 amino
acids for the cell-associated Ftf.
Previous deletion studies have shown that the C-terminal region of the
Ftf directs surface attachment (20). This region displays
high homology with the C termini of other gram-positive surface-bound
polypeptides, such as the M protein of Streptococcus pyogenes (11, 19). In these proteins, the C terminus
contains a cell wall sorting signal which consists of a consensus
pentapeptide motif (LPXTG) followed by a C-terminal hydrophobic domain,
ending with a positively charged tail (8). Secretion of
these proteins is believed to be hindered by the presence of the
charged tail, which allows the LPXTG consensus to be maintained in a
position where it is proteolytically cleaved. In the case of
Staphylococcus aureus, cleavage between the threonine and
glycine results in cross bridge formation between the threonine and
pentaglycine of the peptidoglycan (22). Such a consensus
pentapeptide is absent in the Ftf of S. salivarius, raising
the question of how the Ftf remains attached to the cell surface.
Besides the possible involvement of the hydrophobic domain in
retention, another region common to surface-bound proteins lies directly N-terminal to the sorting signal. This so-called
wall-associated domain is defined as a region spanning 50 to 125 residues with contents of proline-glycine and threonine-serine residues
ranging from 15 to 32 and from 13 to 38%, respectively (8).
The wall-associated domain of the Ftf of S. salivarius ATCC
25975 that directly precedes the hydrophobic C-terminal domain contains
an extended proline-glycine-threonine-serine-rich domain spanning 178 amino acids from Pro-705 to Ser-882, with proline-glycine and
threonine-serine contents of 17 and 22%, respectively. The
wall-associated domain is separated from the catalytic domain of the
Ftf (defined as that region between Gln-198 and Asp-631 possessing high
homology with the SacB's of bacilli [20]) by a spacer
region of 73 amino acids (Fig. 1).
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FIG. 1.
Comparison of the generalized domain structure of
parental and mutated Ftf and their cellular location prior to and
following incubation of washed cells with sucrose. C, catalytic domain;
S, 73-amino-acid spacer; W, wall-associated domain; H, hydrophobic
domain; +, positively charged amino acid C terminus. Numbers refer to
the first or last amino acid present within a given or truncated
domain.
Ftf activity (shown as means ± standard deviations, with the
number of experiments given in parentheses) was quantified as
previously described by using
[U-fructosyl-14C]sucrose (12). One
unit of enzyme activity was defined as the amount of Ftf that catalyzed
the incorporation of 1 µmol of the fructose moiety of sucrose into
75% (vol/vol) ethanol-insoluble fructan per min. In all cases, Ftf
activity was expressed as units per milligram (dry weight) of cells as
a means of indicating the amount of enzyme secreted by a given cell
mass. Ftf activity for S. salivarius was within the range of
580 to 660 mU mg (dry weight) 1, and Ftf activity for
S. gordonii transformed with pKRK2969, pKRK2914, pKRK2816,
pKRK2001, pKRK2003, or pKRK2004 was in the range of 1,220 to 1,720, 700 to 1,360, 610 to 920, 891 to 1095, 644 to 904, or 756 to 958 mU mg (dry
weight)1, respectively.
For cell culture, S. salivarius ATCC 25975 (14)
and S. gordonii LGR2 (24) were grown in
Todd-Hewitt broth supplemented with 1% inactivated horse serum and
0.6% yeast extract, with erythromycin (40 µg ml1)
added where appropriate. At late-exponential phase, cells were
harvested and the amount of Ftf activity in the supernatant was
compared with that bound to the cell.
For sucrose incubation, washed cells were incubated with 10 mM sucrose
as described in the text and the amount of Ftf released from the cell
was determined. Data in this column are shown only where the majority
of Ftf activity was initially cell associated.
|
|
In order to distinguish the roles of the hydrophobic and
wall-associated domains in the attachment of the Ftf of S. salivarius ATCC 25975 to the surface of the cell, regions of the
ftf gene coding for these domains were deleted and the
recombinant proteins were expressed in the heterologous host
Streptococcus gordonii LGR2 (20). S. gordonii LGR2, which does not produce an Ftf, was used as a model
system because S. salivarius ATCC 25975 is refractory to
stable transformation by electroporation in our hands (20).
The phagemids and plasmids used in this study are listed in Table
1.
Effect of proteinase inhibitors on the release of Ftf from S. salivarius.
Surface protein-releasing enzyme activity has been
characterized in some pathogenic streptococci (16). Other
studies have shown that oral streptococci release surface proteins,
particularly antigens such as antigen A and protein P1, by the
action of endogenous proteinases (7, 17). The implication of
these studies is that these streptococci shed antibody-antigen
complexes as a means of evading the host immune system (15).
When washed cells of S. salivarius were incubated for 5 min
at 37°C with 10 mM sucrose in the presence of 10 mM NaF and 50 µg
of chloramphenicol ml1 to inhibit glycolysis and de novo
protein synthesis, respectively (18), phenylmethylsulfonyl
fluoride (35 µg ml1) in conjunction with either
aprotinin (2 µg ml1), chymostatin (100 µg
ml1), leupeptin (500 ng ml1), trypsin
inhibitor (100 µg ml1), EDTA (500 ng
ml1), benzamide (100 µg ml1), or
iodoacetamide (100 µg ml1) failed to prevent the
release of cell-associated Ftf (data not shown). These results
suggested that either the release of the enzyme from the surface of
S. salivarius was independent of a proteolytic event or a
novel proteinase not affected by the inhibitors was responsible for the
secretion process.
Cellular location of mutated Ftf in S. gordonii.
Site-directed mutagenesis of the ftf gene made use of the
previously described mutagenic oligosaccharides in conjunction with the
T7 modification of the Transformer Site-Directed Mutagenesis Kit
supplied by Clontech Laboratories (21). This modified
procedure allowed the construction of an ftf allele that
expressed a truncated Ftf, Ftf with amino acids 880 through 917 deleted
[Ftf(
880-917)], which was devoid of its hydrophobic
domain and its positively charged C terminus (Fig. 1). Site-directed
mutagenesis was also used to introduce BamHI sites into
ftf genes such that in-frame deletions could be constructed
(21). These in-frame deletions expressed Ftf devoid of
portions of the proline-glycine-threonine-serine-rich wall-associated
domain while maintaining their hydrophobic tails and positively charged
C termini intact (Fig. 1). All mutated forms of the enzyme expressed in
Escherichia coli retained the ability to hydrolyze sucrose
and to form fructan except for Ftf(635-875), expressed
by pKRK1005. This inactive form of the enzyme was devoid of its entire
wall-associated domain as well as 96% of the 73-amino-acid C-terminal
spacer region linking it to the catalytic domain but retained its
C-terminal hydrophobic domain with its positively charged C terminus
(Fig. 1). The direct juxtaposition of the hydrophobic domain and the
catalytic domain of the Ftf may have destabilized the tertiary
structure of the enzyme. This hypothesis was supported by a previous
observation that an altered Ftf truncated at Ser-609, and thus having
22 amino acids deleted from the C-terminal region of the `catalytic'
domain, was also inactive (20). These two observations
suggest that the tertiary structure formed by the C-terminal amino
acids of the catalytic domain of the Ftf is critical for the
maintenance of catalytic function.
To study the effect of deletions of various C-terminal domains on the
localization of the Ftf in a gram-positive streptococcus, the mutated
ftf genes were cloned into the E. coli-Streptococcus shuttle vector pVA838 and transformed into
S. gordonii. Cells harvested at various stages throughout
the growth cycle were used to determine the amount of Ftf bound to the
cell, as well as the amount secreted into the culture medium. Where
deemed appropriate, the amount of Ftf activity released following
incubation of washed cells with sucrose was also measured
(18). Irrespective of the stage of growth, the relative
percentage of each mutated Ftf present on the surface of the cell, as
well as that subsequently released from washed cells in the presence of
sucrose, was not significantly different from that measured in
late-exponential phase (data not shown).
Deletion of the C-terminal hydrophobic domain resulted in the secretion
of Ftf(880-917) by the heterologous host, S. gordonii (Fig. 1). This result was in keeping with the data previously obtained with truncated Ftf's expressed by plasmids pKRK2914 and pKRK2816 in S. gordonii that carried 3'
deletions of the ftf gene constructed by random exonuclease
III digestion (20). However, in these two instances,
portions of the C-terminal wall-associated domain of the Ftf had also
been removed along with the hydrophobic domain (Fig. 1). In order to
determine whether there was a specific role for the wall-associated
domain in surface retention of Ftf, Ftf(695-875),
devoid of its wall-associated domain but retaining its hydrophobic C
terminus intact, was expressed by pKRK2003 in S. gordonii.
This mutated Ftf was secreted, indicating that the wall-associated
domain was essential for the stable binding of Ftf to the surface of
S. gordonii irrespective of the presence of the hydrophobic
region (Fig. 1). Inclusion of 22% of the wall-associated domain
together with the C-terminal hydrophobic domain resulted in 54% of the
Ftf remaining attached to the surface of S. gordonii (Fig.
1). This result further supported the hypothesis that the wall-associated domain was as important as the hydrophobic domain in
stabilizing the Ftf on the surface of the cell. The cell-associated Ftf, Ftf(738-875), possessing only 22% of the
wall-associated domain, was released from the surface of S. gordonii by its substrate, sucrose, by an increased (33%) amount
compared with the intact parental enzyme (Fig. 1).
Conclusions.
The release of Ftf from the surface of S. salivarius was not inhibited by any of the proteinase inhibitors
tested. It is possible that the release of the Ftf from the cell
surface is not a proteolytic event but is caused by "tearing" of
the enzyme from the surface by large fructan complexes. Alternatively,
the enzyme may be autoproteolytic in the presence of its substrate,
sucrose, and may be capable of self-cleavage from the surface of the cell.
The absence of the carboxy-terminal pentapeptide anchoring signal
(LPXTG) found in other gram-positive surface proteins implicated the
wall-spanning region and the hydrophobic domain in the attachment of
the Ftf of S. salivarius to the surface of the cell
(20). The results of this study confirmed the
well-documented role of the hydrophobic domain in anchoring proteins to
the plasma membranes of gram-positive bacteria (10). This
study also supported the hypothesis that the wall-associated domain was
just as essential as the hydrophobic C-terminal domain in stabilizing
surface attachment (1, 20). The presence of an extended
wall-associated domain possessing turn-promoting proline-glycine
residues could theoretically allow the C-terminal wall-associated
region of the Ftf to span the cell wall by randomly intercalating
throughout the peptidoglycan-carbohydrate-teichoic acid matrix. The
high serine-threonine content could further stabilize this association
by allowing hydrogen bonding to these constituents of the cell wall.
Whether this is the case or not, it is clear from the results of this
study that both the C-terminal hydrophobic domain and the extended
wall-associated domain of the Ftf of S. salivarius are
required for stable attachment of the enzyme to a gram-positive cell surface.
| |
ACKNOWLEDGMENTS |
This work was supported by a project grant awarded by the
Australian National Health and Medical Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Dental Research, 2 Chalmers St., Surry Hills, NSW 2010, Australia.
Phone: 61-2-9293-3353. Fax: 61-2-9293-3368. E-mail:
nickj{at}dentistry.usyd.edu.au.
| |
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Journal of Bacteriology, December 1998, p. 6400-6403, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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