From the Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain
Received for publication, November 13, 2000, and in revised form, March 9, 2001
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ABSTRACT |
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The type 37 capsule of Streptococcus
pneumoniae is a homopolysaccharide built up from repeating units
of
[ Streptococcus pneumoniae (pneumococcus) is an important
human pathogen and a common etiological agent of community-acquired pneumonia and meningitis in adults and of acute otitis media in children. The capsular polysaccharide has been identified as the main
virulence factor of S. pneumoniae (1). The capsule confers to pneumococcus the advantage to resist phagocytosis and survive in the
host. Pneumococcus has evolved by diversifying its capsule, and up to
90 different capsular types synthesizing polysaccharides with different
immunological properties and chemical structures have been described
(2). Capsular polysaccharide biosynthesis in S. pneumoniae
is usually driven by genes located in the cap/cps locus, and the capsular cluster of 13 pneumococcal types has been sequenced recently (3). In remarkable contrast, only a single gene
(tts) located far apart from the cap cluster,
directs the synthesis of the type 37 capsule (4). Type 37 capsular
polysaccharide is the only homopolysaccharide reported in pneumococcus.
Clinical isolates belonging to this serotype synthesize a conspicuous
capsular envelope that is a branched polysaccharide that has a linear
backbone of Only few gene products involved in pneumococcal capsular formation have
been biochemically characterized, and almost nothing is known about
mechanisms as important as regulation, transport, and assembly of the
polysaccharide chain subunits (3). It is generally thought that these
polysaccharides are synthesized via lipid-linked repeat unit
intermediates because of the biochemical complexity of the repeating
oligosaccharide subunit. In types 14 and 19F, the first step of this
process involves the activity of the protein coded by
cps14(cps19f)E gene (6, 7). This protein catalyzes the selective incorporation of Glc from UDP-Glc to a
membrane lipid-linked acceptor leading to the formation of a complex
where other glycosyltransferases would transfer the sugars present in
the polysaccharide repeating subunit (7). However, in type 3 pneumococci, sugars are transferred directly to the growing
polysaccharide chain without intervention of an anchoring lipid
molecule. We have demonstrated that Cap3B, the type 3 polysaccharide
synthase, is the only protein required to synthesize high molecular
weight type 3 capsular polysaccharide in S. pneumoniae or
Escherichia coli strains provided that UDP-Glc and
UDP-GlcUA, the precursors of type 3 capsular monosaccharides, were
available (8). It has also been shown that Cap3B (also designated as
Cps3S) is a processive enzyme able to transfer alternated residues of
Glc and GlcUA from their respective UDP-sugars to the nonreducing end
of the nascent polysaccharide chain (9). Cap3B possesses a double
We report here the subcellular localization and biochemical
characterization of the type 37 synthase in S. pneumoniae
strains expressing the tts gene. We also show the ability of
Tts to produce a type 37-specific capsule even when expressed in
Gram-positive bacteria other than pneumococcus.
Bacterial Strains, Plasmids, and Growth Conditions--
The
unencapsulated laboratory S. pneumoniae strains used are as
follows: M24 (S3 DNA Techniques and Plasmid Construction--
DNA
manipulations and other standard methods were as described in Sambrook
et al. (22). Primer extension mapping of the transcription
initiation site (4) and polymerase chain reaction amplifications (23)
were carried out as described previously. Conditions for amplification
were chosen according to the G + C content of the corresponding
oligonucleotides. The oligonucleotide primers mentioned in the text are
as follows: (D101) 5'-TTTGACCAAGCTTACACTTCAG-3'; (D112)
5'-TCTCATATTCTAgaCTTCTTTTCAGTTTACAC-3'; (D116)
5'-TCCTTACCATACaTCgATACTAAC-3'; and (D138)
5'-TCAATCTAACATCGTTGCTTCCAC-3'. Lowercase letters indicate nucleotides
introduced to construct appropriate restriction sites; these are shown
underlined (see Fig. 1A).
To construct pDLP50, chromosomal DNA prepared from the 1235/89 strain
was polymerase chain reaction-amplified with oligonucleotide primers
D101 and D112 and made blunt-ended with the Klenow fragment of the
E. coli DNA polymerase I (PolIk). Subsequently, the DNA fragment was digested with XbaI and ligated to pLSE4 that
had previously been digested with SphI, filled in with
PolIk, and then treated with XbaI. The ligation mixture was
used to transform S. pneumoniae M31, and a clone harboring
pDLP50 was isolated by scoring the LnR transformants for
expression of the lytA gene by using a filter technique
described previously (24). Plasmids pDLP48 and pDLP49 were constructed
as follows: 1235/89 DNA was polymerase chain reaction-amplified with
oligonucleotide primers D101 and D116, and the product was digested
with either SphI (for pDLP48) or SacI (for
pDLP49) and filled in with PolIk. After digestion with ClaI
(restriction enzyme target included in the primer D116), the
appropriate fragments were ligated to pLSE1 (previously digested with
EcoRV and MspI) and used to transform competent
M24 cells. Type 37-encapsulated transformants were scored among the
LnR clones, and one clone of each class, i.e.,
harboring either pDLP48 or pDLP49, was selected.
Preparation of Cell-free Extracts and Tts Enzymatic Activity
Measurements--
Exponentially growing cultures (1 liter) of S. pneumoniae M24 harboring pLSE1 or pDLP49 were chilled on ice and
centrifuged (12,000 × g, 20 min, 4 °C), and the
pellet was suspended in 10 ml of TMCa buffer (70 mM
Tris-HCl, pH 7.0, 9 mM MgCl2, 1 mM
CaCl2) containing 0.2 mM phenylmethylsulfonyl
fluoride (PMSF) and centrifuged again (10,000 × g, 10 min, 4 °C). The bacteria, resuspended in the same buffer, were
disrupted by two passages of the suspension through a French pressure
cell (Aminco). The homogenate was centrifuged at 12,000 × g at 4 °C for 15 min, and the supernatant was centrifuged again at 120,000 × g at 4 °C for 1 h. The
pelleted membranes were homogenized in 2 ml of TMCa buffer containing
0.2 mM PMSF, distributed in 100-µl aliquots, and stored
at
Unless otherwise stated, standard reaction mixtures contained 0.5 mg/ml
membrane proteins, 30 µM (0.1 µCi)
UDP-[14C]Glc (specific activity 319 mCi/mmol) (Amersham
Pharmacia Biotech) in a 70 mM Tris-HCl, pH 7.0, buffer
containing 9 mM MgCl2, 1 mM CaCl2, and 50 mM NaCl in a total volume of 100 µl. The reactions, carried out at 30 °C for 15 min, were
terminated by the addition of SDS (0.5% final concentration) and were
incubated at 37 °C for 15 min. Afterwards, bovine serum albumin
(Sigma) at a final concentration of 0.4% and 1 ml of 10%
trichloroacetic acid were added. After incubation for 30 min at
0 °C, the mixtures were passed through Whatman GF/A filters and
extensively washed with 10% trichloroacetic acid. The filters were
dried (65 °C, 20 min) and counted in a 1219 Rackbeta scintillation
counter (LKB Wallack). One unit of Tts activity is expressed as the
amount of enzyme that catalyzed the incorporation into a macromolecular
product of 1 pmol of Glc/mg of protein/min.
Identification of the Reaction Product of the Tts
Synthase--
The total volume of a standard reaction mixture carried
out as described above was treated with SDS and filtered through a Sepharose CL-4B column (20 × 1.5 cm; Amersham Pharmacia Biotech). The products of the reaction were eluted with 20 mM
Tris-HCl, pH 7.5, buffer containing 0.2 M NaCl; 0.5-ml
fractions were collected, and alternate fractions were counted. The
high molecular weight fractions that eluted at
V0 were pooled, dialyzed into water, lyophilized, dissolved in 2.5 M trifluoroacetic acid, and
subjected to hydrolysis for 2.5 h at 120 °C. Then the samples
were analyzed by HPLC as indicated below or subjected to thin layer
chromatography (TLC) after being repeatedly dissolved and lyophilized.
The dried pellet was dissolved in 40% 2-propanol containing 5 mg/ml
unlabeled carrier Glc and Gal. TLC was carried out on HPTLC silica gel
60 plates (Merck), impregnated with phosphate, and activated as
described by Hansen (27) but using the solvent system 2-propanol,
acetone, 0.1 M formic acid (2:2:1) (28). To visualize
unlabeled sugar standards, the TLC plate was sprayed with 5%
H2SO4 in ethanol and heated to 100 °C for
10-30 min. The regions that contain the unlabeled sugar standards were
scraped, added to water, and counted in a liquid scintillation counter.
The radioactive fractions containing the unincorporated sugar
nucleotide precursors that eluted at VT were treated with 10 mM HCl at 100 °C for 10 min and neutralized with
NaOH. Both excluded and retained fractions were then analyzed by HPLC by using an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad) and eluted at 30 °C with 125 µM
H2SO4 at 0.25 or 0.4 ml/min (see below). The
elution of authentic samples of Glc and Gal was monitored with an
in-line 132 refractive index detector (Gilson).
Miscellaneous Techniques--
Type antisera purchased from the
Statens Seruminstitut (Denmark) were used for immunological analyses.
As a potential competitor in immunoprecipitation assays, we used
curdlan, a linear (1 Transcriptional Analysis of the tts Gene--
We have reported
previously the identification of the tts promoter and its
transcription start point (4). The ttsp promoter contains a
Expression of Tts in other Gram-positive Bacteria--
According
to the results described above, we used pDLP49 to transform competent
cells of S. pneumoniae M24, S. oralis NCTC 11427, S. gordonii V288, and B. subtilis YB886.
LnR (or EryR) transformants were isolated, and
selected colonies were grown in broth to test for the production of
type 37 capsule. In every case, expression of tts led to
agglutination of the bacterial cells when incubated in the presence of
type 37-specific antiserum (Fig. 4).
Immunoagglutination never occurred either when the same strains were
incubated with non-type 37 antiserum or when the recipient strains
harbored the vector plasmid pLSE1 and received anti-type 37 serum.
These results demonstrated that only tts is required for the
synthesis of a type 37 capsular polysaccharide in several Gram-positive
species. Furthermore, the above immunoagglutination test using whole
cells indicated that the capsular material is, at least in part, linked
to the outer bacterial surface.
To determine whether a single copy of the tts gene was also
sufficient to direct capsule formation in a heterologous host, we
transformed competent cells of S. oralis with chromosomal
DNA from the pneumococcal strain C2, a type 37 transformant carrying a
single tts copy linked to the ermC resistance
marker. S. oralis LnR transformants agglutinated
in the presence of type 37-specific antiserum (Fig. 4I)
demonstrated that it was possible to transfer tts to this
related species and that its presence in a single copy also leads to
the production of detectable amounts of a capsular polysaccharide
immunologically indistinguishable from the pneumococcal type 37 strains.
Subcellular Localization of the Tts Activity--
To prepare a
homologous system for biochemical assays, we used the type 37 pneumococcal strain M24 [pDLP49] described above. Subcellular
fractions of M24 [pDLP49] were tested for incorporation of
radioactivity into a macromolecular product by using
UDP-[14C]Glc, assuming that UDP-Glc was the natural
substrate for Tts. The membrane fraction turned out to incorporate the
label, whereas the soluble fraction did not (data not shown). SDS-PAGE
analysis of a membrane preparation from M24 [pDLP49] revealed the
presence of an overproduced protein with a molecular mass of ~50 kDa
(Fig. 5). This protein was absent in
membranes prepared from M24 [pLSE1], a strain harboring only the
vector plasmid. Another protein band migrating faster than that
of Tts could also be occasionally observed, and it might have been
originated by proteolysis of Tts, although PMSF was used during the
preparation of the membrane fraction.
Biochemical Properties of the Type 37 Synthase--
Membranes of
the pneumococcal M24 [pDLP49] strain were used to evaluate the
incorporation of [14C]Glc from its precursor
UDP-[14C]Glc into a macromolecular product using
different experimental conditions. Membranes prepared from S. pneumoniae M24 [pLSE1] cells were employed as a negative
control. Tts activity was stimulated in the presence of 10 mM MgCl2 or MnCl2. Moreover, 10 mM EDTA completely inhibited the reaction (Table
I). However, Ca2+ ions
stimulated only slightly Tts activity when added at low concentration
(1 mM) in the absence of Mg2+ (data not shown).
Furthermore, EGTA only produced a small inhibition of the reaction
(Table I). Globally, this behavior is similar to that already described
for several glycosyltransferases like cellulose synthases, HAS, or the
pneumococcal type 3-specific synthase. In addition, 50 mM
NaCl increased 2-fold the incorporation of [14C]Glc into
a macromolecular product (data not shown). Other important properties
of Tts are reported in the composite Fig.
6. The Tts synthase exhibited a
noticeable pH dependence, and the optimal activity was achieved between
6.8 and 7.5 (Fig. 6A). Formation of the radiolabeled
macromolecular product of Tts was proportional to protein concentration
and proceeded linearly with time for up to 15 min and then slowed down
(Fig. 6, B and C). The enzymatic activity reached
a maximum when the reaction was carried out at 30 °C in the presence
of the substrate UDP-[14C]Glc. Tts was relatively stable
when incubated at 0 °C for up to 60 min, but its activity
drastically decreased when preincubation was carried out at 25 °C or
higher temperatures (Fig. 6D).
We have also assayed the effect of sugars or sugar nucleotides on the
Tts synthase activity (Table I). A strong inhibition occurred when
membranes were preincubated in the presence of UTP, UDP, UMP, or TMP,
whereas different sugars (Glc, Gal, GalUA, GlcUA, or Ara) did not
affect noticeably the later incorporation of radiolabeled Glc.
UDP-sugars like UDP-Gal, UDP-Xyl, and UDP-Man completely inhibited the
reaction, whereas CDP-Glc, GDP-Glc, GMP, or CMP only exhibited a
moderate inhibitory effect. These results suggest that the nucleotide
moiety of the substrate, and not the sugar one, would play an important
role in binding and/or activity. On the other hand, detergents were
found to be powerful inhibitors of Tts activity (Table I) suggesting a
close association between Tts and the cell membrane. Interestingly,
titration of the Tts synthase with p-hydroxymercuribenzoate
(pHMB) resulted in a complete loss of enzymatic activity
that could be partially prevented by addition of 2-mercaptoethanol (ME)
(Table I) indicating that there might be sulfhydryl groups implicated
in the folding of the protein, in its enzymatic activity, or both.
Finally, bacitracin added at concentrations of 1 or 100 µg/ml to the
reaction mixture did not inhibit the reaction (Table I), strongly
suggesting that a lipid intermediate is not involved in the
biosynthesis of the type 37 capsular polysaccharide of S. pneumoniae.
Effect of UDP-Gal on the Enzymatic Activity of Tts--
As
reported above (Table I) UDP-Gal is a potent inhibitor of Tts synthase.
Moreover, we have shown that Tts shares conserved motifs with cellulose
synthases and other Characterization of the Polysaccharide Product of Tts
Synthase--
As shown above, the polymer(s) synthesized by using
either UDP-[14C]Glc or UDP-[14C]Gal as
substrate eluted in the void volume of a Sepharose CL-4B column,
whereas non-incorporated radioactive UDP-sugars appeared in the
VT (Fig. 7A). The excluded fractions were
pooled and hydrolyzed with 2.5 M trifluoroacetic acid as
described under "Experimental Procedures," and the samples were
analyzed by HPLC. In addition, fractions containing the
non-incorporated UDP-[14C]sugars were hydrolyzed with 10 mM HCl, neutralized, and also subjected to HPLC analysis.
The radioactivity found in the excluded, hydrolyzed fractions co-eluted
with a Glc standard solution irrespectively of the labeled precursor
used in the reaction (Fig. 7B). Identical results were
obtained when the same fractions were analyzed by TLC; that is,
radioactivity was detected only in the spot corresponding to Glc using
either UDP-[14C]Glc or UDP-[14C]Gal as
substrate (not shown). These results confirmed that, in both cases, Tts
synthesized a polymer composed exclusively of Glc. These findings imply
that UDP-[14C]Gal must be epimerized to
UDP-[14C]Glc before incorporation into the nascent
polysaccharide chain. Some authors (7, 36, 37) had suggested the
presence of a strong UDP-Glc-4'-epimerase activity associated with the
membrane fraction of S. pneumoniae belonging to various
capsular types that did not include type 37. Here we show that this is
also the case for type 37 pneumococcal membranes as fully confirmed
by HPLC analysis of the hydrolyzed UDP-sugars obtained from the
fractions eluted at the VT of the Sepharose CL-4B
column (Fig. 7C). Independently of the radiolabeled
precursor used in the assay, the presence of the pneumococcal membranes
promoted the appearance of both epimers, UDP-[14C]Glc and
UDP-[14C]Gal.
We have recently reported that a single gene (tts)
located outside of the cap/cps locus drives the
synthesis of the capsular polysaccharide in type 37 pneumococci (4). We
have now found that transcription of the tts gene also
initiates at four different points located upstream of the previously
reported promoter ttsp (Figs. 1 and 2). It is important to
point out that three of the additional transcription start points are
located inside a RUP element (Fig. 3). Several features of RUPs led to
the proposal that these small (107 base pairs long) intergenic elements
could be trans-mobilized by the transposase of
IS630-Spn1 insertion sequence (31) and possibly
promote sequence rearrangements (4, 38). If this were the case, the
presence of a RUP element upstream of the structural tts
gene might represent a regulatory mechanism for capsule expression
since transposition (or inversion) of the RUP element should lead to a
variable expression of the capsular polysaccharide in type 37 pneumococci during infection. In addition, the finding that promoter
activity is associated with RUP elements may have other potentially
interesting implications in the physiology of this microorganism. Since
up to 108 copies of this intergenic element are distributed all along
the pneumococcal genome (31), it is conceivable that they could
contribute to the regulation of virulence (and non-virulence) genes.
Interestingly, besides the type 37 tts locus, RUP elements
have been found close to genes coding for several important
pathogenicity factors of S. pneumoniae such as capsular
polysaccharides, neuraminidases, the hyaluronidase, etc. (31).
A type 37 capsule was immunologically detected when several
Gram-positive species were transformed with a recombinant plasmid (pDLP49) harboring the type 37 S. pneumoniae tts gene (Fig.
4). This finding demonstrates that Tts is sufficient for capsular synthesis in heterologous systems. Furthermore, a single copy of the
tts gene inserted into the chromosome of S. oralis also led to capsule formation providing the first example
where a polysaccharide capsule has been described in this species. This
result illustrates how the commensal S. oralis might acquire
the capacity to synthesize this important virulence factor in the
nasopharynx, the natural habitat where many streptococci live. Similar
DNA interchanges have already been reported for other pneumococcal
genes, e.g. the spread of resistance to Hydropathy analysis of Tts predicted six potential transmembrane
domains and a central cytoplasmic region presumably containing the
catalytic site(s) (residues 64-346) (4). We show here that when the
tts gene was overexpressed in S. pneumoniae,
an ~50-kDa active protein was found to be associated with the
membrane fraction (Fig. 5). Furthermore, both ionic and non-ionic
detergents drastically affect the Tts synthase activity associated with
these membranes (Table I). The Mr of the
overproduced Tts deduced from SDS-PAGE analysis was smaller than that
predicted from sequence analysis (~59 kDa), which might be due to an
anomalous migration of the protein as it has been already reported for
two streptococcal HAS (10, 11). Tts contains five Cys residues
presumably located in the cytoplasmic loop (residues at positions 105, 114, 262, 278, and 299), and one more (Cys-470) between the potential
transmembrane regions V and VI. Since ME did not noticeably affect the
enzymatic activity of Tts (Table I), it can be assumed that those Cys
residues are not forming disulfide bonds. However, Cys residues appear to be necessary or important for Tts activity since a complete inhibition of the enzyme was obtained upon titration with the sulfhydryl-reactive agent pHMB (Table I).
The pneumococcal membranes containing Tts incorporate
[14C]Glc from UDP-[14C]Glc into a polymer
immunologically indistinguishable from that of type 37 clinical strains
(Table II). It should be emphasized that, although immunological
cross-reactions have been reported among several anti-pneumococcal
diagnostic sera (41), the type 37 antiserum appears to be very specific
since it only recognizes the homologous polysaccharide. The only
cross-reactivity reported for the type 37 capsule is a slight
precipitin reaction between this polysaccharide and an antiserum raised
against pneumococci of serogroup 12 (41). Types 12F and 12A contains
branches of kojibiosyl residues (42). More recently, the sophorosyl
unit has been demonstrated to be the main immunological determinant of
type 37 capsular polysaccharide by quantitative hapten inhibition
studies (43). Other disaccharides of the isomeric series of Computer analyses have revealed that
To the best of our knowledge, the Tts synthase, which catalyzes
both Our results suggest that a lipid-linked intermediate of the type
that participates in the synthesis of O-antigen and peptidoglycan is
not required for type 37 capsular polysaccharide biosynthesis since
bacitracin did not inhibit the Tts activity (Table I). Bacitracin
inhibits the dephosphorylation of undecaprenyl pyrophosphate by forming
a complex with the lipid (51); this dephosphorylation step is required
to regenerate undecaprenyl pyrophosphate, the lipid carrier in
peptidoglycan biosynthesis. A bacitracin-independent pathway had also
been demonstrated, among others, for the synthesis of group A HAS (52)
and chitin oligosaccharides from Mesorhizobium loti
(53).
The observation that the tts is the only pneumococcal gene
required for the synthesis of a type 37 capsule in different
Gram-positive species strongly suggests that the nascent polysaccharide
chain does not use specific transporters to cross the membrane. The presence of only four potential transmembrane regions at the C-terminal half and two more at the N terminus of Tts (4) might suggest that the
formation by Tts synthase of a membrane pore to facilitate the
extrusion of the polymer is unlikely since the presence of at least 12 transmembrane helices are apparently required to build a channel in
other sugar transporters (54). However, it should be mentioned that in
most of these transporters only four to eight transmembrane helices are
usually involved in sugar transport since there are actually two pores,
a sugar and a cation pore (55). Evidence suggesting that in the HAS
from S. pyogenes only four transmembrane domains and two
membrane-associated regions that, however, do not appear to traverse
the cell membrane are required to create a pore-like structure through
which a nascent HA chain can be extruded to the cell exterior has been
reported recently (56). However, several alternative mechanisms might allow the transport of the hydrophilic type 37 polysaccharide across
the membrane both in pneumococcus and in other Gram-positive species.
These include the use of unspecific transporters, association of
several Tts monomers in the membrane to conform a pore, or interaction
of the synthase with membrane phospholipids, as recently proposed for
HA transport (57). Additional efforts using the experimental tools
developed in this work are required to determine if the current models
for polymerization and transport of linear polysaccharides can be
applied to the synthesis of the branched structure of type 37 polysaccharide that represents the most simplified strategy developed
by pneumococcus to synthesize its main virulence factor.
-D-Glcp-(1
2)]-
-D-Glcp-(1
3). The elements governing the expression of the tts gene,
coding for the glucosyltransferase involved in the synthesis of the
type 37 pneumococcal capsular polysaccharide, have been studied. Primer extension analysis and functional tests demonstrated the presence of
four new transcriptional start points upstream of the previously reported tts promoter (ttsp). Most interesting,
three of these transcriptional start points are located in a RUP
element thought to be involved in recombinational events (Oggioni,
M. R., and Claverys, J. P. (1999) Microbiology
145, 2647-2653). Transformation experiments using either a recombinant
plasmid containing the whole transcriptional unit of tts or
chromosomal DNA from a type 37 pneumococcus showed that tts
is the only gene required to drive the biosynthesis of a type 37 capsule in S. pneumoniae and other Gram-positive bacteria,
namely Streptococcus oralis, Streptococcus gordonii, and
Bacillus subtilis. The Tts synthase was overproduced in
S. pneumoniae and purified as a membrane-associated enzyme. These membrane preparations used UDP-Glc as substrate to catalyze the
synthesis of a high molecular weight polysaccharide immunologically identical to the type 37 capsule. In addition, UDP-Gal was also a
substrate to produce type 37 polysaccharide since a strong
UDP-Glc-4'-epimerase activity is associated to the membrane fraction of
S. pneumoniae. These results indicated that Tts has a dual
biochemical activity that leads to the synthesis of the branched type
37 polysaccharide.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3)-
-D-Glcp-(1
repeating
units with monosaccharide side chains of a
-D-Glc-(1
linked to C2 of each Glc residue (sophorosyl subunits) (5). Several
experimental approaches demonstrated that tts is the only
gene required for the synthesis of the type 37-specific capsular
polysaccharide in S. pneumoniae. The tts gene
encodes a putative glycosyltransferase (Tts) that exhibits significant
sequence similarities with cellulose synthases of bacteria and higher
plants and other
-glycosyltransferases (4).
-1,3- and
-1,4-glycosyltransferase activity in contrast to the
other glycosyltransferases characterized so far among the enzymes
implicated in synthesis of the pneumococcal capsule that only catalyze
the transfer of a single glycosyl residue (8). There is increasing
evidence showing that this property is not so unusual as envisaged
previously. Thus, the family of bacterial hyaluronan synthases
(HAS)1 like those of
Streptococcus pyogenes (10), Streptococcus
equisimilis (11), or Pasteurella multocida (12), and
the KfiC enzyme of E. coli responsible for the synthesis of
the E. coli K5 capsule (13), also provide examples of a dual
enzymatic activity. It should be noted, however, that this enzymatic
activity has only been demonstrated for enzymes that catalyze the
formation of linear polysaccharides, whereas type 37 polysaccharide is
a branched polymer.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (14) and M31 (
lytA)
(S2
) (15). The type 37 clinical isolate 1235/89, kindly
provided by A. Fenoll (Spanish Pneumococcal Reference Laboratory,
Majadahonda, Spain), and the type 37 laboratory transformants DN2 and
DN5 have been described previously (4). Strain C2 is a type 37 lincomycin-resistant (LnR) transformant of the pneumococcal
strain M24 in which the tts gene is genetically linked to
the ermC gene (4). Growth and transformation of laboratory
strains of S. pneumoniae have been described previously
(16). Methods for transformation of Streptococcus oralis
NCTC 11427 (type strain) (17), Streptococcus gordonii V288
(Challis) (18), and Bacillus subtilis YB886 (19, 20) have
also been described elsewhere. Clones obtained by transformation with
derivatives of pLSE1 (tet ermC) (17) were scored on blood agar plates containing 0.7 µg of Ln/ml (for S. pneumoniae
and S. oralis), on brain-heart infusion agar plates (Difco)
supplemented with 10 µg of erythromycin (Ery)/ml (for S. gordonii), or on LB agar plates containing 5 µg of Ery/ml (for
B. subtilis). Plasmid pLSE4 is a promoter-probe vector that
contains a promoterless lytA gene (21). Plasmid pDLP36 (4)
is a pLSE4 derivative expressing the S. pneumoniae LytA
autolytic amidase under the control of the ttsp promoter of
the tts gene.
80 °C. Under these conditions, enzyme activity remained
virtually unaltered for up to 1 month. Determination of protein
concentration was carried out as described previously (25). Analysis of
the membrane fraction for detection of Tts was carried out by 10%
SDS-PAGE (26).
3)-
-D-glucan from
Alcaligenes faecalis (Sigma). This polysaccharide was
suspended in water (10 mg/ml) with a glass homogenizer and centrifuged
(12,000 × g, 30 min, 4 °C), and the insoluble
pellet was discarded. The sugar content of the solution was determined
by using the anthrone reagent (29). Typing by the capsular reaction
(Quellung) was kindly carried out by L. Vicioso (Spanish Pneumococcal
Reference Laboratory, Majadahonda, Spain). The standard assay
conditions for the pneumococcal LytA amidase and the preparation of
[3H]choline-labeled pneumococcal cell walls have been
described elsewhere (21). One unit of LytA amidase activity was defined as the amount of enzyme that catalyzed the hydrolysis (solubilization) of 1 µg of pneumococcal cell wall material in 10 min.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 consensus sequence with an extended TtTG motif characteristic of
the
16 region of S. pneumoniae (30) and transcription initiates 9 nucleotides after the
10 consensus sequence (4). However,
we have now observed that unencapsulated pneumococcal cells transformed
with a recombinant plasmid (pDLP49) containing the region upstream of
ttsp formed colonies noticeably more mucous than those from
cells transformed with pDLP48, an equivalent plasmid that only contains
ttsp and the structural tts gene. To determine the promoter strength of both constructs, we compared the cell wall
lytic activity (see "Experimental Procedures") expressed in a
pneumococcal
lytA strain (M31) transformed
either with pDLP36 (4) (Fig.
1A), which contains the
reporter lytA gene under the control of ttsp, or
with pDLP50, a construct that also includes the upstream region of
ttsp (Fig. 1A). Sonicated cell extracts prepared
from M31 [pDLP50] showed 6 times more LytA activity than those from
M31 [pDLP36] (Fig. 1B). In addition, M31 [pDLP50]
exhibited a faster autolysis at the end of exponential phase of growth
than M31 [pDLP36] (Fig. 1C). Furthermore, primer extension
analysis using total RNA extracted from M31 [pDLP50] revealed the
presence of at least four additional transcription start points
upstream of ttsp (Fig. 2).
Interestingly, three of them lie in a RUP element present in this
position in the clinical type 37 strains (4) (Fig.
3). RUP elements are thought to be
insertion sequence derivatives that facilitate recombinational events
(31), but a promoter activity had never been described in these
elements.
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Fig. 1.
Analysis of the expression of the
tts gene and functional characterization of its
promoter region. A, the upper part of the
figure shows a schematic representation of the DNA fragments of the
pneumococcal strain 1235/89 (type 37) cloned into pLSE1 (pDLP48 and
pDLP49) and pLSE4 (pDLP36 and pDLP50) to study the expression of
tts. Pertinent restriction sites and oligonucleotide primers
(black triangles) employed to construct the corresponding
recombinant plasmids are described under "Experimental Procedures."
The black and white box corresponds to the
location of ttsp. B, lytic activity of sonicated extracts
prepared from the lytA pneumococcal strain M31
harboring different plasmids assayed on
[3H]choline-labeled pneumococcal cell walls.
ND, not detectable. C, growth (and lysis) curves
of the S. pneumoniae M31 strain harboring plasmids pLSE4
(
), pDLP36 (
), or pDLP50 (×). Cells were incubated in C+Y medium
containing Ln (0.7 µg/ml), and growth was followed by nephelometry
(N). One N unit corresponds to about 2 × 106 colony-forming units/ml.
View larger version (32K):
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Fig. 2.
Primer extension analysis of the
tts gene. Total RNA was extracted
from a culture of M31 [pDLP50], and primer extension analysis was
performed using the oligonucleotide primer D138 to map additional
transcription start points upstream of ttsp. The final
products were loaded onto a 6% polyacrylamide 7 M urea
sequencing gel, in parallel with a sequencing reaction using
oligonucleotide D138 and pDLP50 (left). Extended products
are indicated by arrows and numbers 1-4.
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Fig. 3.
Localization of transcription start points of
the tts gene. The sequence corresponds to a
fragment of the upstream region of the tts gene (4).
Numbers on the right indicate the nucleotide
position corresponding to the sequence included in the EMBL data base
under GenBankTM accession number AJ131985. This
fragment contains the initiation ATG codon of tts, the
promoter ttsp (consensus 10 and
35 boxes are
boxed), and the transcription start point previously
reported (4) (black arrow). The figure also shows the RUP
element (black box) present in the type 37 clinical isolates
of S. pneumoniae. The numbered white vertical
arrows correspond to the transcription start points shown in Fig.
2. Putative extended
10 sites are overlined and the bases
that coincide with the consensus sequence (TaTGgTATAAT) (30) are
indicated by asterisks. The horizontal arrow
corresponds to the oligonucleotide primer D138 used for primer
extension.
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Fig. 4.
Type 37 capsule production in several
Gram-positive bacterial species. From top to
bottom, late exponentially growing cells of S. pneumoniae (rows 1 and 2), S. oralis (row 3), S. gordonii (row
4), or B. subtilis (row 5) were
incubated with type 37-specific antiserum at 4 °C for 1-2 h and
examined with a phase-contrast microscope. Agglutination occurs only
when type 37 polysaccharide is present at the cell surface.
A, type 37 clinical strain 1235/89; B and
C, type 37 laboratory transformant strains DN2 and DN5,
respectively; D, unencapsulated strain M24; E,
M24 [pLSE1]; F, M24 [pDLP49]; G, S. oralis NCTC 11427 [pLSE1]; H, S. oralis
[pDLP49]; I, an LnR isolate of S. oralis obtained by transformation with chromosomal DNA from the
pneumococcal type 37 LnR strain C2; J, S. gordonii V288; K, S. gordonii [pLSE1];
L, S. gordonii [pDLP49]; M, B. subtilis YB886; N, B. subtilis [pLSE1];
Ñ, B. subtilis [pDLP49].
View larger version (75K):
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Fig. 5.
Overproduction and membrane localization of
the Tts synthase. SDS-PAGE analysis of the membrane fraction of
strain M24 [pLSE1] (lane 1) and of M24 [pDLP49]
(lane 2). Black arrow indicates the presence of
an overproduced ~50-kDa protein. Molecular mass markers
(S) are also shown.
Effect of different compounds on the Tts enzymatic activity
View larger version (15K):
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Fig. 6.
Some biochemical properties of the Tts
synthase. A, pH dependence of the enzymatic activity of
Tts. The following buffers were used: 70 mM Tris/maleic
NaOH ( ), 70 mM sodium phosphate (
), 70 mM
Tris-HCl (
), 70 mM glycine-NaOH (
).
14C incorporation assays were carried out as
described under "Experimental Procedures." Effect of protein
concentration (B) and incubation time (C) on Tts
activity. Thermal stability (D) was studied by preincubating
the membranes at the indicated temperatures before adding the
substrate. Aliquots were withdrawn at different times, and Tts activity
was assayed as described under "Experimental Procedures." The data
represent the amount of product synthesized during the assay
period.
-glucosyltransferases (4) that are presumably
implicated in substrate binding (UDP-Glc) (32, 33). These motifs might
be specific for UDP-Glc, although we cannot rule out the possibility
that they only recognize the nucleotide part of the molecule, as
already suggested for the mechanism of action of this family of enzymes
(34, 35). If this were the case, it might account for the inhibitory
effect found when adding UTP, UDP, or UMP to the reaction mixture
(Table I). It is also conceivable that UDP-Gal (and perhaps any other UDP-sugar showing an inhibitory effect) may serve as substrate of the
Tts synthase for polysaccharide biosynthesis. Interestingly, we were
able to detect the formation of a radiolabeled high molecular weight
product by gel filtration through a Sepharose CL-4B column in
experiments where either UDP-[14C]Glc or
UDP-[14C]Gal was used as substrate (Fig.
7A). The macromolecular
product(s) of these reactions that eluted in the
V0 of the column was immunoprecipitated with an
anti-type 37 polysaccharide serum but not with a heterologous antiserum directed against type 3 pneumococci (Table
II). Interestingly, curdlan, a linear
(1
3)-
-D-glucan, did not preclude the recognition of
the type 37 polysaccharide by its antiserum. These results demonstrated
that the Tts-containing pneumococcal membranes are capable of
incorporating the 14C label to a polymer immunologically
indistinguishable from type 37 polysaccharide using either
UDP-[14C]Glc or UDP-[14C]Gal as
substrate.
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Fig. 7.
Chemical characterization of the products of
Tts activity. A, the products resulting from the
incubation of pneumococcal membranes prepared from strain M24
[pDLP49] with either UDP-[14C]Glc ( ) or
UDP-[14C]Gal (
) under the conditions described under
"Experimental Procedures" were filtered through a Sepharose CL-4B
column (20 × 1.5 cm), and the radioactivity of alternate
fractions (0.5 ml) was determined. Afterwards, fractions excluded from
B or retained in C the column were pooled,
acid-hydrolyzed, and analyzed by HPLC as described under
"Experimental Procedures." Elution was carried out with 125 µM H2SO4 at 0.25 ml/min
(B) or 0.4 ml/min (C). Net cpm means that
background counts (about 35 cpm) were subtracted from the radioactivity
of each fraction. Other symbols correspond to those in
A.
Immunological characterization of the product of Tts synthase
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactam
antibiotics has been attributed to horizontal transfer events involving
fragments of the genes coding for penicillin-binding protein(s) of
pneumococcus and other related streptococcal species (39). Moreover,
compelling evidence for recombination events between the
galU gene of S. pneumoniae and that of several
streptococcal species has also been provided recently (40).
- and
-(1
2), -(1
3), -(1
4), and -(1
6) were poorly active as
competitive inhibitors of antibody precipitation (43). Here we have
shown (Table II) that when a linear (1
3)-
-D-glucan
(curdlan) was employed, no inhibition of the immunoprecipitation
reaction was observed (Table II), which fully confirmed that the
anti-type 37 serum preferentially recognizes the branched part of the
type 37 polysaccharide. Since the type 37 polysaccharide contains two
different
-glucosidic bonds (
-1,3 and
-1,2), Tts should be
responsible for the formation of both linkages according to our
findings that tts is the only gene required for a type 37 capsule synthesis. The polysaccharide synthesized by Tts was composed
exclusively by Glc, as revealed by HPLC analysis (Fig. 7)
and TLC (not shown). Combined similar HPLC and TLC analyses and
immunological tests revealed that when UDP-Gal was used in vitro as substrate, the polymer synthesized was indistinguishable from that formed by using UDP-Glc. This finding implies the presence of
an epimerase that converts UDP-Gal to UDP-Glc (Fig. 7C).
-glycosyltransferases share conserved sequences and structural
features (34). The processive transferases contain a
D(X)40-130D(X)90-140 D(X)30-40QXXRW
motif distributed over two domains, named "A" and "B," whereas
nonprocessive enzymes lack domain B, and so have only the first two Asp
residues of the motif (34, 44). Both domains have also been identified
in Tts since this enzyme contains the conserved motif
D(X)53D(X)88D(X)36RXXKW
(4). A classification of glycosyltransferases using nucleotide
diphospho-sugars, nucleotide monophospho-sugars, and sugar phosphates
(EC 2.4.1.x), and related proteins into 48 distinct
sequence-based families has been proposed (45). Tts belongs to family 2 that includes, among other inverting glycosyltransferases, cellulose
synthases, HAS, and
-1,3-glucan synthases. Although the HAS from
P. multocida is currently a member of this family, it
appears to be structurally distinct from other HAS (46). Experimental
evidence for the role of carboxyl residues in
-glycan synthases
comes from site-directed mutagenesis of chitin synthase 2 from
Saccharomyces cerevisiae (47) and of the AcsAB cellulose
synthase from Acetobacter xylinum (44) as well as from the
use of amino acid-modifying reagents on a
-(1,3)-glucan synthase
from ryegrass (48). Based on these and other results it was assumed
that Asp residues are involved in the acid-base catalytic mechanism of
this kind of glycosyltransferases (49). Nevertheless, the recent
elucidation of the three-dimensional crystal structure of SpsA, a
member of family 2 of glycosyltransferases implicated in the synthesis
of the mature spore coat of B. subtilis, has allowed us to
shed light on the mechanisms of this ubiquitous family of inverting
glycosyltransferases (50). It has been found that the invariant Asp
residues of domain A are intimately involved with UDP binding, whereas
a candidate for the general base has not been identified with
certainty. It should be noted, however, that the glycosyltransferase
specificity of SpsA has not been characterized as yet and that this
enzyme lacks the domain B characteristic of the processive
transferases. Nevertheless, the observed inhibitory effects of UDP,
UTP, UMP, or UDP-sugars on Tts activity (Table I) are in agreement with
the involvement of the conserved Asp residues in binding to the
nucleotide rather than to the sugar moiety of the UDP-sugar substrate.
-1,2 and
-1,3 linkages, is the first inverting
glucosyltransferase able to synthesize a branched polysaccharide.
Perhaps the most intriguing characteristic of Tts is that it shares
sequence similarities with other enzymes that produced various types of
linear polymers, either homo- or heteropolysaccharides. Therefore, only
hypothetical models can be proposed for polymerization of the type 37 polysaccharide of S. pneumoniae. The formation of
-1,3-
and
-1,2-glycosidic bonds may occur either simultaneously or
consecutively. Mutational studies should be carried out in the future
to determine whether the synthesis of a curdlan-like polysaccharide
(
-1,3-glucan) precedes that of sophorose or formation of
-1,2-
and
-1,3- bonds takes place simultaneously as the polysaccharide
chain grows.
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ACKNOWLEDGEMENTS |
---|
We thank P. García and E. Díaz for helpful discussion and for critical reading of the manuscript. We also acknowledge J. A. Leal and O. Ahrazem for their advice on the hydrolysis of type 37 polysaccharide and TLC methodology.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants PB96-0809 and BMC2000-1002 from the Dirección General de Investigación Científica y Técnica.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.
Recipient of a doctoral fellowship from Programa Sectorial de
Formación de Profesorado Universitario y Personal Investigador (Ministerio de Educación y Cultura).
§ To whom correspondence should be addressed. Tel.: 34-91-561-1800; Fax: 34-91-562-7518; E-mail: e.garcia@cib.csic.es.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M010287200
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ABBREVIATIONS |
---|
The abbreviations used are: HAS, HA synthase(s); Ery, erythromycin; GalUA, galacturonic acid; HA, hyaluronan, hyaluronate, or hyaluronic acid; HPLC, high performance liquid chromatography; Ln, lincomycin; ME, 2-mercaptoethanol; PAGE, polyacrylamide gel electrophoresis; pHMB, p-hydroxymercuribenzoate; PMSF, phenylmethylsulfonyl fluoride; PolIk, Klenow (large) fragment of the Escherichia coli DNA polymerase I; ttsp, promoter of the tts gene; [ ], plasmid-carrier state.
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