Tts, a Processive beta -Glucosyltransferase of Streptococcus pneumoniae, Directs the Synthesis of the Branched Type 37 Capsular Polysaccharide in Pneumococcus and Other Gram-positive Species*

Daniel LlullDagger, Ernesto García§, and Rubens López

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [beta -D-Glcp-(1right-arrow2)]-beta -D-Glcp-(1right-arrow3). 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow3)-beta -D-Glcp-(1right-arrow repeating units with monosaccharide side chains of a beta -D-Glc-(1right-arrow 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 beta -glycosyltransferases (4).

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 beta -1,3- and beta -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.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains, Plasmids, and Growth Conditions-- The unencapsulated laboratory S. pneumoniae strains used are as follows: M24 (S3-) (14) and M31 (Delta 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.

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 -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).

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 (1right-arrow3)-beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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 Delta 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.


View larger version (20K):
[in this window]
[in a new window]
 
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 Delta 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 (open circle ), 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):
[in this window]
[in a new window]
 
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.


View larger version (107K):
[in this window]
[in a new window]
 
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.

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.


View larger version (146K):
[in this window]
[in a new window]
 
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].

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.


View larger version (75K):
[in this window]
[in a new window]
 
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.

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).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of different compounds on the Tts enzymatic activity
The reaction mixtures contained membranes prepared from strain M24 [pDLP49], as described under "Experimental Procedures," and the different compounds detailed below. In experiment A, the additions were done immediately before the substrate. In experiment B, nucleoside mono-, di, or triphosphates and sugar nucleotides (10 mM) or free sugars (20 mM) were incubated with the membranes for 30 min at 25 °C before addition of UDP-[14C]Glc. In experiment C, the reaction mixtures were preincubated with the indicated detergents for 10 min at 37 °C prior to addition of the substrate. Results are the mean of three independent determinations. ND, not detectable. DOC, sodium deoxycholate.


View larger version (15K):
[in this window]
[in a new window]
 
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 (open circle ), 70 mM Tris-HCl (black-square), 70 mM glycine-NaOH (black-triangle). 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.

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 beta -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 (1right-arrow3)-beta -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.


View larger version (17K):
[in this window]
[in a new window]
 
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 (open circle ) 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.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Immunological characterization of the product of Tts synthase
Standard reactions were carried out as described under "Experimental Procedures." The reaction mixtures were filtered through a Sepharose CL-4B column, and the excluded fractions (0.5 ml each) containing about 10 ng of [14C]Glc-labeled type 37 polysaccharide were incubated for 2-3 h with protein A from Staphylococcus aureus (Sigma) sensitized either with type 3- or type 37-specific antiserum. When curdlan was employed, this polysaccharide was preincubated with protein A from S. aureus at room temperature for 1 h before addition of the type 37 polysaccharide. Mixtures were centrifuged (10,000 × g, 15 min) to separate the immunoprecipitated material, and radioactivity was determined. Values are the mean of three independent experiments.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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).

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 alpha - and beta -(1right-arrow2), -(1right-arrow3), -(1right-arrow4), and -(1right-arrow6) were poorly active as competitive inhibitors of antibody precipitation (43). Here we have shown (Table II) that when a linear (1right-arrow3)-beta -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 beta -glucosidic bonds (beta -1,3 and beta -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).

Computer analyses have revealed that beta -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 beta -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 beta -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 beta -(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.

To the best of our knowledge, the Tts synthase, which catalyzes both beta -1,2 and beta -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 beta -1,3- and beta -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 (beta -1,3-glucan) precedes that of sophorose or formation of beta -1,2- and beta -1,3- bonds takes place simultaneously as the polysaccharide chain grows.

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.

    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.

Dagger 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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Griffith, F. (1928) J. Hyg. 27, 113-159
2. Henrichsen, J. (1995) J. Clin. Microbiol. 33, 2759-2762[Abstract]
3. García, E., Llull, D., Muñoz, R., Mollerach, M., and López, R. (2000) Res. Microbiol. 151, 429-435[CrossRef][Medline] [Order article via Infotrieve]
4. Llull, D., Muñoz, R., López, R., and García, E. (1999) J. Exp. Med. 190, 241-251[Abstract/Free Full Text]
5. Adeyeye, A., Jansson, P.-E., Lindberg, B., and Henrichsen, J. (1988) Carbohydr. Res. 180, 295-299[CrossRef]
6. Guidolin, A., Morona, J. K., Morona, R., Hansman, D., and Paton, J. C. (1994) Infect. Immun. 62, 5385-5396
7. Kolkman, M. A., Morrison, D. A., Van Der Zeijst, B. A., and Nuijten, P. J. (1996) J. Bacteriol. 178, 3736-3741[Abstract]
8. Arrecubieta, C., López, R., and García, E. (1996) J. Exp. Med. 184, 449-455[Abstract]
9. Cartee, R. T., Forsee, W. T., Schutzbach, J. S., and Yother, J. (2000) J. Biol. Chem. 275, 3907-3914[Abstract/Free Full Text]
10. DeAngelis, P. L., and Weigel, P. H. (1994) Biochemistry 33, 9033-9039[Medline] [Order article via Infotrieve]
11. Kumari, K., and Weigel, P. H. (1997) J. Biol. Chem. 272, 32539-32546[Abstract/Free Full Text]
12. Jing, W., and DeAngelis, P. L. (2000) Glycobiology 10, 883-889[Abstract/Free Full Text]
13. Griffiths, G., Cook, N. J., Gottfridson, E., Lind, T., Lidholt, K., and Roberts, I. S. (1998) J. Biol. Chem. 273, 11752-11757[Abstract/Free Full Text]
14. García, E., García, P., and López, R. (1993) Mol. Gen. Genet. 239, 188-195[Medline] [Order article via Infotrieve]
15. Sánchez-Puelles, J. M., Ronda, C., García, J. L., García, P., López, R., and García, E. (1986) Eur. J. Biochem. 158, 289-293[Abstract]
16. Mollerach, M., López, R., and García, E. (1998) J. Exp. Med. 188, 2047-2056[Abstract/Free Full Text]
17. Ronda, C., García, J. L., and López, R. (1988) Mol. Gen. Genet. 215, 53-57[Medline] [Order article via Infotrieve]
18. Pozzi, G., Musmanno, R. A., Lievens, P. M.-J., Oggioni, M. R., Plevani, P., and Manganelli, R. (1990) Res. Microbiol. 141, 659-670[CrossRef][Medline] [Order article via Infotrieve]
19. Canosi, U., Iglesias, A., and Trautner, T. A. (1981) Mol. Gen. Genet. 181, 434-440[Medline] [Order article via Infotrieve]
20. Yasbin, R. E., Wilson, G. A., and Young, F. E. (1975) J. Bacteriol. 121, 296-304[Medline] [Order article via Infotrieve]
21. Díaz, E., and García, J. L. (1990) Gene (Amst.) 90, 163-167[Medline] [Order article via Infotrieve]
22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
23. Llull, D., López, R., García, E., and Muñoz, R. (1998) Biochim. Biophys. Acta 1443, 217-224[Medline] [Order article via Infotrieve]
24. García, E., Ronda, C., García, J. L., and López, R. (1985) FEMS Microbiol. Lett. 29, 77-81[CrossRef]
25. Bradford, M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
26. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
27. Hansen, S. A. (1975) J. Chromatogr. 107, 224-226[CrossRef]
28. Roy, A. B., and Harwood, J. L. (1999) Biochem. J. 344, 185-187[CrossRef][Medline] [Order article via Infotrieve]
29. Herbert, D., Phipps, P. J., and Strange, R. E. (1971) Methods Microbiol. 5, 209-344
30. Sabelnikov, A. G., Greenberg, B., and Lacks, S. A. (1995) J. Mol. Biol. 250, 144-155[CrossRef][Medline] [Order article via Infotrieve]
31. Oggioni, M. R., and Claverys, J. P. (1999) Microbiology 145, 2647-2653[Abstract/Free Full Text]
32. Delmer, D. P. (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 245-276[CrossRef]
33. Pear, J. R., Kawagoe, Y., Schreckengost, W. E., Delmer, D. P., and Stalker, D. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12637-12642[Abstract/Free Full Text]
34. Saxena, I. M., Brown, R. M., Jr., Fevre, M., Geremia, R. A., and Henrissat, N. (1995) J. Bacteriol. 177, 1419-1424[Free Full Text]
35. Koyama, M., Helbert, W., Imai, T., Sugiyama, J., and Henrissat, B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9091-9095[Abstract/Free Full Text]
36. Distler, J., and Roseman, S. (1964) Proc. Natl. Acad. Sci. U. S. A. 48, 2187-2193
37. Kolkman, M. A., van der Zeijst, B. A., and Nuijten, P. J. (1998) J. Biochem. (Tokyo) 123, 937-945[Abstract]
38. Claverys, J. P., Prudhomme, M., Mortier-Barrière, I., and Martin, B. (2000) Mol. Microbiol. 35, 251-259[CrossRef][Medline] [Order article via Infotrieve]
39. Dowson, C. G., Barcus, V., King, S., Pickerill, P., Whatmore, A., and Yeo, M. (1997) J. Appl. Microbiol. 83, 42-51
40. Mollerach, M., and García, E. (2000) Gene (Amst.) 260, 77-86[CrossRef][Medline] [Order article via Infotrieve]
41. Heidelberger, M. (1983) Infect. Immun. 41, 1234-1244[Medline] [Order article via Infotrieve]
42. Kamerling, J. P. (2000) in Molecular Biology and Mechanisms of Disease (Tomasz, A., ed) , pp. 81-114, Mary Ann Liebert, Inc., Larchmont, NY
43. Allen, P. Z., and Bowen, W. H. (1988) Mol. Immunol. 25, 1011-1017[Medline] [Order article via Infotrieve]
44. Saxena, I. M., and Brown, R. M., Jr. (1997) Cellulose 4, 33-49[CrossRef]
45. Campbell, J. A., Davies, G. J., Bulone, V., and Henrissat, B. (1997) Biochem. J. 326, 929-939[Medline] [Order article via Infotrieve]
46. DeAngelis, P. L. (1999) Cell. Mol. Life Sci. 56, 670-682[CrossRef][Medline] [Order article via Infotrieve]
47. Nagahashi, S., Sudoh, M., Ono, N., Sawada, R., Yamaguchi, E., Uchida, Y., Moi, T., Takagi, M., Arisawa, M., and Yamada-Okabe, H. (1995) J. Biol. Chem. 270, 13961-13967[Abstract/Free Full Text]
48. Bulone, V., Lam, B.-T., and Stone, B. A. (1999) Phytochemistry 50, 9-15[CrossRef]
49. Brown Jr, R. M., and Saxena, I. M. (2000) Plant Physiol. Biochem. 38, 57-67[CrossRef]
50. Charnock, S. J., and Davies, G. J. (1999) Biochemistry 38, 6380-6385[CrossRef][Medline] [Order article via Infotrieve]
51. Siewert, G., and Strominger, J. L. (1967) Proc. Natl. Acad. Sci. U. S. A. 57, 767-773
52. Stoolmiller, A. C., and Dorfman, A. (1969) J. Biol. Chem. 244, 236-246[Abstract/Free Full Text]
53. Kamst, E., Bakkers, J., Quaedvlieg, N. E. M., Pilling, J., Kijne, J. W., Lugtenberg, B. J. J., and Spaink, H. P. (1999) Biochemistry 38, 4045-4052[CrossRef][Medline] [Order article via Infotrieve]
54. Deves, R., and Boyd, C. A. (1998) Physiol. Rev. 78, 487-545[Abstract/Free Full Text]
55. Frillingos, S., Sahin-Tóthe, M., Wu, J., and Kaback, H. R. (1998) FASEB J. 12, 1281-1299[Abstract/Free Full Text]
56. Heldermon, C., DeAngelis, P. L., and Weigel, P. H. (2001) J. Biol. Chem. 276, 2037-2046[Abstract/Free Full Text]
57. Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239-4245[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.