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

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m010287200

1083-351X

Daniel Llull, Ernesto Garcı́a, Rubens López,

Pneumonia and Respiratory Infections

The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [β-d-Glcp-(1→2)]-β-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) Microbiology145, 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 ttsis 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, andBacillus subtilis. The Tts synthase was overproduced inS. 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 ofS. pneumoniae. These results indicated that Tts has a dual biochemical activity that leads to the synthesis of the branched type 37 polysaccharide. The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [β-d-Glcp-(1→2)]-β-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) Microbiology145, 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 ttsis 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, andBacillus subtilis. The Tts synthase was overproduced inS. 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 ofS. pneumoniae. These results indicated that Tts has a dual biochemical activity that leads to the synthesis of the branched type 37 polysaccharide. HA synthase(s) erythromycin galacturonic acid hyaluronan, hyaluronate, or hyaluronic acid high performance liquid chromatography lincomycin 2-mercaptoethanol polyacrylamide gel electrophoresis p-hydroxymercuribenzoate phenylmethylsulfonyl fluoride Klenow (large) fragment of the Escherichia coli DNA polymerase I promoter of thetts gene plasmid-carrier state 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 (1Griffith F. J. Hyg. 1928; 27: 113-159Crossref PubMed Scopus (674) Google Scholar). 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 (2Henrichsen J. J. Clin. Microbiol. 1995; 33: 2759-2762Crossref PubMed Google Scholar). Capsular polysaccharide biosynthesis in S. pneumoniaeis usually driven by genes located in the cap/cpslocus, and the capsular cluster of 13 pneumococcal types has been sequenced recently (3Garcı́a E. Llull D. Muñoz R. Mollerach M. López R. Res. Microbiol. 2000; 151: 429-435Crossref PubMed Scopus (48) Google Scholar). In remarkable contrast, only a single gene (tts) located far apart from the cap cluster, directs the synthesis of the type 37 capsule (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar). 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 →3)-β-d-Glcp-(1→ repeating units with monosaccharide side chains of a β-d-Glc-(1→ linked to C2 of each Glc residue (sophorosyl subunits) (5Adeyeye A. Jansson P.-E. Lindberg B. Henrichsen J. Carbohydr. Res. 1988; 180: 295-299Crossref Scopus (26) Google Scholar). 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 β-glycosyltransferases (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar). 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 (3Garcı́a E. Llull D. Muñoz R. Mollerach M. López R. Res. Microbiol. 2000; 151: 429-435Crossref PubMed Scopus (48) Google Scholar). 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 bycps14(cps19f)E gene (6Guidolin A. Morona J.K. Morona R. Hansman D. Paton J.C. Infect. Immun. 1994; 62: 5385-5396Crossref Google Scholar, 7Kolkman M.A. Morrison D.A. Van Der Zeijst B.A. Nuijten P.J. J. Bacteriol. 1996; 178: 3736-3741Crossref PubMed Google Scholar). 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 (7Kolkman M.A. Morrison D.A. Van Der Zeijst B.A. Nuijten P.J. J. Bacteriol. 1996; 178: 3736-3741Crossref PubMed Google Scholar). 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 orEscherichia coli strains provided that UDP-Glc and UDP-GlcUA, the precursors of type 3 capsular monosaccharides, were available (8Arrecubieta C. López R. Garcı́a E. J. Exp. Med. 1996; 184: 449-455Crossref PubMed Scopus (48) Google Scholar). 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 (9Cartee R.T. Forsee W.T. Schutzbach J.S. Yother J. J. Biol. Chem. 2000; 275: 3907-3914Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Cap3B possesses a double β-1,3- and β-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 (8Arrecubieta C. López R. Garcı́a E. J. Exp. Med. 1996; 184: 449-455Crossref PubMed Scopus (48) Google Scholar). 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 ofStreptococcus pyogenes (10DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar), Streptococcus equisimilis (11Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), or Pasteurella multocida (12Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar), and the KfiC enzyme of E. coli responsible for the synthesis of the E. coli K5 capsule (13Griffiths G. Cook N.J. Gottfridson E. Lind T. Lidholt K. Roberts I.S. J. Biol. Chem. 1998; 273: 11752-11757Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), 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. pneumoniaestrains 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. The unencapsulated laboratory S. pneumoniae strains used are as follows: M24 (S3−) (14Garcı́a E. Garcı́a P. López R. Mol. Gen. Genet. 1993; 239: 188-195Crossref PubMed Scopus (35) Google Scholar) and M31 (ΔlytA) (S2−) (15Sánchez-Puelles J.M. Ronda C. Garcı́a J.L. Garcı́a P. López R. Garcı́a E. Eur. J. Biochem. 1986; 158: 289-293Crossref PubMed Scopus (106) Google Scholar). 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 (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar). 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 (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar). Growth and transformation of laboratory strains of S. pneumoniae have been described previously (16Mollerach M. López R. Garcı́a E. J. Exp. Med. 1998; 188: 2047-2056Crossref PubMed Scopus (76) Google Scholar). Methods for transformation of Streptococcus oralisNCTC 11427 (type strain) (17Ronda C. Garcı́a J.L. López R. Mol. Gen. Genet. 1988; 215: 53-57Crossref PubMed Scopus (33) Google Scholar), Streptococcus gordonii V288 (Challis) (18Pozzi G. Musmanno R.A. Lievens P.M.-J. Oggioni M.R. Plevani P. Manganelli R. Res. Microbiol. 1990; 141: 659-670Crossref PubMed Scopus (57) Google Scholar), and Bacillus subtilis YB886 (19Canosi U. Iglesias A. Trautner T.A. Mol. Gen. Genet. 1981; 181: 434-440Crossref PubMed Scopus (70) Google Scholar, 20Yasbin R.E. Wilson G.A. Young F.E. J. Bacteriol. 1975; 121: 296-304Crossref PubMed Google Scholar) have also been described elsewhere. Clones obtained by transformation with derivatives of pLSE1 (tet ermC) (17Ronda C. Garcı́a J.L. López R. Mol. Gen. Genet. 1988; 215: 53-57Crossref PubMed Scopus (33) Google Scholar) were scored on blood agar plates containing 0.7 μg of Ln/ml (for S. pneumoniaeand 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 (forB. subtilis). Plasmid pLSE4 is a promoter-probe vector that contains a promoterless lytA gene (21Dı́az E. Garcı́a J.L. Gene ( Amst. ). 1990; 90: 163-167Crossref PubMed Scopus (12) Google Scholar). Plasmid pDLP36 (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar) is a pLSE4 derivative expressing the S. pneumoniae LytA autolytic amidase under the control of the ttsp promoter of the tts gene. DNA manipulations and other standard methods were as described in Sambrooket al. (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Primer extension mapping of the transcription initiation site (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar) and polymerase chain reaction amplifications (23Llull D. López R. Garcı́a E. Muñoz R. Biochim. Biophys. Acta. 1998; 1443: 217-224Crossref PubMed Scopus (29) Google Scholar) 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. 1 A). 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 theE. 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 (24Garcı́a E. Ronda C. Garcı́a J.L. López R. FEMS Microbiol. Lett. 1985; 29: 77-81Crossref Google Scholar). 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 withEcoRV 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. 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 mmTris-HCl, pH 7.0, 9 mm MgCl2, 1 mmCaCl2) 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 (25Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217487) Google Scholar). Analysis of the membrane fraction for detection of Tts was carried out by 10‥ SDS-PAGE (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207516) Google Scholar). 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 mmCaCl2, 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. 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 mmTris-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 atV 0 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 (27Hansen S.A. J. Chromatogr. 1975; 107: 224-226Crossref Scopus (171) Google Scholar) but using the solvent system 2-propanol, acetone, 0.1 m formic acid (2:2:1) (28Roy A.B. Harwood J.L. Biochem. J. 1999; 344: 185-187Crossref PubMed Google Scholar). 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 V T 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 μmH2SO4 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). 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→3)-β-d-glucan fromAlcaligenes 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 (29Herbert D. Phipps P.J. Strange R.E. Methods Microbiol. 1971; 5: 209-344Crossref Scopus (1478) Google Scholar). 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 (21Dı́az E. Garcı́a J.L. Gene ( Amst. ). 1990; 90: 163-167Crossref PubMed Scopus (12) Google Scholar). 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. We have reported previously the identification of the tts promoter and its transcription start point (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar). The ttsp promoter contains a −10 consensus sequence with an extended TtTG motif characteristic of the −16 region of S. pneumoniae (30Sabelnikov A.G. Greenberg B. Lacks S.A. J. Mol. Biol. 1995; 250: 144-155Crossref PubMed Scopus (127) Google Scholar) and transcription initiates 9 nucleotides after the −10 consensus sequence (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar). However, we have now observed that unencapsulated pneumococcal cells transformed with a recombinant plasmid (pDLP49) containing the region upstream ofttsp formed colonies noticeably more mucous than those from cells transformed with pDLP48, an equivalent plasmid that only containsttsp 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 ΔlytA strain (M31) transformed either with pDLP36 (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar) (Fig.1 A), which contains the reporter lytA gene under the control of ttsp, or with pDLP50, a construct that also includes the upstream region ofttsp (Fig. 1 A). Sonicated cell extracts prepared from M31 [pDLP50] showed 6 times more LytA activity than those from M31 [pDLP36] (Fig. 1 B). In addition, M31 [pDLP50] exhibited a faster autolysis at the end of exponential phase of growth than M31 [pDLP36] (Fig. 1 C). 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 (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar) (Fig.3). RUP elements are thought to be insertion sequence derivatives that facilitate recombinational events (31Oggioni M.R. Claverys J.P. Microbiology. 1999; 145: 2647-2653Crossref PubMed Scopus (85) Google Scholar), but a promoter activity had never been described in these elements.Figure 3Localization of transcription start points of the tts gene. The sequence corresponds to a fragment of the upstream region of the tts gene (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar).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 areboxed), and the transcription start point previously reported (4Llull D. Muñoz R. López R. Garcı́a E. J. Exp. Med. 1999; 190: 241-251Crossref PubMed Scopus (90) Google Scholar) (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) (30Sabelnikov A.G. Greenberg B. Lacks S.A. J. Mol. Biol. 1995; 250: 144-155Crossref PubMed Scopus (127) Google Scholar) are indicated by asterisks. The horizontal arrowcorresponds to the oligonucleotide primer D138 used for primer extension.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 4 I) 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. 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. 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 (TableI). 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 mmNaCl 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. 6 A). 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. 6 D).Table IEffect of different compounds on the Tts enzymatic activityAdditionEnzymatic activityunits‥Experiment ANone90.7100EDTA (10 mm)NDNDEGTA (1 mm)85.394EGTA (2 mm)83.191.6pHMB (5 μm)NDNDMB (2 mm)76.284pHMB (5 μm) + ME (2 mm)72.479.8Bacitracin (1 μg/ml)89.598.7Bacitracin (100 μg/ml)90.199.3Experiment BNone67.5100TMP8.712.9UMP8.412.2UDPNDNDUTPNDNDGMP44.165.3CMP51.676.4UDP-Gal1.42.1UDP-XylNDNDUDP-Man3.45CDP-Glc36.153.5GDP-Glc43.864.9Glc60.890.1Gal61.491GalUA53.679.4GlcUA51.976.9Ara61.691.3Experiment CNone58.5100Brij 58 (1‥)16.127.5Brij 58 (0.1‥)51.988.7DOC (1‥)1.83.1DOC (0.1‥)6.410.9Triton X-100 (1‥)12.821.9Triton X-100 (0.1‥)15.326.1The 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 deox