Purified, Recombinant TagF Protein from Bacillus subtilis 168 Catalyzes the Polymerization of Glycerol Phosphate onto a Membrane Acceptor in Vitro

We report the first characterization of a recombinant protein involved in the polymerization of wall teichoic acid. Previously, a study of the teichoic acid polymerase activity associated with membranes from Bacillus subtilis 168 strains bearing thermosensitive mutations in tagB, tagD,and tagF implicated TagF as the poly(glycerol phosphate) polymerase (Pooley, H. M., Abellan, F. X., and Karamata, D. (1992) J. Bacteriol. 174, 646–649). In the work reported here, we have demonstrated an unequivocal role for tagF in the thermosensitivity of one such mutant (tagF1) by conditional complementation at the restrictive temperature withtagF under control of the xylose promoter at theamyE locus. We have overexpressed and purified recombinantB. subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B. subtilis168. Recombinant hexahistidine-tagged TagF protein was purified fromEscherichia coli and was used to develop a novel membrane pelleting assay to monitor poly(glycerol phosphate) polymerase activity. Purified TagF was shown to incorporate radioactivity from its substrate CDP-[14C]glycerol into a membrane fractionin vitro. This activity showed a saturable dependence on the concentration of CDP-glycerol (Km of 340 μm) and the membrane acceptor (half-maximal activity at 650 μg of protein/ml of purified B. subtilis membranes). High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, ∼35 glycerol phosphate units in length. We report the first characterization of a recombinant protein involved in the polymerization of wall teichoic acid. Previously, a study of the teichoic acid polymerase activity associated with membranes from Bacillus subtilis 168 strains bearing thermosensitive mutations in tagB, tagD,and tagF implicated TagF as the poly(glycerol phosphate) polymerase (Pooley, H. M., Abellan, F. X., and Karamata, D. (1992) J. Bacteriol. 174, 646–649). In the work reported here, we have demonstrated an unequivocal role for tagF in the thermosensitivity of one such mutant (tagF1) by conditional complementation at the restrictive temperature withtagF under control of the xylose promoter at theamyE locus. We have overexpressed and purified recombinantB. subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B. subtilis168. Recombinant hexahistidine-tagged TagF protein was purified fromEscherichia coli and was used to develop a novel membrane pelleting assay to monitor poly(glycerol phosphate) polymerase activity. Purified TagF was shown to incorporate radioactivity from its substrate CDP-[14C]glycerol into a membrane fractionin vitro. This activity showed a saturable dependence on the concentration of CDP-glycerol (Km of 340 μm) and the membrane acceptor (half-maximal activity at 650 μg of protein/ml of purified B. subtilis membranes). High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, ∼35 glycerol phosphate units in length. thermosensitive high pressure liquid chromatography Wall teichoic acids are a chemically diverse group of linear, hydrophilic, anionic polymers of polyol and/or sugar residues some 30–50 units long. The major wall teichoic acid of Bacillus subtilis 168 is a linear 1,3-linked poly(glycerol phosphate) polymer that is anchored to peptidoglycan through a “linkage unit” disaccharide (Scheme FS1). TheN-acetylglucosamine-β-(1–4)-N-acetylmannosamine disaccharide is attached to peptidoglycan through a phosphodiester linkage between the anomeric carbon of N-acetylglucosamine and the 6-hydroxyl of N-acetylmuramic acid of peptidoglycan. The current understanding of teichoic acid biosynthesis is derived from biochemical work dating from almost 40 years ago and from more recent genetic analyses of B. subtilis. The latter studies focused largely on the tag (teichoic acidglycerol phosphate) gene cluster of B. subtilis168 (1Soldo B. Lazarevic V. Karamata D. Microbiology. 2002; 148: 2079-2087Crossref PubMed Scopus (108) Google Scholar, 2Lazarevic V. Karamata D. Mol. Microbiol. 1995; 16: 345-355Crossref PubMed Scopus (118) Google Scholar, 3Mauel C. Young M. Margot P. Karamata D. Mol. Gen. Genet. 1989; 215: 388-394Crossref PubMed Scopus (59) Google Scholar, 4Mauel C. Young M. Karamata D. J. Gen. Microbiol. 1991; 137: 929-941Crossref PubMed Scopus (73) Google Scholar, 5Briehl M. Pooley H.M. Karamata D. J. Gen. Microbiol. 1989; 135: 1325-1334Google Scholar, 6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar) and have also defined the tar(teichoic acid ribitol phosphate) loci in strain W23 (7Lazarevic V. Abellan F.X. Moller S.B. Karamata D. Mauel C. Microbiology. 2002; 148: 815-824Crossref PubMed Scopus (70) Google Scholar). In aggregate, the biochemical and genetic work suggested that the synthesis of the poly(glycerol phosphate) chain inB. subtilis 168 would require the stepwise conversion of lipid intermediates to produce a membraneanchored prenolpyrophosphate-linked disaccharide that would undergo the addition of a single glycerol phosphate residue in a priming reaction followed by poly(glycerol phosphate) polymerization. Burger and Glaser (8Burger M.M. Glaser L. J. Biol. Chem. 1964; 239: 3168-3177Abstract Full Text PDF PubMed Google Scholar) first described teichoic acid polymerization using crude membrane preparations. They demonstrated transfer of glycerol phosphate from CDP-glycerol to an unknown membrane acceptor resulting in poly(glycerol phosphate) synthesis. Those studies and others of poly(ribitol phosphate) synthesis (9Glaser L. J. Biol. Chem. 1964; 239: 3178-3186Abstract Full Text PDF PubMed Google Scholar, 10Leaver J. Hancock I.C. Baddiley J. J. Bacteriol. 1981; 146: 847-852Crossref PubMed Google Scholar, 11Ishimoto N. Strominger J.L. J. Biol. Chem. 1966; 241: 639-650Abstract Full Text PDF PubMed Google Scholar) first established that the teichoic acid synthetic machinery was membrane-associated. Soon after their discovery, the poly(glycerol phosphate) and poly(ribitol phosphate) polymerases were shown to be readily extractable with detergents to produce active, soluble enzymes (12Mauck J. Glaser L. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2386-2390Crossref PubMed Scopus (24) Google Scholar, 13Fiedler F. Glaser L. J. Biol. Chem. 1974; 249: 2684-2689Abstract Full Text PDF PubMed Google Scholar, 14Fiedler F. Glaser L. J. Biol. Chem. 1974; 249: 2690-2695Abstract Full Text PDF PubMed Google Scholar), but these were never purified presumably due to difficulties in recovering wild type levels of enzyme. Pooley et al. (6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar) assayed poly(glycerol phosphate) activities from membranes of wild type B. subtilis 168 and 11 strains bearing thermosensitive (ts)1 mutations mapped to the tag cluster, and they found that the polymerase deficiency was associated only with mutant tagFalleles. This study strongly suggested that tagF encodes the main chain polymerase (Scheme FS2). In the work reported here, we have further characterized one such mutant,tagF1, by inserting a wild type copy of tagFunder the control of a xylose-based expression system (15Bhavsar A.P. Zhao X. Brown E.D. Appl. Environ. Microbiol. 2001; 67: 403-410Crossref PubMed Scopus (124) Google Scholar) atamyE. We have thus demonstrated an unequivocal role fortagF in the thermosensitivity of this strain through xylose-dependent complementation of the mutant at the non-permissive temperature. Apart from TagD, the CDP-glycerol pyrophosphorylase that has a role in providing activated glycerol phosphate for teichoic acid synthesis, the enzymes encoded by the tag cluster of B. subtilis168 remain uncharacterized biochemically. Reasonable functional predictions based on sequence homology can be made for the enzymes involved in the synthesis of the prenolpyrophosphate-linked disaccharide including TagO, a probableN-acetylglucosamine-1-phosphate transferase (1Soldo B. Lazarevic V. Karamata D. Microbiology. 2002; 148: 2079-2087Crossref PubMed Scopus (108) Google Scholar), and TagA, a credible N-acetylmannosamine transferase (7Lazarevic V. Abellan F.X. Moller S.B. Karamata D. Mauel C. Microbiology. 2002; 148: 815-824Crossref PubMed Scopus (70) Google Scholar). GenestagGH appear to encode a membrane transporter of the ABC-2 family, with homology to a number of bacterial lipopolysaccharide and capsule transport systems (16Paulsen I.T. Beness A.M. Saier Jr., M.H. Microbiology. 1997; 143: 2685-2699Crossref PubMed Scopus (135) Google Scholar), presumably with a role in exporting intracellularly synthesized teichoic acid. TagE is clearly a glycosyltransferase involved in glucosylation of poly(glycerol phosphate) (17Pooley H.M. Karamata D. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishing Co., Inc., New York1994: 187-198Google Scholar). In contrast, the protein(s) required for poly(glycerol phosphate) synthesis remain speculative. Candidates for this function are TagB and TagF, which have no meaningful homology to characterized proteins, differ considerably in size (381 and 746 amino acids, respectively), and show pairwise sequence identity of about 30% over the C terminus of TagF. Indeed, the work of Pooley et al.(6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar) suggested that TagF encodes a poly(glycerol phosphate) polymerase. Chemical logic suggests that a primer of glycerol phosphate residue(s) might be required for elongation by a polymerase. This rationale and the conservation of TagB and TagF homologs in the poly(ribitol phosphate) synthesis cluster of B. subtilis W23 led Lazarevic et al. (7Lazarevic V. Abellan F.X. Moller S.B. Karamata D. Mauel C. Microbiology. 2002; 148: 815-824Crossref PubMed Scopus (70) Google Scholar) to propose TagB as a likely primase that would attach a single glycerol phosphate residue to prenolpyrophosphate-linked disaccharide as a primer for polymerization by TagF. Here we describe the first purification and functional characterization of recombinant TagF protein in order to confirm its predicted role as the poly(glycerol phosphate) polymerase of B. subtilis 168. The strains, plasmids, and oligonucleotides used in this work are described in Tables I andII. All cultures were grown in LB medium (18Sambrook J. Fritsch E.F. Maniatis T. 2nd Ed. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 1.47-1.84Google Scholar) supplemented with antibiotics or sugars where necessary. Antibiotics were used at the following concentrations: 50 μg/ml ampicillin, 20 (Escherichia coli) and 5 μg/ml (B. subtilis) chloramphenicol. Cloning was performed in the E. coli strain Novablues (Novagen) according to established protocols (18Sambrook J. Fritsch E.F. Maniatis T. 2nd Ed. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 1.47-1.84Google Scholar). Transformations in B. subtilis were carried out according to procedures described previously (19Cutting S.M. Vander Horn P.B. Harwood C.R. Cutting S.M. Molecular Biological Methods for Bacillus. John Wiley & Sons, Inc., New York1990: 175-209Google Scholar). Restriction enzymes, T4 DNA ligase, and Vent polymerase were purchased from New England Biolabs (Beverly, MA). Hotstar Taq polymerase was purchased from Qiagen (Mississauga, Ontario, Canada). The GatewayTMcloning system was purchased from Invitrogen. [U-14C]glycerol 3-phosphate, Ni2+-chelating columns, and SuperdexTM 200 columns were purchased from Amersham Biosciences. HPLC columns and scintillation fluids were purchased from Waters (Mississauga, Ontario, Canada). Chromatography was carried out using either an Amersham Biosciences ÄKTATM FPLC system or Waters HPLC system. Filters were purchased from Millipore (Nepean, Ontario, Canada). Mouse anti-His antibodies were purchased from Amersham Biosciences; donkey anti-mouse horseradish peroxidase antibodies were purchased from BIO/CAN (Mississauga, Ontario, Canada). Dithiothreitol, isopropyl-β-d-thiogalactopyranoside, imidazole, and antibiotics were purchased from Bioshop (Burlington, Ontario, Canada). Potassium phosphate (mono- and dibasic) was purchased from EM Science (Darmstadt, Germany). Protease Inhibitor Cocktail Set III was purchased from Calbiochem. CDP-glycerol and CDP-[U-14C]glycerol (18 Ci/mol) were synthesized using glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus and previously described methods (20Badurina D.S. Zolli-Juran M. Brown E.D. Biochim. Biophys. Acta. 2003; 1646: 196-206Crossref PubMed Scopus (36) Google Scholar). All other were purchased from and used in this or subtilis coli work subtilis for at A.P. Zhao X. Brown E.D. Appl. Environ. Microbiol. 2001; 67: 403-410Crossref PubMed Scopus (124) Google with work for Bacillus in a used in this primer primer primer primer primer primer primer primer primer primer primer primer in a The tagF gene was from the of using and with a of Taq and Vent The resulting was into the of to which was using and to confirm its was used as DNA for was by with to the found of in of tagF and tagF appear to a of The resulting was into (15Bhavsar A.P. Zhao X. Brown E.D. Appl. Environ. Microbiol. 2001; 67: 403-410Crossref PubMed Scopus (124) Google Scholar), the and the resulting was using The was used to strain to a copy of tagF at the resulting in the of strain was confirmed using a assay (19Cutting S.M. Vander Horn P.B. Harwood C.R. Cutting S.M. Molecular Biological Methods for Bacillus. John Wiley & Sons, Inc., New York1990: 175-209Google Scholar), and using and established the the The system and and were used to the for expression of hexahistidine-tagged TagF in E. coli The sequence of the was confirmed using and of E. coli with were grown at to an of using and grown for 20 at were by for and with were in 20 of and Inhibitor Cocktail Set and by through a pressure at The was by at for This was on a using and and was over a from to only the protein by were and further purified by chromatography over SuperdexTM 200 with in were and assayed for activity using the poly(glycerol phosphate) polymerase assay into and at membranes were from strain to be used as poly(glycerol phosphate) acceptor in the polymerase assay cultures were grown at in LB medium to an of and at for were as described previously (8Burger M.M. Glaser L. J. Biol. Chem. 1964; 239: 3168-3177Abstract Full Text PDF PubMed Google Scholar). were in of were by through a pressure at and were by at for The was at The membrane was in of the into and at membranes were at for to in the assay to teichoic acid polymerase activity. of B. subtilis membranes of with of purified TagF protein in 40 were addition of CDP-glycerol to reaction were to at temperature for by the addition of to were from the by at for 40 The was and by liquid scintillation The membrane was by and pelleting in the reaction after which the of radioactivity in and the were also In order to confirm polymerase teichoic acids synthesized by TagF were from the membrane acceptor and for TagF was with CDP-[U-14C]glycerol and of protein/ml B. subtilis membranes for at temperature were by at for 40 and in for at to synthesized poly(glycerol phosphate) from the linkage N. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of teichoic acid was also in scintillation for a radioactivity used to the of were into soluble and by as described and the soluble was for further A of the soluble was by size chromatography using a Waters in at of the soluble further in for at to poly(glycerol phosphate) to its H. Baddiley J. J. 1972; PubMed Scopus (73) Google Scholar). The were and by size chromatography using a Waters in at oligonucleotides of and residues and respectively), 3-phosphate, and were also under to as In order to confirm a role for the tagF gene in the synthesis of teichoic we to the wild type gene in single copy on the of the thermosensitive mutant strain the The wild type tagF gene was under the control of a promoter and on the of strain using the (15Bhavsar A.P. Zhao X. Brown E.D. Appl. Environ. Microbiol. 2001; 67: 403-410Crossref PubMed Scopus (124) Google Scholar) to strain the of strains type of and on LB or xylose and grown at or the non-permissive temperature of which only the was to at of xylose This is with the and that the of xylose in the medium is of the The wild type strain at in the and of strain the wild type copy of the tagF gene was to the and at the non-permissive temperature. of the was only this strain was grown on a medium supplemented with with conditional complementation by tagF at We also the of these strains in liquid to the non-permissive temperature The strain showed a in after temperature with a that has for tag gene A.P. Brown E.D. J. Bacteriol. 2001; PubMed Scopus Google Scholar, J. Bacteriol. PubMed Google Scholar). of the in supplemented with In the of was temperature following a to that of the thermosensitive mutant strain the show that expression from the wild gene is to the that this is responsible for the E. from the strain showed a on with the of the hexahistidine-tagged TagF protein We found that this be with the E. coli membrane in a with work that poly(glycerol phosphate) polymerase activity was associated with B. subtilis membranes (6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar, M.M. Glaser L. J. Biol. Chem. 1964; 239: 3168-3177Abstract Full Text PDF PubMed Google Scholar, J. Glaser L. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2386-2390Crossref PubMed Scopus (24) Google Scholar). in a high was to this and the of protein into The TagF protein was purified to by of an by using and size TagF activity was from the Ni2+-chelating over a of concentration where a that was with an hexahistidine-tagged TagF protein in at of was from the The identity of the protein was confirmed by analysis using the in the of purified TagF protein be at for with no of activity. III of the poly(glycerol phosphate) polymerase activity of the the A purification of that activity from the E. coli was with the in the of required of TagF and a on the protein of protein be purified with this more for characterization of the enzyme of hexahistidine-tagged in a Purified recombinant TagF was found to incorporate radioactivity from CDP-[14C]glycerol into B. subtilis membranes using a membrane pelleting We also activity in this assay in the of recombinant TagF protein, likely to subtilis TagF of membranes for was found to this activity and was as in of recombinant TagF that the activity in the membrane pelleting assay of the TagF protein was linear with and enzyme analysis of the dependence of reaction on enzyme concentration the of a number of under where membranes and CDP-glycerol were and 5 the dependence of the TagF activity on its and that the reaction was a saturable function of CDP-glycerol and membrane acceptor The for CDP-glycerol was 340 and activity was at 650 μg of protein/ml of B. subtilis The for TagF from these studies were and 20 confirm that TagF was glycerol phosphate residues into a polymeric the of the reaction was by size work has established that the poly(glycerol phosphate) chain can be from the disaccharide linkage by with the polymeric chain largely N. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Hancock I.C. Baddiley J. PubMed Scopus Google Scholar, E. J. PubMed Scopus Google Scholar). In this in of from the membrane In the of of poly(glycerol we the size of the polymer by size using of We found that the polymer in a and on a to a of to a poly(glycerol phosphate) polymer of ∼35 of poly(glycerol phosphate) with acid is to the phosphodiester between glycerol phosphate residues H. Baddiley J. J. 1972; PubMed Scopus (73) Google Scholar). Chromatography of the polymer synthesized by TagF confirmed that be to that were as and 20 on the size with the of phosphate and which is in with the of al. H. Baddiley J. J. 1972; PubMed Scopus (73) Google Scholar) reported that acid of poly(glycerol phosphate) in a of glycerol and glycerol In size analysis of the reaction on of recombinant TagF with CDP-glycerol and B. is with the role of the protein as the poly(glycerol phosphate) teichoic acid polymerase. The by Karamata and V. Karamata D. Mol. Microbiol. 1995; 16: 345-355Crossref PubMed Scopus (118) Google Scholar, 5Briehl M. Pooley H.M. Karamata D. J. Gen. Microbiol. 1989; 135: 1325-1334Google Scholar, 6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar, V. Abellan F.X. Moller S.B. Karamata D. Mauel C. Microbiology. 2002; 148: 815-824Crossref PubMed Scopus (70) Google Scholar, H.M. Karamata D. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishing Co., Inc., New York1994: 187-198Google Scholar) of the gene involved in the biosynthesis of teichoic acid in B. subtilis and the study of the associated with thermosensitive mutations in the tag gene cluster have the of for the encoded with the of the glycerol-3-phosphate cytidylyltransferase encoded by C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. S. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Google Scholar), of the enzymes have characterized biochemically. In this we to confirm the role for tagF in the temperature of one such and to the function of recombinant TagF We show that expression of a wild type copy of tagF on the of a B. subtilis strain is to the of this This the made by Pooley et al. (6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar) between the of the strain and the thermosensitivity of the poly(glycerol phosphate) polymerase activity in of the of this with wild is with work Mol. Microbiol. 1989; PubMed Scopus (36) Google Scholar) a in the sequence the complementation analysis is in with a of that teichoic acid biosynthesis is for the of B. subtilis V. Karamata D. Mol. Microbiol. 1995; 16: 345-355Crossref PubMed Scopus (118) Google Scholar, 3Mauel C. Young M. Margot P. Karamata D. Mol. Gen. Genet. 1989; 215: 388-394Crossref PubMed Scopus (59) Google Scholar, 4Mauel C. Young M. Karamata D. J. Gen. Microbiol. 1991; 137: 929-941Crossref PubMed Scopus (73) Google Scholar, 5Briehl M. Pooley H.M. Karamata D. J. Gen. Microbiol. 1989; 135: 1325-1334Google Scholar, A.P. Zhao X. Brown E.D. Appl. Environ. Microbiol. 2001; 67: 403-410Crossref PubMed Scopus (124) Google Scholar, A.P. Brown E.D. J. Bacteriol. 2001; PubMed Scopus Google Scholar). and J. Bacteriol. PubMed Scopus Google Scholar) reported that they were to the tagF gene in Staphylococcus the that the nature of teichoic acid biosynthesis to other including We have purified recombinant TagF protein to and in for biochemical studies of and This is the first of the TagF protein and has to the poly(glycerol phosphate) polymerase function of the purified protein in vitro. this we a novel polymerase assay where the were from the reaction by by acid (6Pooley H.M. Abellan F.X. Karamata D. J. Bacteriol. 1992; 174: 646-649Crossref PubMed Google Scholar, M.M. Glaser L. J. Biol. Chem. 1964; 239: 3168-3177Abstract Full Text PDF PubMed Google Scholar, J. Glaser L. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2386-2390Crossref PubMed Scopus (24) Google Scholar). assay the transfer of phosphate from CDP-[14C]glycerol an acceptor in membrane and poly(glycerol phosphate) that was for further The activity was linear with and enzyme concentration and was a saturable function of CDP-glycerol concentration (Km of 340 This is in with of 200 (12Mauck J. Glaser L. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2386-2390Crossref PubMed Scopus (24) Google Scholar) and (8Burger M.M. Glaser L. J. Biol. Chem. 1964; 239: 3168-3177Abstract Full Text PDF PubMed Google Scholar) reported more years ago using purified preparations. The activity was the concentration of B. subtilis membranes with activity at 650 μg of the of and Glaser (12Mauck J. Glaser L. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2386-2390Crossref PubMed Scopus (24) Google Scholar) that the enzyme a acceptor for activity. we a number for the TagF enzyme of The in this work that the TagF protein subtilis 168 is responsible for the polymerization of poly(glycerol phosphate) teichoic acid. of the synthesized polymer from its membrane acceptor confirmed the polymeric nature of the of the TagF Molecular analysis that TagF synthesized polymers of ∼35 glycerol phosphate of this polymer to its further confirmed the polymeric of the TagF reaction A polymer of residues is with the the first years ago (8Burger M.M. Glaser L. J. Biol. Chem. 1964; 239: 3168-3177Abstract Full Text PDF PubMed Google Scholar, 14Fiedler F. Glaser L. J. Biol. Chem. 1974; 249: 2690-2695Abstract Full Text PDF PubMed Google Scholar), to poly(glycerol phosphate) and poly(ribitol phosphate) in using crude enzyme preparations. teichoic acids from the of are found to be on the order of residues J. Bacteriol. PubMed Google Scholar, E. J. PubMed Scopus Google Scholar). was of a of reaction residues where the of synthesized polymer was between 20 and 50 This the that is a in the polymerization reaction that likely with TagF the of membranes in The study of teichoic acid has largely by genetic the of CDP-glycerol and the purification of of TagF, the poly(glycerol phosphate) the is for in biochemical studies of teichoic acid synthesis. is in to the of teichoic acid polymerization by TagF as as the synthesis and priming of the teichoic acid linkage

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