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

Jeffrey W. Schertzer and Eric D. BrownDagger

From the Antimicrobial Research Centre, the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

Received for publication, January 21, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 with tagF under control of the xylose promoter at the amyE locus. We have overexpressed and purified recombinant B. subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B. subtilis 168. Recombinant hexahistidine-tagged TagF protein was purified from Escherichia 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 fraction in 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 1). The N-acetylglucosamine-beta -(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 acid glycerol phosphate) gene cluster of B. subtilis 168 (1-6) and have also defined the tar (teichoic acid ribitol phosphate) loci in strain W23 (7). In aggregate, the biochemical and genetic work suggested that the synthesis of the poly(glycerol phosphate) chain in B. 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.


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Scheme 1.   Peptidoglycan-linked poly(glycerol phosphate) teichoic acid.

Burger and Glaser (8) 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 (9-11) 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 (12-14), but these were never purified presumably due to difficulties in recovering wild type levels of enzyme.

Pooley et al. (6) 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 tagF alleles. This study strongly suggested that tagF encodes the main chain polymerase (Scheme 2). In the work reported here, we have further characterized one such mutant, tagF1, by inserting a wild type copy of tagF under the control of a xylose-based expression system (15) at amyE. We have thus demonstrated an unequivocal role for tagF in the thermosensitivity of this strain through xylose-dependent complementation of the mutant at the non-permissive temperature.


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Scheme 2.   Reaction catalyzed by TagF.

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. subtilis 168 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 probable N-acetylglucosamine-1-phosphate transferase (1), and TagA, a credible N-acetylmannosamine transferase (7). Genes tagGH appear to encode a membrane transporter of the ABC-2 family, with homology to a number of bacterial lipopolysaccharide and capsule transport systems (16), presumably with a role in exporting intracellularly synthesized teichoic acid. TagE is clearly a glycosyltransferase involved in glucosylation of poly(glycerol phosphate) (17). 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. (6) 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. (7) 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

General Methods-- The strains, plasmids, and oligonucleotides used in this work are described in Tables I and II. All cultures were grown in LB medium (18) 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 (18). Transformations in B. subtilis were carried out according to procedures described previously (19). 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 GatewayTM cloning 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-beta -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 (20). All other chemicals were purchased from Sigma.


                              
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Table I
Strains and plasmids used in this study


                              
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Table II
Oligonucleotides used in this study

Strain Construction-- The tagF gene was PCR-amplified from the chromosome of EB6 using primers JS01 and JS02 with a 25:1 mixture of Taq and Vent polymerases. The resulting blunt-ended product was cloned into the EcoRV site of pET-Blue-1 to create pET-Blue-1-tagF which was sequenced using primers JS19 and JS21-JS25 to confirm its identity. Plasmid pET-Blue-1-tagF was then used as template DNA for subsequent cloning.

Plasmid pSWEET-tagF was constructed by amplification of tagF with primers JS03-JS05 to place the ribosome-binding site found upstream of tagE in front of tagF (the tagE and tagF genes appear to share a strong ribosome-binding site located upstream of tagE). The resulting PCR product was cloned into PacI and BamHI restriction sites within pSWEET-bgaB (15), displacing the bgaB gene, and the resulting insert was sequenced using primers JS20-JS25. The insertional plasmid pSWEET-tagF was used to engineer strain EB247 to contain a second complementing copy of tagF at the amyE locus resulting in the creation of strain EB311. Disruption of amyE was confirmed using a starch utilization plate assay (19), and PCR using primers JS20 and JS05 established the presence of tagF behind the xylA promoter.

The GatewayTM recombination-based cloning system (Invitrogen) and primers JS07 and JS08 were used to create the T7-based pDEST17-tagF plasmid for expression of N-terminal hexahistidine-tagged TagF in E. coli BL21(DE3) cells. The sequence of the inserted gene, tagF, was confirmed using primers JS19 and JS21-25.

TagF Overexpression and Purification-- Cultures of E. coli BL21(DE3) transformed with pDEST17-tagF were grown at 37 °C to an A600 of 0.7, induced using 1 mM isopropyl-beta -D-thiogalactopyranoside, and grown for 20 h at 16 °C. Subsequently, cells were harvested by centrifugation (7,000 × g for 10 min) and washed with 0.85% NaCl. Cells were resuspended in 20 ml of buffer L (20 mM sodium phosphate, 500 mM NaCl, 20% glycerol, 0.1 mg/ml DNase I, 0.1 mg/ml RNase A, pH 7.2, and Calbiochem Protease Inhibitor Cocktail Set III) and lysed by three passes through a French pressure cell at 20,000 pounds/square inch. The lysate was clarified by centrifugation at 111,000 × g for 60 min. This sample was then chromatographed on a chelating SepharoseTM nickel affinity column (Amersham Biosciences) using buffer L minus DNase, RNase, and protease inhibitors and was eluted over a discontinuous gradient going from 0 to 500 mM imidazole. Fractions containing only the expressed 90-kDa protein when visualized by Coomassie-stained SDS-PAGE were pooled, concentrated, and further purified by gel exclusion chromatography over SuperdexTM 200 resin with isocratic elution in buffer GF1 (20 mM Tris-HCl, 20% glycerol, 150 mM NaCl, pH 7.5). Protein-containing fractions were pooled and assayed for activity using the poly(glycerol phosphate) polymerase assay (see below), separated into appropriate aliquots, and frozen at -80 °C.

Preparation of Acceptor Membranes-- Inverted membranes were prepared from strain EB6 to be used as poly(glycerol phosphate) acceptor in the polymerase assay (below). 4-Liter cultures were grown at 30 °C in LB medium to an A600 of 0.8 and then pelleted at 7000 × g for 10 min. Membranes were prepared essentially as described previously (8). Pellets were resuspended in 25 ml of buffer MR (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.2). Cells were lysed by three passages through a French pressure cell at 20,000 pounds/square inch. Unlysed cells and insoluble aggregates were separated by centrifugation at 40,000 × g for 15 min. The supernatant was centrifuged again at 111,000 × g for 60 min. The membrane pellet was then resuspended in 7.33 ml of the same buffer, separated into appropriate aliquots, and frozen at -80 °C.

Poly(glycerol Phosphate) Polymerase Assay-- Acceptor membranes were incubated at 65 °C for 30 min prior to use in the assay to eliminate endogenous teichoic acid polymerase activity. 250-µl reactions containing varying concentrations of B. subtilis membranes (0.1-1.5 mg of protein/ml) with various concentrations of purified TagF protein (30-60 nM) in buffer PR (10 mM Tris, 40 mM MgCl2, pH 7.5) were initiated upon addition of CDP-glycerol to 150-1200 µM. Each reaction contained 0.2 µCi CDP-[U-14C]glycerol. Reactions were allowed to proceed at room temperature for appropriate time intervals before being quenched by the addition of urea to 4 M. Membranes were then separated from the supernatant by centrifugation at 257,000 × g for 40 min. The supernatant was removed and analyzed by liquid scintillation counting. The membrane pellet was then washed twice by resuspension and pelleting in the reaction buffer after which the level of radioactivity in both wash supernatants and the final pellet itself were also counted.

Confirmation of Polymeric State of Synthesized Teichoic Acid-- In order to confirm polymerase activity, teichoic acids synthesized by TagF were hydrolyzed from the membrane acceptor and analyzed for size. TagF (50 nM) was incubated with 1200 µM CDP-[U-14C]glycerol (0.4 µCi) and 3.33 mg of protein/ml heat-treated B. subtilis membranes for 16 h at room temperature (250 µl). Membranes were then sedimented by ultracentrifugation at 257,000 × g for 40 min and resuspended in 0.5 N NaOH for 25 min at 37 °C to release synthesized poly(glycerol phosphate) from the lipid-associated linkage unit (21). A portion of synthesized, labeled teichoic acid was also resuspended in scintillation mixture for a total radioactivity count used to calculate the yield of hydrolyzed radioactive polymer. Base-treated samples were separated into soluble and insoluble fractions by centrifugation as described above, and the soluble fraction was neutralized for further analysis. A portion of the soluble fraction was analyzed by size exclusion chromatography using a Waters Protein-Pak 300SW column in buffer GF2 (50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, pH 7.5) at 0.5 ml/min. Another portion of the soluble fraction underwent further treatment in 1 N HCl for 3 h at 100 °C to hydrolyze poly(glycerol phosphate) to its monomeric constituents (22). The acid-treated samples were neutralized and analyzed by size exclusion chromatography using a Waters Ultrahydrogel 120 column in buffer GF2 at 0.5 ml/min. For comparison, polycytidine oligonucleotides of 5, 10, 15, 20, 25 and 30 residues (1615, 3230, 4845, 6460, 8075 and 9690 g/mol, respectively), CDP-[14C]glycerol, [14C]glycerol 3-phosphate, and [3H]glycerol were also run under identical chromatographic conditions to serve as molecular weight standards.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Second Copy of tagF on the Chromosome Can Suppress the Lethal Temperature-sensitive tagF1 (rodC1) Defect-- In order to confirm a role for the tagF gene in the synthesis of teichoic acid, we sought to test whether the wild type gene provided in single copy on the chromosome of the thermosensitive tagF1 (rodC1) mutant strain (EB247) could suppress the lethal phenotype. The wild type tagF gene was cloned under the control of a xylose-inducible promoter and deposited on the chromosome (at locus amyE) of strain EB247 using the pSWEET plasmid (15) to create strain EB311. Fig. 1 shows the growth of strains EB263 (wild type parent of EB247), EB247, and EB311, plated on LB agar containing or lacking xylose (2%), and grown at 30 °C or the non-permissive temperature of 47 °C. Strain EB247, which contains only the tagF1 allele, was unable to survive at 47 °C regardless of xylose concentration. This observation is consistent with the ts phenotype and indicates that the presence of xylose in the medium alone is incapable of rescuing the cells. The wild type strain EB263 grew well at both temperatures in the presence and absence of xylose. For strain EB311, the wild type copy of the tagF gene present at amyE was able to suppress the ts mutation and permit growth at the non-permissive temperature. Suppression of the lethal ts phenotype was only observed when this strain was grown on a medium supplemented with 2% xylose, consistent with conditional complementation by tagF at amyE.


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Fig. 1.   Conditional complementation of the tagF1 defect using wild type tagF. Strains EB263 (wild type), EB247 (tagF1), and EB311 (tagF1 amyE::xylR PxylA tagF) were plated on LB agar or LB agar supplemented with xylose (2% w/v) and were allowed to grow at 30 or 47 °C as indicated. 47 °C is the non-permissive temperature for the tagF1 mutant strain. A, cells were grown at 30 °C on LB. B, cells were grown at 30 °C on LB supplemented with 2% xylose. C, cells were grown at 47 °C on LB. D, cells were grown at 47 °C on LB supplemented with 2% xylose. Strain labels are as in A for all panels.

We also examined the growth of these strains in liquid culture upon shift to the non-permissive temperature (Fig. 2). The ts strain EB247 showed a reduction in A600 beginning ~3 h after temperature shift, consistent with a cell lysis phenotype that has been observed for tag gene lesions (23, 24). Strain EB311 exhibited clear suppression of the lethal phenotype when cultured in media supplemented with xylose. In the absence of xylose, however, cell lysis was observed upon temperature increase following a time frame equivalent to that of the thermosensitive mutant strain EB247. Together the above results show that expression from the wild type tagF gene is sufficient to complement the tagF1 defect, confirming that this defect alone is responsible for the observed thermosensitivity.


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Fig. 2.   Conditional complementation of tagF1 in liquid culture. Growth curves are depicted for strains EB263 (wild type), EB247 (tagF1), and EB311 (tagF1 amyE::xylR PxylA tagF). Samples were incubated at 30 °C for 6 h before the temperature was increased to 47 °C (indicated with a vertical arrow). Shown are strains EB263 (), EB247 (open circle ), and EB311 (down-triangle) grown in LB and EB311 (black-down-triangle ) grown in LB supplemented with 2% xylose. The growth of strains EB263 and EB247 in LB supplemented with 2% xylose was indistinguishable from growth in media lacking xylose (data not shown).

Overexpression and Purification of Recombinant TagF-- E. colilysates from the overproducing strain showed a prominent band on SDS-polyacrylamide gels consistent with the theoretical molecular mass (90.4 kDa) of the hexahistidine-tagged TagF protein (Fig. 3). We found that this species could be pelleted with the E. coli membrane fraction in a low ionic strength buffer (data not shown) consistent with previous work indicating that poly(glycerol phosphate) polymerase activity was associated with B. subtilis membranes (6, 8, 12). Lysis in a high ionic strength buffer was sufficient to disrupt this interaction and release the vast majority of protein into solution. The TagF protein was purified to near-homogeneity, judged by Coomassie staining of an SDS-PAGE gel (Fig. 3), by using Ni2+ affinity and size exclusion chromatographies. TagF activity was eluted from the Ni2+-chelating column over a broad range of imidazole concentration spanning 40-225 mM where most fractions contained a smaller peptide (50 kDa) that was reactive with an anti-hexahistidine monoclonal antibody (data not shown). Nonetheless, hexahistidine-tagged TagF protein contained in fractions eluting at concentrations of imidazole greater than 175 mM was recovered free from the lower molecular weight contaminant. The identity of the fusion protein was confirmed by Western blot analysis using the anti-hexahistidine antibody (data not shown). When frozen in the presence of 20% glycerol, purified TagF protein could be stored at -80 °C for several months with no measurable loss of activity.


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Fig. 3.   Purification of hexahistidine-tagged TagF. Protein samples were analyzed by discontinuous SDS-PAGE using 3% stacking and 12% separating gels visualized with Coomassie Blue stain. Lanes contained the clarified cell lysate (15 µg, lane 1), Ni2+ affinity-purified protein (3 µg, lane 2), and size exclusion purified protein (3 µg, lane 3). The purified protein migrated during electrophoresis with an apparent molecular mass of 88 kDa.

Table III provides details of the poly(glycerol phosphate) polymerase activity of the preparation throughout the purification. A modest 5.3-fold purification of that activity from the E. coli lysate was consistent with the large overexpression evident in Fig. 3; however, the persistence of contaminating degraded species required judicious selection of fractions containing pure TagF and had a negative impact on the ultimate protein yield (0.6%). Nonetheless, near milligram quantities of protein could be purified with this protocol, more than enough for steady-state characterization of the enzyme activity.


                              
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Table III
Purification of hexahistidine-tagged TagF

Purified TagF Incorporates Radioactivity from CDP-[14C]Glycerol into B. subtilis Membranes-- Purified recombinant TagF was found to incorporate radioactivity from CDP-[14C]glycerol into B. subtilis membranes using a membrane pelleting assay. We also noted significant background activity in this assay in the absence of recombinant TagF protein, likely attributable to endogenous B. subtilis TagF (data not shown). Prior heat treatment of membranes (65 °C for 30 min) was found to completely eliminate this background activity and was adopted as standard procedure in our assays of recombinant TagF protein. Fig. 4 shows that the activity measured in the membrane pelleting assay of the TagF protein was linear with both time and enzyme concentration. Our analysis of the dependence of reaction velocity on enzyme concentration (Fig. 4, inset) facilitated the determination of a turnover number of 16 min-1 under conditions where both membranes and CDP-glycerol were saturating (3.33 mg/ml and 1.2 mM, respectively).


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Fig. 4.   Time and TagF dependence of the poly(glycerol phosphate) polymerase assay. TagF protein at concentrations of 30 (), 40 (open circle ), 50 (black-down-triangle ), and 60 nM (down-triangle) was incubated in the presence of 1.2 mM CDP-[14C]glycerol (0.2 µCi) and 3.33 mg of protein/ml of B. subtilis membranes for 5, 15, and 25 min (250-µl reaction volume). Reactions were quenched by the addition of urea to 4 M after which the membrane fraction was pelleted by ultracentrifugation. The amount of glycerol phosphate incorporated into the membrane fraction was determined by liquid scintillation counting and was plotted against the time. Each data point represents the average of two measurements. The inset shows the initial velocity of the reaction plotted as a function of enzyme concentration. The slope of this plot represents the turnover of the enzyme under experimental conditions (16 min-1).

TagF Activity Is Dependent on CDP-glycerol and Membrane Acceptor Concentrations-- Fig. 5 shows the dependence of the TagF activity on its substrates and indicates that the reaction velocity was a saturable function of both CDP-glycerol and membrane acceptor concentrations. The Km for CDP-glycerol was 340 µM, and half-maximal activity was observed at 650 µg of protein/ml of heat-inactivated B. subtilis membranes. The calculated turnover numbers for TagF from these two studies were 16 and 20 min-1, respectively.


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Fig. 5.   The dependence of reaction velocity on substrate concentration. A, TagF (50 nM) was incubated in the presence of 3.33 mg of protein/ml of B. subtilis membranes and varying concentrations of CDP-[14C]glycerol (150-1200 µM, 0.2 µCi). B, TagF (50 nM) was incubated in the presence of 1200 µM CDP-[14C]glycerol (0.2 µCi) and varying concentrations of heat-inactivated B. subtilis membranes (0.1-1.5 mg of protein/ml). Reactions (250 µl) were quenched after 20 min by the addition of urea to 4 M. The data were fit to the equation: V = Vmax [S]/Km + [S] using Sigma Plot 2000 (SPSS, Chicago). Analysis of the data yielded a Km for CDP-glycerol of 340 ± 50 µM (A) and half-maximal velocity at 650 ± 60 µg of protein/ml of purified B. subtilis membranes (B). The calculated turnover numbers for TagF from these two studies were 16 ± 0.8 and 20 ± 0.8 min-1, respectively.

Confirmation of Polymeric State of Synthesized Teichoic Acid-- To confirm that TagF was incorporating glycerol phosphate residues into a polymeric structure, the product of the reaction was analyzed by size exclusion chromatography. Previous analytical work has established that the poly(glycerol phosphate) chain can be selectively cleaved from the disaccharide linkage unit by treatment with mild alkali, leaving the polymeric chain largely intact (21, 25, 26). In our experiments, this treatment resulted in greater than 99% extraction of radioactive material from the membrane (data not shown). In the absence of standards of poly(glycerol phosphate), we analyzed the size of the released polymer by size exclusion using polycytidine standards of known molecular weight. We found that the radioactive polymer eluted in a broad and symmetric peak centered on a retention time corresponding to a molecular mass of 6200 g/mol (Fig. 6A). That mass corresponds to a poly(glycerol phosphate) polymer of ~35 residues.


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Fig. 6.   Confirmation of the polymeric nature of the synthesized teichoic acid. A, size exclusion (Waters Protein-Pak 300SW) chromatogram of alkali-treated (0.5 N NaOH, 37 °C, 25 min) products of the polymerase reaction. The inset to A shows a standard curve of the elution of polycytidine molecular weight standards (1615, 3230, 4845, 6460, 8075, and 9690 g/mol) run under the same chromatographic conditions. B, size exclusion (Waters Ultrahydrogel 120) chromatogram of acid-treated (1 N HCl, 100 °C, 3 h) products of the polymerase reaction. Samples were analyzed by size exclusion chromatography using an HPLC apparatus equipped with in-line liquid scintillation counting.

Harsher treatment of poly(glycerol phosphate) with acid is known to hydrolyze the phosphodiester linkages between glycerol phosphate residues (22). Chromatography of the acid-treated polymer synthesized by TagF confirmed that it could be digested to low molecular weight species that were resolved as two predominant peaks (15 and 20 ml) on the size exclusion column (Fig. 6B). These low molecular weight species coincided with the elution of standards [14C]glycerol phosphate and [3H]glycerol, respectively, which is in agreement with the findings of Anderson et al. (22) who reported that acid treatment of poly(glycerol phosphate) resulted in a mixed population of glycerol, glycerol monophosphates, and glycerol diphosphates being released. In sum, size analysis of the reaction product on incubation of recombinant TagF with CDP-glycerol and B. subtilis membranes is consistent with the proposed role of the protein as the poly(glycerol phosphate) teichoic acid polymerase.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The identification by Karamata and colleagues (2, 5-7, 17) of the gene clusters involved in the biosynthesis of teichoic acid in B. subtilis and the study of the phenotypes associated with thermosensitive mutations in the tag gene cluster have facilitated the assignment of putative functions for the encoded proteins. Nonetheless, with the exception of the glycerol-3-phosphate cytidylyltransferase encoded by tagD (27-29), none of the Tag enzymes have been characterized biochemically. In this contribution we undertook to confirm the role for tagF in the temperature sensitivity of one such mutant, tagF1 (rodC), and to investigate the function of recombinant TagF protein.

We show here that expression of a wild type copy of tagF on the chromosome of a B. subtilis strain harboring the tagF1 temperature-sensitive mutation is sufficient to suppress the lethal phenotype of this strain. This finding solidifies the connection made by Pooley et al. (6) between the phenotype of the tagF1 strain and the thermosensitivity of the poly(glycerol phosphate) polymerase activity observed in extracts of the mutant. Complementation of this defect with wild type tagF is consistent with previous work (30) describing a missense mutation in the tagF1 sequence (S644F). Furthermore, the complementation analysis is in line with a growing body of research indicating that teichoic acid biosynthesis is essential for the viability of B. subtilis (2-5, 15, 23). Interestingly, Fitzgerald and Foster (31) reported that they were unable to disrupt the tagF gene in Staphylococcus epidermidis, raising the prospect that the essential nature of teichoic acid biosynthesis may extend to other Gram-positive bacteria, including pathogens.

We have purified recombinant TagF protein to near-homogeneity and in quantities necessary for biochemical studies of structure and function. This is the first ever isolation of the TagF protein and has allowed us to unambiguously examine the poly(glycerol phosphate) polymerase function of the purified protein in vitro. To this end we developed a novel membrane-pelleting polymerase assay where the products were separated from the reaction mixture by ultracentrifugation rather than by acid precipitation (6, 8, 12). Our assay monitored the transfer of [14C]glycerol phosphate from CDP-[14C]glycerol onto an acceptor resident in inverted membrane vesicles and yielded acceptor-linked poly(glycerol phosphate) that was then usable for further analysis. The activity was linear with time and enzyme concentration and was a saturable function of CDP-glycerol concentration (Km of 340 µM). This value is in agreement with values of 200 (12) and 830 µM (8) reported more than 30 years ago using partially purified preparations. The observed activity was dependent upon the concentration of B. subtilis membranes present with half-maximal activity occurring at 650 µg of protein/ml. These data support the suggestion of Mauck and Glaser (12) that the enzyme requires a membrane-bound acceptor for activity. With saturating substrates we calculate a turnover number for the TagF enzyme of ~20 min-1.

The results presented in this work demonstrate that the TagF protein of B. subtilis 168 is responsible for the polymerization of poly(glycerol phosphate) teichoic acid. Base hydrolysis of the synthesized polymer from its membrane acceptor confirmed the polymeric nature of the radiolabeled product of the TagF reaction. Molecular weight analysis indicated that TagF synthesized polymers of ~35 glycerol phosphate units. Acid hydrolysis of this polymer to its radiolabeled constituent monomers further confirmed the polymeric character of the TagF reaction product. A polymer length of 35 residues is consistent with the 25-35-residue range seen during the first attempts, many years ago (8, 14), to synthesize poly(glycerol phosphate) and poly(ribitol phosphate) in vitro using crude enzyme preparations. Nevertheless, teichoic acids extracted from the cell walls of Gram-positive bacteria are often found to be somewhat longer, on the order of 55-60 residues (24, 26). Perhaps most remarkable was our finding of a normal distribution of reaction products around 35 residues where the vast majority of synthesized polymer was between 20 and 50 residues. This leaves open the intriguing possibility that there is a length-sensing ability in the polymerization reaction that likely resides with TagF given the use of heat-treated membranes in our assay.

The study of teichoic acid biogenesis has largely been dominated by genetic studies. With the ready availability of TagD-synthesized CDP-glycerol and the purification of milligram quantities of TagF, the poly(glycerol phosphate) polymerase, the stage is now set for in vitro biochemical studies of teichoic acid synthesis. Work is ongoing in our laboratory to define the mechanism of teichoic acid polymerization by TagF as well as the synthesis and priming of the teichoic acid linkage unit.

    FOOTNOTES

* This work was supported by the Canadian Institutes of Health Research Grant MOP-15496 and by a Canada Research Chair in Microbial Biochemistry (to E. D. B.).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 To whom correspondence should be addressed: Antimicrobial Research Centre, Dept. of Biochemistry, McMaster University, 1200 Main St. W., Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22392); Fax: 905-522-9033; E-mail: ebrown@mcmaster.ca.

Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M300706200

    ABBREVIATIONS

The abbreviations used are: ts, thermosensitive; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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