Late-stage polyribitol phosphate wall teichoic acid biosynthesis in Staphylococcus aureus

Wall teichoic acids are cell wall polymers that maintain the integrity of the cellular envelope and contribute to the virulence of Staphylococcus aureus. Despite the central role of wall teichoic acid in S. aureus virulence, details concerning the biosynthetic pathway of the predominant wall teichoic acid polymer are lacking, and workers have relied on a presumed similarity to the putative polyribitol phosphate wall teichoic acid pathway in Bacillus subtilis. Using high-resolution polyacrylamide gel electrophoresis for analysis of wall teichoic acid extracted from gene deletion mutants, a revised assembly pathway for the late-stage ribitol phosphate-utilizing enzymes is proposed. Complementation studies show that a putative ribitol phosphate polymerase, TarL, catalyzes both the addition of the priming ribitol phosphate onto the linkage unit and the subsequent polymerization of the polyribitol chain. It is known that the putative ribitol primase, TarK, is also a bifunctional enzyme that catalyzes both ribitol phosphate priming and polymerization. TarK directs the synthesis of a second, electrophoretically distinct polyribitol-containing teichoic acid that we designate K-WTA. The biosynthesis of K-WTA in S. aureus strain NCTC8325 is repressed by the accessory gene regulator (agr) system. The demonstration of regulated wall teichoic acid biosynthesis has implications for cell envelope remodeling in relation to S. aureus adhesion and pathogenesis.     

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.     

Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.     

Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23

The stepwise formation and characterization of linkage unit intermediates and their functions in ribitol teichoic acid biosynthesis were studied with membranes obtained from Staphylococcus aureus H and Bacillus subtilis W23. The formation of labeled polymer from CDP-[14C]ribitol and CDP-glycerol in each membrane system was markedly stimulated by the addition of N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine (ManNAc-GlcNAc) linked to pyrophosphorylyisoprenol. Whereas incubation of S. aureus membranes with CDP-glycerol and ManNAc-[14C]GlcNAc-PP-prenol led to synthesis of (glycerol phosphate) 1-3-ManNAc-[14C]GlcNAc-PP-prenol, incubation of B. subtilis membranes with the same substrates yielded (glycerol phosphate)1-2-ManNAc-[14C]GlcNAc-PP-prenol. In S. aureus membranes, (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol as well as (glycerol phosphate)3-ManNAc-[14C]GlcNAc-PP-prenol served as an acceptor for ribitol phosphate units, but (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol did not. In B. subtilis W23 membranes, (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol served as a better acceptor for ribitol phosphate units than (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol. In this membrane system (ribitol phosphate)-(glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol was formed from ManNAc-[14C]GlcNAc-PP-prenol, CDP-glycerol and CDP-ribitol. The results indicate that (glycerol phosphate)1-3-ManNAc-GlcNAc-PP-prenol and (glycerol phosphate)1-2-ManNac-GlcNAc-PP-prenol are involved in the pathway for the synthesis of wall ribitol teichoic acids in S. aureus H and B. subtilis W23 respectively.     

Phosphate-containing cell wall polymers of bacilli

Anionic phosphate-containing cell wall polymers of bacilli are represented by teichoic acids and poly(glycosyl 1-phosphates). Different locations of phosphodiester bonds in the main chain of teichoic acids as well as the nature and combination of the constituent structural elements underlie their structural diversity. Currently, the structures of teichoic acids of bacilli can be classified into three types, viz. poly(polyol phosphates) with glycerol or ribitol as the polyol; poly(glycosylpolyol phosphates), mainly glycerol-containing polymers; and poly(acylglycosylglycerol phosphate), in which the components are covalently linked through glycosidic, phosphodiester, and amide bonds. In addition to teichoic acids, poly(glycosyl 1-phosphates) with mono- and disaccharide residues in the repeating units have been detected in cell walls of several Bacillus subtilis and Bacillus pumilus strains. The known structures of teichoic acids and poly(glycosyl 1-phosphates) of B. subtilis, B. atrophaeus, B. licheniformis, B. pumilus, B. stearothermophilus, B. coagulans, B. cereus as well as oligomers that link the polymers to peptidoglycan are surveyed. The reported data on the structures of phosphate-containing polymers of different strains of B. subtilis suggest heterogeneity of the species and may be of interest for the taxonomy of bacilli to allow differentiation of closely related organisms according to the “structures and composition of cell wall polymers” criterion     

Studies on the linkage between teichoic acid and peptidoglycan in a bacteriophage-resistant mutant of Staphylococcus aureus H.

1. In addition to poly(ribitol phosphate) the walls of a bacteriophage-resistant mutant of Staphylococcus aureus H contain glycerol phosphate residues that are not removed on digestion with trypsin or extraction with phenol. 2. The glycerol phosphate is present in a chain, containing three or four glycerol phosphate residues, which is covalently attached to the peptidoglycan through a phosphodiester linkage to muramic acid; this linkage is readily hydrolysed by dilute alkali. 3. The degradative studies described suggest that the poly(ribitol phosphate) chains of the wall teichoic acid may be attached to the wall by linkage to this glycerol phosphate oligomer.     

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.     

Influence of phosphate supply on teichoic acid and teichuronic acid content of Bacillus subtilis cell walls

Bacillus subtilis 168 was grown in chemostat culture in fully defined media containing a constant concentration of magnesium and concentrations of phosphate that varied from those giving phosphate-limited growth to those in which phosphate was present in excess and magnesium was limiting. Phosphate-limited bacteria were deficient in wall teichoic acid and contained less than half as much cellular phosphate as did bacteria grown in excess of phosphate. Approximately 70% of the additional phosphate in the latter bacteria was present as wall teichoic acid, indicating that the ability of the bacteria to discontinue teichoic acid synthesis when grown under phosphate limitation permits a substantial increase in their growth yield. Since not all of the additional phosphate is present as wall teichoic acid other cellular phosphates may also be present in reduced amounts in the phosphate-limited bacteria. The content of phosphate groups in walls of magnesium-limited bacteria was similar to the content of uronic acid groups in walls of phosphate-limited bacteria, and walls of bacteria grown in media of intermediate composition contained intermediate proportions of the two anionic polymers. Phage SP50, used as a marker for the presence of teichoic acid, bound densely to nearly all of the bacteria in samples containing down to 22% of the maximum content of teichoic acid. Apparently, therefore, nearly all of these bacteria contain teichoic acid, and the population does not consist of a mixture of individuals having exclusively one kind of anionic polymer. Bacteria containing less than 22% of the maximum content of teichoic bound in a nonuniform manner, and possible explanations for this are discussed.     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

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 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-[(14)C]glycerol into a membrane fraction in vitro. This activity showed a saturable dependence on the concentration of CDP-glycerol (K(m) of 340 microm) and the membrane acceptor (half-maximal activity at 650 microg of protein/ml of purified B. subtilis membranes). High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, approximately 35 glycerol phosphate units in length.     

The cell wall teichoic acids of streptomycetes from the "Streptomyces cyaneus" cluster

The cell wall anionic polymers of the 13 species of the "Streptomyces cyaneus" cluster have a similar structure and contain beta-glucosylated 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate). In the degree of glucosylation of the ribitol phosphate units of their teichoic acids, the cluster members can be divided into two groups. The streptomycetes of the first group (S. afghaniensis, S. janthinus, S. purpurascens, S. roseoviolaceus, and S. violatus) are characterized by a very similar structure of their cell walls, completely glucosylated 1,5-poly(ribitol phosphate) chains, and a high degree of DNA homology (67-88%). The cell wall teichoic acids of the second group (S. azureus, S. bellus, S. caelestis, S. coeruleorubidus, S. curacoi, and S. violarus) differ in the degree of beta-glucosylation of their 1,5-poly(ribitol phosphate) chains and have a lower level of DNA homology (54-76%). Two streptomycetes of the cluster (S. cyaneus and S. hawaiiensis) are genetically distant from the other cluster members but have the same composition and structure of the cell wall teichoic acids as the second-group streptomycetes. The data obtained confirm the genetic relatedness of the "S. cyaneus" cluster members and suggest that the structure of the cell wall teichoic acids may serve as one of the taxonomic criteria of the species-level status of streptomycetes.     

Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.     

Synthesis of teichoic acid by Bacillus subtilis protoplasts

Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane. During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane. No evidence could be obtained for a transphosphorylation mechanism for the translocation process. It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle.     

Effects of carbon source and growth rate on cell wall composition of Bacillus subtilis subsp. niger.

A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation of synthesis of teichoic and teichuronic acids in Bacillus subtilis subsp. niger WM. First, the nature of the carbon source was varied while the dilution rate was maintained at about 0.3 h-1. Irrespective of whether the carbon source was glucose, glycerol, galactose, or malate, teichoic acid was the main anionic wall polymer whenever phosphorus was present in excess of the growth requirement, and teichuronic acid predominated in the walls of phosphate-limited cells. The effect of growth rate was studied by varying the dilution rate. However, only under phosphate limitation did the wall composition change with the growth rate: walls prepared from cells grown at dilution rates above 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culture still being phosphate limited. The wall content of the cells did not vary with the nature of the growth limitation, but a correlation was observed between the growth rate and wall content. No indications were obtained that the composition of the peptidoglycan of B. subtilis subsp. niger WM was phenotypically variable.     

Duplication of teichoic acid biosynthetic genes in Staphylococcus aureus leads to functionally redundant poly(ribitol phosphate) polymerases

Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI′J′ and tarIJ) as well as poly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI′J′) could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI′J′ is due to poor translation of the TarJ′ enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.     

Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyglycerol phosphate teichoic acids in Bacillus subtilis strains 168 and W23

A 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis. No homology was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly-(rboP)) biosynthesis. Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly-(groP) into their cell walls revealed that, in every case, integral substitution of the W23 tar genes for the 168 tag genes had occurred. Interstrain hybrids of the 'W23-like' type have inherited larger segments of W23 DNA than interstrain hybrids of the 'mixed' type. The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair. They provide the first example of a pseudo-allelic relationship between non-homologous genes in B. subtilis.     

The occurrence of teichoic acids in streptomycetes

The presence of teichoic acids in a number of streptomycetes led to the conclusion that these biopolymers were widely spread in genus Streptomyces. The nature of the teichoic acid present in the mycelium was determined by extracting it with 10% trichloroacetic acid, precipitating it with ethanol and identifying the precipitated polymer by partial acid and alkali hydrolysis to alditol, alditol phosphates and glycosylalditol phosphates. Most strains examined in this survey contained glycerol or ribitol teichoic acids; in some cases neither type was detected.Structurally teichoic acids closely resemble those of other genera of gram-positive bacteria and in many cases represent poly(glycerol phosphate) and poly(ribitol phosphate) chains. The proportion of alditol residues bearing sugar substituents varied widely.Three species of genus Streptoverticillium contained glycerol teichoic acids. It is belived that some of the data presented in this paper might be used with some success in taxonomic studies of streptomycetes.     

The effects of growth conditions on cell wall composition and cell morphology in a temperature-sensitive tag-B mutant of Bacillus subtilis

The relationship between wall anionic polymer synthesis and cell morphology has been studied in Bacillus subtilis 168 and its temperature-sensitive tagB mutant strain BR19-200B. The amount and type of anionic polymer synthesized varied under different growth conditions, as did the morphology of the bacteria. Anionic polymer synthesis was affected by the phosphate supply. It was also found that teichuronic acid synthesis was temperature-sensitive in wild-type bacteria. Teichuronic acid synthesis was affected by the tagB lesion, previously thought to affect only teichoic acid synthesis. A relationship was observed between synthesis of the alternative polymers, such that suppression of teichuronic acid synthesis is accompanied by an increase in the synthesis of teichoic acid. Variation in anionic polymer content was accompanied by variation in cell shape. Differences in shape were related to differences in total anionic polymer rather than to differences in individual polymer type.     

A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis

Bacterial cell wall homeostasis is an intricately coordinated process that ensures that envelope integrity is maintained during cell growth and division, but can also adequately respond to growth‐limiting conditions such as phosphate starvation. In Bacillus subtilis, biosynthesis of the two major cell wall components, peptidoglycan and anionic polymers, is controlled by a pair of paralogous two‐component systems, WalRK and PhoPR respectively. Favorable growth conditions allow for a fast rate of cell wall biosynthesis (WalRK‐ON) and the incorporation of the phosphate‐containing anionic polymer teichoic acids (PhoPR‐OFF). In contrast, growth‐restricted cells under phosphate‐limiting conditions reduce the incorporation of peptidoglycan building blocks (WalRK‐OFF) and switch from the phosphate‐containing teichoic acids to the phosphate‐free anionic polymer teichuronic acid (PhoPR‐ON). Botella et al. (2014) deepen our knowledge on the PhoPR system by identifying one signal that is perceived by its histidine kinase PhoR. In fast‐growing cells, intracellular intermediates of teichoic acid biosynthesis are sensed by the cytoplasmic Per‐Arnt‐Sim domain as an indicator of favorable conditions, thereby inhibiting the autokinase activity of PhoR and keeping the system inactive. Depletion of teichoic acid building blocks under phosphate‐limiting conditions relieves this inhibition, activates PhoPR‐dependent signal transduction and hence the switch to teichuronic acid biosynthesis.     

The TagB protein in Bacillus subtilis 168 is an intracellular peripheral membrane protein that can incorporate glycerol phosphate onto a membrane-bound acceptor in vitro

Genes involved in the synthesis of poly(glycerol phosphate) wall teichoic acid have been identified in the tag locus of the model Gram-positive organism Bacillus subtilis 168. The functions of most of these gene products are predictable from sequence similarity to characterized proteins and have provided limited insight into the intracellular synthesis and translocation of wall teichoic acid. Nevertheless, critical steps of poly(glycerol phosphate) teichoic acid polymerization continue to be a puzzle. TagB and TagF, encoded in the tag locus, do not show sequence similarity to characterized proteins. We recently showed that recombinant TagF could catalyze glycerol phosphate polymerization in vitro. Based largely on homology to TagF, the TagB protein has been proposed to catalyze either an intracellular glycerophosphotransfer reaction or the extracellular teichoic acid/peptidoglycan ligation reaction. Here we have taken steps to characterize TagB, particularly through in vivo localization studies and in vitro biochemical assay, in order to make a case for either role in teichoic acid biogenesis. We have shown that TagB associates peripherally with the intracellular face of the cell membrane in vivo. We have also produced recombinant TagB and used it to demonstrate the enzymatic incorporation of labeled glycerol phosphate onto a membrane-bound acceptor. The data collected from this and the accompanying study are strongly supportive of a role for TagB in B. subtilis 168 teichoic acid biogenesis as the CDP-glycerol:N-acetyl-beta-d-mannosaminyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenyl glycerophosphotransferase. Here we use the trivial name "Tag primase."