Synthesis of CDP-Activated Ribitol for Teichoic Acid Precursors in Streptococcus pneumoniae

Streptococcus pneumoniae has unusually complex cell wall teichoic acid and lipoteichoic acid, both of which contain a ribitol phosphate moiety. The lic region of the pneumococcal genome contains genes for the uptake and activation of choline, the attachment of phosphorylcholine to teichoic acid precursors, and the transport of these precursors across the cytoplasmic membrane. The role of two other, so far uncharacterized, genes, spr1148 and spr1149, in the lic region was determined. TarJ (spr1148) encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol 5-phosphate from ribulose 5-phosphate. TarI (spr1149) encodes a cytidylyl transferase for the synthesis of cytidine 5′-diphosphate (CDP)-ribitol from ribitol 5-phosphate and cytidine 5′-triphosphate. We also present the crystal structure of TarI with and without bound CDP, and the structures present a rationale for the substrate specificity of this key enzyme. No transformants were obtained with insertion plasmids designed to interrupt the tarIJ genes, indicating that their function could be essential for cell growth. CDP-activated ribitol is a precursor for the synthesis of pneumococcal teichoic acids and some of the capsular polysaccharides. Thus, all eight genes in the lic region have a role in teichoic acid synthesis.     

Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.     

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.     

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.     

Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ?29 Appendage Protein GP12 Using Synthetic Substrate Analogs

The genetics and enzymology of the biosynthesis of wall teichoic acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage ϕ29 has been implicated as a wall teichoic acid hydrolase. We have studied the wall teichoic acid hydrolase activity of pure, recombinant GP12 using chemically defined wall teichoic acid analogs. The GP12 protein had potent wall teichoic acid hydrolytic activity in vitro and demonstrated ∼13-fold kinetic preference for glycosylated poly(glycerol phosphate) teichoic acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.     

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.     

Biosynthesis of wall polymers in Bacillus subtilis.

Preparations of membrane plus wall derived from Bacillus subtilis W23 were used to study the in vitro synthesis of peptidoglycan and teichoic acid and their linkage to the preexisting cell wall. The teichoic acid synthesis showed an ordered requirement for the incorporation of N-acetylglucosamine from uridine 5'-diphosphate (UDP)-N-acetylglucosamine followed by addition of glycerol phosphate from cytidine 5'-diphosphate (CDP)-glycerol and finally by addition of ribitol phosphate from CDP-ribitol. UDP-N-acetylglucosamine was not only required for the synthesis of the teichoic acid, but N-acetylglucosamine residues formed an integral part of the linkage unit attaching polyribitol phosphate to the cell wall. Synthesis of the teichoic acid was exquisitely sensitive to the antibiotic tunicamycin, and this was shown to be due to the inhibition of incorporation of N-acetylglucosamine units from UDP-N-acetylglucosamine.     

Cell wall teichoic acids from Streptomyces daghestanicus VKM Ac-1722T and Streptomyces murinus INA-00524T

The structure of cell wall teichoic acids was studied by chemical methods and NMR spectroscopy in the type strains of two actinomycete species of the "Streptomyces griseoviridis" phenetic cluster: Streptomyces daghestanicus and Streptomyces murinus. S. daghestanicus VKM Ac-1722T contained two polymers having a 1,5-poly(ribitol phosphate) structure. In one of them, the ribitol units had alpha-rhamnopyranose and 3-O-methyl-alpha-rhamnopyranose substituents; in the other, each ribitol unit was carrying 2,4-ketal-bound pyruvic acid. Such polymers were earlier found in the cell walls of Streptomyces roseolus and Nocardiopsis albus, respectively; however, their simultaneous presence in the cell wall has never been reported. The cell wall teichoic acid of Streptomyces murinus INA-00524T was is a 1,5-poly(glucosylpolyol phosphate), whose repeating unit was [-6)-beta-D-glucopyranosyl-(1 --> 2)-glycerol phosphate-(3-P-]. Such a teichoic acid was earlier found in Spirilliplanes yamanashiensis. The 13C NMR spectrum of this polymer is presented for the first time. The results of the present investigation, together with earlier published data, show that the type strains of four species of the "Streptomyces griseoviridis" phenetic cluster differ in the composition and structure of their teichoic acids; thus, teichoic acids may serve as chemotaxonomic markers of the species.     

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 -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, the completely glucosylated 1,5-poly(ribitol phosphate) chains, and a high degree of DNA homology (67–88% according to literature data). 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 -glucosylation of their 1,5-poly(ribitol phosphate) chains and have a lower level of DNA homology (54–76% according to literature data). 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.     

Classification of staphylococci based on chemical and biochemical properties

Summary In the present work the chemical cell wall composition and some other biochemical characteristics were studied in staphylococci with the intention of utilizing the data obtained in their classification.According to the cell wall peptidoglycans and teichoic acids, the 130 strains of staphylococci studied are divided into 10 major groups. This division of staphylococci into groups is in good agreement with their present classification only in some cases. All of the 47Staphylococcus aureus strains contain a cell wall peptidoglycan of thel-Lys-Gly_5–6 type and ribitol teichoic acid. Coagulase-negative staphylococci are more heterogeneous and are divided according to their cell wall composition into 9 major groups. 21 strains of them are classified asS. epidermidis sensu stricto. They form a natural group and are distinguished by the occurrence of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 peptidoglycan type, glycerol teichoic acid and anl-lactate dehydrogenase which is activated by fructose-1,6-diphosphate. 8 strains with peptidoglycan of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 type and ribitol teichoic acid are labeled asS. saprophyticus. The remaining groups have not been given species names and require further extensive comparative study.     

Glycoepitopes of Staphylococcal Wall Teichoic Acid Govern Complement-mediated Opsonophagocytosis via Human Serum Antibody and Mannose-binding Lectin

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or β-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, β-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that β-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact β-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-β-GlcNAc-specific WTA IgGs, anti-β-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.     

Cell wall teichoic acids ofNocardiopsis prasina VKM Ac-1880T

The cell wall ofNocardiopsis prasina VKM Ac-1880^T was found to contain two structurally different teichoic acids: unsubstituted 3,5-poly(ribitol phosphate) and l,3-poly(glycerol phosphate) substituted at position 2 by 10% with α-N-acetylglucosamine and by 5% withO-acetyl groups. The structure of the polymers was studied by chemical analysis and NMR spectroscopy. The results obtained correlate wellwith 16S rRNA sequence data and confirm the species-specificity of teichoic acids in the genusNocardiopsis.     

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.     

Cell wall teichoic acids from Streptomyces daghestanicus VKM Ac-1722T and streptomyces murinus INA-00524T

The structure of cell wall teichoic acids was studied by chemical methods and NMR spectroscopy in the type strains of two actinomycete species of the Streptomyces griseoviridis phenetic cluster: streptomyces daghestanicus and streptomyces murinus. S. daghestanicus VKM Ac-1722^t contained two polymers having a 1,5-poly(ribitol phosphate) structure. In one of them, the ribitol units had -rhamnopyranose and 3-O-methyl--rhamnopyranose substituents; in the other, each ribitol unit was carrying 2,4-ketal-bound pyruvic acid. Such polymers were earlier found in the cell walls of Streptomyces roseolus and Nocardiopsis albus, respectively; however, their simultaneous presence in the cell wall has never been reported. The cell wall teichoic acid of Streptomyces murinus INA-00524^T was a 1,5-poly(glucosylpolyol phosphate), whose repeating unit was [-6)--D-glucopyranosyl-(12)-glycerol phosphate-(3-P-]. Such a teichoic acid was earlier found in Spirilliplanes yamanashiensis. The ¹³C NMR spectrum of this polymer is presented for the first time. The results of the present investigation, together with earlier published data, show that the type strains of four species of the Streptomyces griseoviridis phenetic cluster differ in the composition and structure of their teichoic acids; thus, teichoic acids may serve as chemotaxonomic markers of the species.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 48–54.Original Russian Text Copyright © 2005 by Streshinskaya, Kozlova, Alferova, Shashkov, Evtushenko.     

Pleiotropic roles of polyglycerolphosphate synthase of lipoteichoic acid in growth of Staphylococcus aureus cells

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus ΔltaS mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, ΔltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of ΔltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the ΔltaS mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the ΔltaS mutation was found to be synthetically lethal with the ΔtagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function.     

Teichoic acids from the cell wall of Nocardiopsis prasina BKM Ac-1880T

The cell wall of Nocardiopsis prasina VKM Ac-1880T was found to contain two structurally different teichoic acids: unsubstituted 3,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), substituted at position 2 by 10% with alpha-N-acetylglucosamine and by 5% with O-acetyl groups. The structure of the polymers was studied by chemical analysis and NMR spectroscopy. The results obtained correlate well with 16S rRNA sequence data and confirm the species-specificity of teichoic acids in the genus Nocardiopsis.     

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.     

The N-Acetylmannosamine Transferase Catalyzes the First Committed Step of Teichoic Acid Assembly in Bacillus subtilis and Staphylococcus aureus

There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the N-acetylmannosamine transferase (tagA in Bacillus subtilis; tarA in Staphylococcus aureus), which adds N-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the N-acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable gene in the pathway, suggesting that tagA (or tarA) encodes the first committed step in wall teichoic acid synthesis.The cell wall of gram-positive bacteria is composed of not only peptidoglycan, but also a significant proportion of the polyol phosphate polymer known as teichoic acid. Wall teichoic acid has long been held as an essential component of the cell wall architecture (2-5, 19). However, recently, our group has demonstrated a complex pattern of dispensability for wall teichoic acid biosynthetic genes of both Bacillus subtilis and Staphylococcus aureus (9, 10).The synthesis of wall teichoic acid polymers occurs through the sequential action of several enzymes (14, 17). The action of no less than seven enzymes is thought to synthesize the completed polymer on the cytoplasmic face of the cell membrane for export to the outside of the cell. Once outside, the completed polymer is covalently attached to the C-6 of the N-acetylmuramic acid of peptidoglycan through the action of an uncharacterized transferase. The best-characterized wall teichoic acid biosynthetic machinery is that for polymers composed of glycerol phosphate and ribitol phosphate. In the last several years, biochemical experiments have characterized the activities of nearly all of the enzymes responsible for the synthesis of both glycerol phosphate and ribitol phosphate polymers (6, 11, 18).Work on the essential nature of wall teichoic acid dates back many years to the discovery and characterization of temperature-sensitive B. subtilis tag mutants for poly(glycerol phosphate) synthesis by D. Karamata''s lab (4, 5, 19). That work and follow-up studies by our research group (2, 3, 20) showed convincingly that genetic lesions in several wall teichoic acid biosynthetic steps led to cell death in vitro. Recently, however, we uncovered some remarkable complexity in the dispensability pattern of wall teichoic acid synthetic genes. Working with both B. subtilis and S. aureus, we showed that viable deletions could be generated in the first gene of the pathway, encoding the N-acetylglucosamine-1-phosphate transferase (tagO in B. subtilis; tarO in S. aureus), while deletions could not be made for late-acting genes, including those encoding the glycerol phosphate primase (tagB in B. subtilis; tarB in S. aureus) and downstream enzymes. This apparent paradox was resolved when it was discovered that all of the indispensable genes became dispensable in a tagO (or tarO) deletion background and suggested that lesions in late steps of wall teichoic acid synthesis lead to a premature termination of the pathway, causing a buildup of toxic intermediates or the sequestration of a common and vital precursor molecule (i.e., undecaprenol phosphate).While extensive investigations have charted the complex genetics of wall teichoic acid synthesis in both B. subtilis 168 (2-5, 9, 15, 16, 19, 21) and S. aureus (10, 23), no experiments have so far been reported to characterize the dispensability phenotype of the N-acetylmannosamine transferase encoded by tagA (B. subtilis) and tarA (S. aureus). Indeed, tagA from B. subtilis was recently shown to catalyze the addition of N-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide, a core component of the “linkage unit” of wall teichoic acid (6, 11, 25). This places TagA (TarA) as an enzyme catalyzing the second step in wall teichoic biosynthesis after TagO (TarO), the N-acetylglucosamine-1-phosphate transferase. Given the dispensable phenotype of tagO (tarO) and the capacity of this deletion for suppression of downstream, essential, late-acting genes, we were motivated to explore the dispensability phenotype of this as-yet-unexplored step of wall teichoic acid synthesis. Here, we analyzed the dispensability of the N-acetylmannosamine transferase genes of both B. subtilis and S. aureus (tagA and tarA, respectively) for growth in vitro.Gene tarA from S. aureus COL was identified as SACOL0693, using BLAST analysis. Dispensability testing of tarA was done in S. aureus strain SA178RI, using an allelic replacement system developed by us (pSAKO) and described previously (10). Using this methodology (see the supplemental material for detailed methods), we demonstrated that in a wild-type background, S. aureus tarA could be readily replaced with an erythromycin resistance cassette, allowing for mutant generation at a high frequency (Table ​(Table1).1). Thus, our data reveal that this locus is dispensable for growth in vitro. In B. subtilis, we were likewise able to replace the tagA gene with a spectinomycin resistance cassette after the generation and transformation of a PCR product containing the flanking regions of tagA surrounding the resistance cassette. To confirm that the deletion of tagA was not the result of a suppressor mutation elsewhere in the chromosome, we performed an analysis of congression to compare the efficiency of recombination of the Spec resistance determinant (replacing tagA) into wild-type B. subtilis to that of the Erm resistance determinant (replacing tagO). We also compared these with that of a control Chl resistance cassette at the amy locus. The frequencies of recombination for all of these experiments were very similar (data not shown). These findings indicated that the loss of tagA was not the result of a concomitant suppressor mutation. The resulting colonies (ΔtagA) were small, smooth, and very similar in morphology to the tagO mutant that we have described previously (9).

TABLE 1.

Allelic replacement for testing gene dispensability in S. aureus

Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.     

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