Spatial regulation of protein A in Staphylococcus aureus.

Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Phosphoglycerol-type wall and lipoteichoic acids are enantiomeric polymers differentiated by the stereospecific glycerophosphodiesterase GlpQ.

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.

The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by ß-lactam antibiotics or inhibitors of WTA biosynthesis.

ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.

A new antibiotic kills pathogens without detectable resistance.

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

Lipoteichoic Acid Biosynthesis Inhibitors as Potent Inhibitors of S. aureus and E. faecalis Growth and Biofilm Formation.

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) have been deemed as serious threats by the CDC. Many chronic MRSA and VRE infections are due to biofilm formation. Biofilm are considered to be between 10-10,000 times more resistant to antibiotics, and therefore new chemical entities that inhibit and/or eradicate biofilm formation are needed. Teichoic acids, such as lipoteichoic acids (LTAs) and wall teichoic acids (WTAs), play pivotal roles in Gram-positive bacteria's ability to grow, replicate, and form biofilms, making the inhibition of these teichoic acids a promising approach to fight infections by biofilm forming bacteria. Here, we describe the potent biofilm inhibition activity against MRSA and VRE biofilms by two LTA biosynthesis inhibitors HSGN-94 and HSGN-189 with MBICs as low as 0.0625 µg/mL against MRSA biofilms and 0.5 µg/mL against VRE biofilms. Additionally, both HSGN-94 and HSGN-189 were shown to potently synergize with the WTA inhibitor Tunicamycin in inhibiting MRSA and VRE biofilm formation.

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation.

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Role of the msaABCR Operon in Cell Wall Biosynthesis, Autolysis, Integrity, and Antibiotic Resistance in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen in both community and health care settings. One of the challenges with S. aureus as a pathogen is its acquisition of antibiotic resistance. Previously, we showed that deletion of the msaABCR operon reduces cell wall thickness, resulting in decreased resistance to vancomycin in vancomycin-intermediate S. aureus (VISA). In this study, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR mutant cells had decreased cross-linking in both strains. This defect is typically due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased protease activity in mutant cells. The defect was enhanced by a decrease in teichoic acid content in the msaABCR mutant. Therefore, we propose that deletion of the msaABCR operon results in decreased peptidoglycan cross-linking, leading to increased susceptibility toward cell wall-targeting antibiotics, such as ß-lactams and vancomycin. Moreover, we also observed significantly downregulated transcription of early cell wall-synthesizing genes, supporting the finding that msaABCR mutant cells have decreased peptidoglycan synthesis. More specifically, the msaABCR mutant in the USA300 LAC strain (MRSA) showed significantly reduced expression of the murA gene, whereas the msaABCR mutant in the Mu50 strain (VISA) showed significantly reduced expression of glmU, murA, and murD Thus, we conclude that the msaABCR operon controls the balance between cell wall synthesis and cell wall hydrolysis, which is required for maintaining a robust cell wall and acquiring resistance to cell wall-targeting antibiotics, such as vancomycin and the ß-lactams.

Lipoteichoic acid synthesis and function in gram-positive bacteria.

Lipoteichoic acid (LTA) is an important cell wall polymer found in gram-positive bacteria. Although the exact role of LTA is unknown, mutants display significant growth and physiological defects. Additionally, modification of the LTA backbone structure can provide protection against cationic antimicrobial peptides. This review provides an overview of the different LTA types and their chemical structures and synthesis pathways. The occurrence and mechanisms of LTA modifications with D-alanyl, glycosyl, and phosphocholine residues will be discussed along with their functions. Similarities between the production of type I LTA and osmoregulated periplasmic glucans in gram-negative bacteria are highlighted, indicating that LTA should perhaps be compared to these polymers rather than lipopolysaccharide, as is presently the case. Lastly, current efforts to use LTAs as vaccine candidates, synthesis proteins as novel antimicrobial targets, and LTA mutant strains as improved probiotics are highlighted.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.

Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

Wall teichoic acids of gram-positive bacteria.

The peptidoglycan layers of many gram-positive bacteria are densely functionalized with anionic glycopolymers known as wall teichoic acids (WTAs). These polymers play crucial roles in cell shape determination, regulation of cell division, and other fundamental aspects of gram-positive bacterial physiology. Additionally, WTAs are important in pathogenesis and play key roles in antibiotic resistance. We provide an overview of WTA structure and biosynthesis, review recent studies on the biological roles of these polymers, and highlight remaining questions. We also discuss prospects for exploiting WTA biosynthesis as a target for new therapies to overcome resistant infections.

Membrane Translocation and Assembly of Sugar Polymer Precursors.

Bacterial polysaccharides play an essential role in cell viability, virulence, and evasion of host defenses. Although the polysaccharides themselves are highly diverse, the pathways by which bacteria synthesize these essential polymers are conserved in both Gram-negative and Gram-positive organisms. By utilizing a lipid linker, a series of glycosyltransferases and integral membrane proteins act in concert to synthesize capsular polysaccharide, teichoic acid, and teichuronic acid. The pathways used to produce these molecules are the Wzx/Wzy-dependent, the ABC-transporter-dependent, and the synthase-dependent pathways. This chapter will cover the initiation, synthesis of the various polysaccharides on the cytoplasmic face of the membrane using nucleotide sugar precursors, and export of the nascent chain from the cytoplasm to the extracellular milieu. As microbial glycobiology is an emerging field in Gram-positive bacteria research, parallels will be drawn to the more widely studied polysaccharide biosynthesis systems in Gram-negative species in order to provide greater understanding of these biologically significant molecules.

Envelope Structures of Gram-Positive Bacteria.

Gram-positive organisms, including the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, have dynamic cell envelopes that mediate interactions with the environment and serve as the first line of defense against toxic molecules. Major components of the cell envelope include peptidoglycan (PG), which is a well-established target for antibiotics, teichoic acids (TAs), capsular polysaccharides (CPS), surface proteins, and phospholipids. These components can undergo modification to promote pathogenesis, decrease susceptibility to antibiotics and host immune defenses, and enhance survival in hostile environments. This chapter will cover the structure, biosynthesis, and important functions of major cell envelope components in gram-positive bacteria. Possible targets for new antimicrobials will be noted.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

Dissecting the regulation of bile-induced biofilm formation in Staphylococcus aureus.

Aspiration of bile into the cystic fibrosis (CF) lung has emerged as a prognostic factor for reduced microbial lung biodiversity and the establishment of often fatal, chronic pathogen infections. Staphylococcus aureus is one of the earliest pathogens detected in the lungs of children with CF, and once established as a chronic infection, strategies for its eradication become limited. Several lung pathogens are stimulated to produce biofilms in vitro in the presence of bile. In this study, we further investigated the effects of bile on S. aureus biofilm formation. Most clinical S. aureus strains and the laboratory strain RN4220 were stimulated to form biofilms with sub-inhibitory concentrations of bovine bile. Additionally, we observed bile-induced sensitivity to aminoglycosides, which we exploited in a bursa aurealis transposon screen to isolate mutants reduced in aminoglycoside sensitivity and augmented in bile-induced biofilm formation. We identified five mutants that exhibited hypersensitivity to bile with respect to bile-induced biofilm formation, three of which carried transposon insertions within gene clusters involved in wall teichoic acid (WTA) biosynthesis or transport. Strain TM4 carried an insertion between the divergently oriented tagH and tagG genes, which encode the putative WTA membrane translocation apparatus. Ectopic expression of tagG in TM4 restored a wild-type bile-induced biofilm response, suggesting that reduced translocation of WTA in TM4 induced sensitivity to bile and enhanced the bile-induced biofilm formation response. We propose that WTA may be important for protecting S. aureus against exposure to bile and that bile-induced biofilm formation may be an evolved response to protect cells from bile-induced cell lysis.

Benzimidazole analogs as WTA biosynthesis inhibitors targeting methicillin resistant Staphylococcus aureus.

A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria.

The current epidemic of infections caused by antibiotic-resistant gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.

Structural and mechanistic insight into the Listeria monocytogenes two-enzyme lipoteichoic acid synthesis system.

Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaP(Lm) initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaS(Lm) extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaP(Lm) and LtaS(Lm). Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaS(Lm) structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain.