Lipoteichoic acid polymer length is determined by competition between free starter units

Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (Glc₂DAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstituted Staphylococcus aureus LtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred Glc₂DAG starter unit, yet the use of Glc₂DAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.

All cell surfaces are rich in carbohydrate polymers that act as structural components, scaffolds for other molecules, and participants in signaling processes (1). The biological functions of a carbohydrate polymer are often greatly affected by its length. For example, depending on molecular weight, hyaluronic acid polymers can promote cell migration, differentiation, and inflammation or can inhibit these processes (2, 3). Similarly, the number of repeat units in bacterial O-antigen has a profound effect on complement activation and host cell uptake (4, 5). Unlike protein and nucleic acid polymers, which are assembled on a template that determines both length and composition, carbohydrate polymers are assembled without the use of a template. Template-independent length regulation is not as precise as template-directed polymerization, but physiological lengths of carbohydrate polymers typically fall into a defined range that is important for function (6). How different polymerases achieve length control is a fundamental question in the field.Several mechanisms for carbohydrate polymer length determination have been described. Some polymerases include a “molecular ruler” domain that measures the polymer against a portion of the enzyme (7), some use a dedicated “termination enzyme” to control length (8), and others rely on repeat unit concentration to control polymerization (9). These mechanisms are not mutually exclusive and can act together to control length (10, 11). The degree to which a polymerase is processive also influences product length. Processivity, a fundamental property of polymerases, refers to the number of elongation steps that occur without release of the growing polymer (12). A polymerase may be partially processive, in that more than one monomer addition occurs while the polymer is bound to the enzyme, but the polymer can be released and then rebind to continue elongation. A polymerase may also act in a distributive manner, where the growing polymer is released after each round of monomer addition. While some general mechanisms and aspects of length control for carbohydrate polymerases are known, here we describe a previously unknown mechanism for length regulation of a common type of lipoteichoic acid (LTA), a cell surface polymer that is crucially important to the physiology of most Gram-positive bacteria (13, 14).In the Gram-positive pathogen Staphylococcus aureus (Sa), LTA is a membrane-anchored poly(glycerol-phosphate) polymer involved in virulence (15–19), regulation of cell size and division (20–23), and osmotic stability (24, 25) (Fig. 1A). Sa LTA is assembled by the conserved lipoteichoic acid synthase (LtaS) on the cell surface using glucose(β1,6)-glucose(β1,3)-diacylglycerol (Glc₂DAG) as the membrane-anchored “starter unit” (20, 26). The polymer elongates in a process that involves the repeated transfer of phosphoglycerol units from phosphatidylglycerol (PG) to a catalytic threonine in LtaS (T300) and then to the tip of the growing polymer (Fig. 1B) (27–29). Repeat units may be modified by D-alanyl esters or, less commonly, GlcNAc moieties (24, 30). Because LTA is so important for Sa survival (13, 14, 21, 22), LtaS is a proposed target for antibiotics, and understanding its behavior may facilitate inhibitor development.Open in a separate windowFig. 1.LTA is a lipid-anchored polymer assembled from Glc₂DAG and PG on the bacterial cell surface. (A) Chemical structure of LTA from Sa. Phosphoglycerol repeat units may be modified with D-alanine esters or GlcNAc moieties. (B) Mechanism of LTA synthesis by LtaS. Phosphoglycerol units are transferred from PG to residue T300 to form a covalent intermediate, releasing DAG. Phosphoglycerol is then transferred to a Glc₂DAG starter unit to form GroP-Glc₂DAG. Additional repeat units are added to the glycerol tip of the polymer. (C) In Sa, PgcA and GtaB synthesize UDP-glucose from glucose-6-phosphate. UgtP uses UDP-glucose and DAG to make Glc₂DAG. LtaA exports Glc₂DAG to the cell surface. LtaS transfers phosphoglycerol units derived from PG to T300, releasing DAG for recycling. (D) Anti-LTA Western blot of Sa RN4220 wild-type (wt) or ΔugtP lysates. ΔugtP mutants lack Glc₂DAG, and LTA is instead polymerized directly on PG (20).Glc₂DAG, the starter unit for LTA polymerization, is biosynthesized on the cytoplasmic leaflet of the membrane by the sequential action of three enzymes: the phosphoglucose mutase PgcA, the UTP-glucose-1-phosphate uridylyltransferase GtaB, and the diacylglycerol β-glucosyltransferase UgtP (also called YpfP) (15, 20). Glc₂DAG is exported to the cell surface by the flippase LtaA (Fig. 1C) (15). An interesting feature of LtaS is that it can use PG as an alternative starter unit if Glc₂DAG synthesis or export is blocked (20). However, polymers formed on this alternative starter unit (PG-LTA) are significantly longer than polymers formed on Glc₂DAG (Glc₂DAG-LTA, Fig. 1D) (15, 23), and cells that make these longer polymers have cell division defects (20, 23), are much less virulent (15, 16), and are more sensitive to beta-lactam antibiotics and other cell envelope stresses (23). Whether the shorter polymers assembled on Glc₂DAG reflect the intrinsic behavior of LtaS or the action of other cellular factors is an important question that cannot be definitively answered with genetic approaches.Here we used in vitro reconstitution to test whether the identity of the LTA membrane anchor determines the length of the polymers that LtaS synthesizes. We show that the length differences observed between wild-type and mutant cells lacking Glc₂DAG are recapitulated in a proteoliposome system that contains only purified LtaS, PG, and either Glc₂DAG or an alternative anchor. Based on our studies, we propose a model for how polymer length can be controlled in polymerases that operate without a template.