Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry inEscherichia coliO9a

Abstract

The lengths of bacterial polysaccharides can be critical for their biological function. Unlike DNA or protein synthesis, where polymer length is implicit in the nucleic acid template, the molecular mechanisms for regulating polysaccharide length are poorly understood. Two models are commonly cited: a "molecular clock" regulates length by controlling the duration of the polymer extension process, whereas a "molecular ruler" determines length by measurement against a physical structure in the biosynthetic complex. Escherichia coli O9a is a prototype for the biosynthesis of O polysaccharides by ATP-binding cassette transporter-dependent processes. The length of the O9a polysaccharide is determined by two proteins: an extension enzyme, WbdA, and a termination enzyme, WbdD. WbdD is known to self-oligomerize and also to interact with WbdA. Changing either enzyme's concentration can alter the polysaccharide length. We quantified the O9a polysaccharide length distribution and the enzyme concentration dependence in vivo, then made mathematical models to predict the polymer length distributions resulting from hypothetical length-regulation mechanisms. Our data show qualitative features that cannot be explained by either a molecular clock or a molecular ruler model. Therefore, we propose a "variable geometry" model, in which a postulated biosynthetic WbdA-WbdD complex assembles with variable stoichiometry dependent on relative enzyme concentration. Each stoichiometry produces polymers with a distinct, geometrically determined, modal length. This model reproduces the enzyme concentration dependence and modality of the observed polysaccharide length distributions. Our work highlights limitations of previous models and provides new insight into the mechanisms of length control in polysaccharide biosynthesis.chain length | mathematical modeling | template-independent polymerization W hereas nucleic acids and proteins are synthesized from a template of specified length, carbohydrates are constructed template-free, implying that the mechanisms for length control must lie in the biosynthetic machinery itself.Lipopolysaccharide (LPS) is a glycolipid which constitutes the major and characteristic component of the outer leaflet of the outer membrane of most Gram negative bacterial cell envelopes (reviewed in ref. 1). Most bacteria produce LPS molecules that contain a conserved lipid A moiety linked to a short (core) oligosaccharide. In many species, a proportion of LPS molecules have an additional domain known as O polysaccharide (OPS), O antigen or O chain. OPS is a carbohydrate polymer in which the repeating unit (O unit) consists of a short oligosaccharide or sometimes a single sugar. OPS is synthesized template-free by glycosyltransferases and is found in lengths that vary from one repeat unit to several hundred sugars.The biological roles of OPS vary from species to species, but for bacterial pathogens, it commonly confers resistance to the bactericidal effects of host complement. The mechanisms of resistance can vary, so that ...

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism

“…the ABC transporter-dependent pathway (56,57). What is clear from NMR spectra is that the recombinant system in E. coli lacks the phase variation that results in a combination of modified and unmodified chains in some Salmonella isolates (8,55).…”

The Klebsiella pneumoniae O12 ATP-binding Cassette (ABC) Transporter Recognizes the Terminal Residue of Its O-antigen Polysaccharide Substrate

“…WbdD recruits the O-PS polymerase, WbdA, to active biosynthesis complexes in the membrane (12). The termination reaction occurs when the chain reaches a certain length and is affected by the stoichiometry of the WbdD-WbdA complex (13). In addition, WbdD possesses an extended coiled-coil region separating its membrane-anchoring amphipathic helix from the catalytic domains; this serves as the molecular ruler to fine-tune glycan chain length (11,14).…”

“…However, there is currently no evidence for a conserved mechanism. To address this, we investigated export of an O-PS structure composed of a disaccharide repeat unit [34)-␣-L-Rha-(133)-D-GlcNAc- (13] produced by Raoultella terrigena ATCC 33257 and K. pneumoniae O12 (Fig. 1) (24,25).…”

Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

“…A reduction in the amount of WbdA may explain the slight downward shift in the size distribution of the ␣-(132)-polymannose glycans they generate (Fig. 3A) because alterations in stoichiometry of WbdA:WbdD are known to effect chain length (14,20). …”

“…The WbdD terminator plays an additional pivotal structural role in recruiting WbdA to the membrane (19). The stoichiometry of WbdA:WbdD in active complexes is a critical factor in establishing the chain length distribution of the resulting glycans, and the "variable geometry" model has been developed to explain this process (20). The key element in the model is that complexes with different stoichiometries have different physical sizes that generate a range of glycan chain lengths.…”