Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control

Abstract

Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-β-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a β-linked Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.microbial glycobiology | lipopolysaccharides | glycosyltransferases | glycan biosynthesis | molecular ruler B acterial surfaces possess an array of complex glycoconjugates (sugar-containing macromolecules) that play varied and vital roles in the biology of these organisms. In pathogens, glycoconjugates participate in adhesion, biofilm formation, and interaction with innate and adaptive immune responses. For example, in gram-negative bacteria such as Escherichia coli, long glycan chains in capsular polysaccharides and the O-antigen polysaccharide (OPS) components of lipopolysaccharide (LPS) molecules typically confer resistance to opsonophagocytosis and complement-mediated killing (1, 2). Effective protection depends on the amount and surface distribution of glycan, as well as glycan chain lengths suited for a given purpose. As an example, shorter OPS chains offer no resistance to complementmediated killing, whereas chains longer than an optimal size potentially represent an unnecessary energy cost while offering no additional advantage (3, 4). Despite its fundamental importance in colonization and virulence of bacterial pathogens, the molecular mechanisms of glycan chain-length regulation are often poorly understood (5). However, OPS biosynthesis provides influential prototypes for understanding the guiding principles underpinning chainlength regulatory mechanisms in bacterial glycoconjugates in general.The LPS glycolipid is a major component of the outer membrane of gram-negative bac...

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility

“…Many polysaccharides, including the galactan, are assembled in the cytoplasm and then transported into the extracellular space; therefore, export and chain length control are tightly coupled ( 25 , 26 ). Our ability to isolate a mycobacterial strain with a truncated galactan indicates that there is flexibility in chain length required for transport.…”

Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism

“…High conservation of nucleotide sequence with other L. johnsonii strains and the presence of analogous genes in other lactobacilli suggest that this might be part of a novel mechanism for homopolysaccharide EPS biosynthesis in Gram-positive bacteria. EPS/O-PS biosynthetic pathways have been studied in detail, but many questions remain unanswered, and new enzymes are still being discovered (43). Given the potential technological applications of EPS, there is significant interest in engineering novel forms (32), and their important roles in protection and biofilm formation make EPS biosynthesis a valid target for novel strategies to control pathogens.…”

Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification

“…Additionally, the O12 and O4 serotypes possess single terminal 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues on repeat-unit structures that do not contain Kdo (38,42). The addition of the terminal residue(s) is determined by a molecular ruler in the terminating enzyme to ensure OPS chain lengths fall within a discrete range (43,44). Only properly terminated OPSs are exported due to the essential participation of a carbohydrate-binding module attached to the ABC transporter nucleotide-binding domain, which recognizes the terminal modification (45,46).…”

“…O1 uses a dual-GT polymerase (WbbY), which is common for these systems. For example, multidomain polymerases are recognized for O8 and O9 (50) and in O12 (where the polymerase and terminating enzymes are combined in one protein) (43). In contrast, O2c requires two independent GTs, which seem to require interactions for proper function.…”