Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units

Abstract

The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective a or b configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzydependent pathway is responsible for much of the diversity.

Progress in Our Understanding of Wzx Flippase for Translocation of Bacterial Membrane Lipid-Linked Oligosaccharide

“…This was confirmed in an S. enterica group C2 mutant that produces an O unit lacking a side-branch Abe and experiences lethality, where the growth defect was shown to be abolished when translocation was improved by overexpression of the wzx gene (49). It should be noted that Xayarath and Yother (64) reported lethal mutations in the capsule Wzy polymerase, although this is not the case for O-antigen synthesis (57,68,69).…”

“…Deleterious O-antigen mutants can be maintained in strains in which O-antigen synthesis is controlled at or before the reaction that commits the product to O-unit synthesis, allowing cultures to grow without difficulty until O-unit synthesis is initiated. This approach was pioneered by Osborn et al, who utilized an S. enterica group B1 ΔgalE mutant in several studies to act as a parental stain that suppressed O-antigen production prior to the addition of exogenous Gal (47,68,69). Several derivatives of this parental strain with deletions of O-antigen genes (68) have been shown to stop growing soon after activation of O-antigen synthesis and exhibit clear signs of cell lysis within 1 h. Interestingly, it appears that mutations affecting cytoplasmic RU assembly (Δabe and ΔwbaV) and flipping (Δwzx) are lethal, whereas those that affect synthesis after the RU is flipped to the periplasmic face (Δwzy and ΔwaaL) have no effect on growth (68).…”

“…If so, it is perhaps Wzx that represents the key to the extent of diversity in prokaryote surface polysaccharides. The diversity in Wzy is easily accounted for, as Wzy is effectively a GT that makes a typical glycosyl linkage but differs from a GT in using an RU on Und-PP as the donor instead of a sugar on a nucleotide triphosphate (69). There is no obvious reason why Wzy cannot be as diverse as typical GTs.…”

Whole-Genome Sequencing of Lactobacillus helveticus D75 and D76 Confirms Safety and Probiotic Potential

“…The gene is not part of the genetic cluster for the synthesis of exopolysaccharides, but plays a key role in their synthesis along the WZX-WZY-dependent pathway. Flippase transporters transfer polysaccharide precursor molecules from the cytoplasmic space to the external pre-membrane region [37][38][39]. To synthesise exopolysaccharides via the WZX-WZY-dependent pathway, the wzy gene is also required, which encodes a polysaccharide polymerase protein that, together with the polysaccharide co-polymerase protein (encoded by the wzz gene), polymerises the growing chain of the exopolysaccharide.…”

Attenuated Salmonella Typhimurium expressing Salmonella Paratyphoid A O-antigen induces protective immune responses against two Salmonella strains

“…However, Wzx in fact exhibits a clear, and often strong, preference for its native substrate beyond the first sugar [23][24][25]. Furthermore, it is assumed that the diversity of the wzy sequence reflects both the variation in adjacent O-unit linkage, which is synthesized by Wzy, and the variation in other parts of the acceptor O-unit [26].…”