Bacterial outer membrane polysaccharides play key roles in a range of bacterial activities from homeostasis to virulence. Two such OM polysaccharide populations are ECA and LPS Oag, which are synthesized by separate homologs of the Wzy-dependent pathway.

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales. In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyESF’s periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.

ECA has been shown to play major roles in the homeostasis of cells as mutants along its biosynthetic pathway, particularly its glycosyltransferases have been shown to induce pleiotropic phenotypes as well as induce cell wall stress pathways (Castelli & Vescovi, 2011;Jorgenson et al., 2016). Due to this, research into the roles that the ECA glycosyltransferases play in these pleiotropic phenotypes has dominated ECA research with little research being performed in understanding the enzymes themselves. WecG research has not progressed since 1988 when it was first identified by Barr et al. who described WecG as a membrane protein. Here, we show that WecG is a peripheral membrane protein, maintained to the IM via interactions facilitated by its C-terminal tail. We describe a new model of how WecG is maintained to the IM and present WecG as the second protein in the novel glycosyltransferase-fold family, GT-E. | INTRODUC TI ONEnterobacterales, an order of bacteria belonging to Gram-negatives, have developed unique adaptations which allow them to persist and