A. baumannii is a Gram-negative opportunistic bacteria that is listed as a critical-priority pathogen by the World Health Organisation due to the emergence of strains that are resistant to all commercially available antibiotics. The CPS of A. baumannii is a major virulence factor and a number of strains have been shown to incorporate Pse glycans into their capsule, including highly virulent A. baumannii strain ACICU, which bears a K2 type CPS repeat unit 4 (Figure ), reportedly encoded within >10% of deposited A. baumannii genomes. NMR characterisation of 4 had previously identified the presence of a terminal Pse5Ac7Ac attached to a glucopyranoside through an a(2,6)-linkage. This axial a-Pse5Ac7Ac glycosidic linkage is in contrast to the presentation of Neu5Ac 2 in nature, which exists exclusively linked to other glycans through an equatorial a-linkage (note for Pse5Ac7Ac glycosides: a = axial and b = equatorial linkages, whilst for Neu5Ac glycosides: a = equatorial and b = axial linkages). The enzymatic construction of this a-Pse5Ac7Ac linkage would therefore require a PseT that proceeds with a retention of anomeric configuration if using a-CMP-Pse5Ac7Ac 3 as a donor. Analysis of the A. baumannii ACICU KL2 biosynthetic locus reveals the presence of a cluster of six genes (pseBCFGHI) responsible for the biosynthesis of a-CMP-Pse5Ac7Ac 3 (Figure ). In addition, downstream from this cluster of genes, a number of GTs are encoded that are likely responsible for the enzymatic assembly of the CPS trisaccharide core in the repeat unit, and an as yet uncharacterised kpsS1 gene. Although no retaining sialyltransferases that utilise a Neu5Ac substrate have been discovered to date, KpsS1 shares 30% sequence identity to Escherichia coli KpsS, which was recently characterised as the first retaining b-Kdo (3-deoxy-dmanno-oct-2-ulosonic acid) transferase, and a member of the GT107 family of carbohydrate active enzymes. It was therefore tentatively predicted that KpsS1 may be responsible for construction of the Pse5Ac7Ac-a(2,6)-Glcp linkage in 4, and act as a dedicated retaining PseT. We set out to test this proposed function through expression and in vitro characterisation of the enzyme.