With crude Im9-KpsS1 lysate in hand we undertook preliminary activity screens using the predicted a-CMP-Pse5Ac7Ac donor 3, and a series of glycosyl acceptors (Supplementary Figure ). Reaction samples were then subjected to negative ion ESI LC-MS analysis to determine if any Pse5Ac7Ac glycoside products had formed. Considering the structure of the A. baumannii CPS repeat unit 4, we anticipated KpsS1 might utilise β-D-glucopyranoside structures as substrates, and therefore initially screened the simple Me-β-D-Glcp as an acceptor. However, under the reaction conditions screened no turnover of the donor 3 was observed. It has been reported that GTs in A. baumannii gene clusters are often distributed in inverse order to function. As kpsS1 is the first GT in the KL2 gene cluster, upstream from the remaining putative GTs, it therefore likely uses a di, tri, or tetrasaccharide acceptor in vivo. So next we screened para-nitrophenyl (PNP)-β-D-Glcp 5 as an acceptor, in the hope that the aryl aglycone would occupy the region of the enzyme active site reserved for binding to a larger acceptor. Pleasingly, after 18 h, all CMP-donor 3 was consumed and a peak for the expected disaccharide product was observed in LCMS. To test enzyme specificity towards the Glcp in the acceptor, PNP-β-D-Xylp 6 which lacks the nucleophilic 6-OH group present in Glcp was also screened and unsurprisingly no turnover was observed, reaffirming the hypothesis that KpsS1 is a PseT that forms a linkage to the 6-OH of Glcp in vivo. We also sought to determine whether the enzyme could utilise the alternative b-CMP glycosyl donor of Neu5Ac 2. Under otherwise identical conditions using PNP-b-D-Glcp 6 as an acceptor and b-CMP-Neu5Ac as a donor, no turnover to product was catalysed by the enzyme (Supplementary Figure ). An internal competition reaction between a-CMP-Pse5Ac7Ac 3 and b-CMP-Neu5Ac for the acceptor 6 under the same conditions also yielded only Pse5Ac7Ac-Glcp disaccharide, with the b-CMP-Neu5Ac donor remaining.