K17 capsular polysaccharide produced by Acinetobacter baumannii isolate G7 contains an amide of 2-acetamido-2-deoxy-D-galacturonic acid with D-alanine

The K17 capsular polysaccharide (CPS) produced by Acinetobacter baumannii G7, which carries the KL17 configuration at the capsule biosynthesis locus, was isolated and studied by chemical methods along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy. Selective cleavage of the glycosidic linkage of a 2,4-diacetamido-2,4,6-trideoxy-d-glucose (d-QuiNAc4NAc) residue by (i) trifluoroacetic acid solvolysis or (ii) alkaline β-elimination (NaOH-NaBH₄) of the 4-linked D-alanine amide of a 2-acetamido-2-deoxy-d-galacturonic acid residue (d-GalNAcA6DAla) yielded trisaccharides that were isolated by Fractogel TSK HW-40 gel-permeation chromatography and identified by using NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The following structure was established for the trisaccharide repeat (K unit) of the CPS: →4)-α-d-GalpNAcA6dAla-(1→4)-α-d-GalpNAcA-(1→3)-β-d-QuipNAc4NAc-(1→ . The presence of the itrA1 gene coding for the initial glycosylphosphotransferase in the KL17 gene cluster established the first sugar of the K unit as d-QuipNAc4NAc. KL17 includes genes for three transferases that had been annotated previously as glycosyltransferases (Gtrs). As only two Gtrs are required for the K17 structure and one d-GalpNAcA residue is modified by a d-alanine amide, these assignments were re-assessed. One transferase was found to belong to the ATPgrasp_TupA protein family that includes d-alanine-d-alanine ligases, and thus was renamed Alt1 (alanine transferase). Alt1 represents a novel family that amidate the carboxyl group of d-GalpNAcA or d-GalpA.