Bacterial capsules are surface area layers made of long-chain polysaccharides. the

Bacterial capsules are surface area layers made of long-chain polysaccharides. the first full polysaccharide gene cluster cloned and it opened up biochemical and molecular genetic strategies to investigate these and other bacterial glycans. Since then the K1 and K5 systems have been influential prototypes for studying CPS assembly via ABC transporter-dependent pathways (3 4 K1 CPS consists of polysialic acid (PSA) a homopolymer of α-(2→8)-linked sialic acid (NeuAc) and K5 is composed of a heparosan-like glycan made up of glucuronic acid (GlcA) and serogroup B and serogroup A2 (9 10 whereas type D produces a nonsulfated heparosan CPS polymer (11). Biosynthesis of these CPSs occurs at the cytoplasmic (inner) membrane before its export to the periplasm by KU-57788 the system-defining ABC transporter (comprising proteins KpsM and KpsT in BAD nomenclature) (3 4 Translocation of CPS from your periplasm to the cell surface requires the periplasmic and outer-membrane proteins KpsE and KpsD. Jointly KpsMTED are forecasted to create KU-57788 a transenvelope complicated (3 4 12 13 KpsMTED features are not restricted to confirmed CPS repeat-unit framework and one feasible description of their wide substrate specificity may be the presence KU-57788 of the conserved lipid terminus which may be acknowledged by the ABC transporter (3 5 14 15 This lipid continues to be implicated in anchoring CPSs towards the external membrane (16). Mass spectrometry evaluation of acid-hydrolyzed PSA from K1 and K92 aswell as group B discovered dipalmitoylglycerol as an element (17-20). However immediate covalent linkage between your CPS which lipid is not established. As an extra complication tests with K5 CPS recommended a 3-deoxy-d-wild-type strains need cytidine-5′-monophosphate (CMP)-Kdo being a precursor for the biosynthesis of lipopolysaccharide which is vital for viability (22) however the hereditary loci encoding ABC transporter-dependent CPS set up pathways in contain extra copies of KU-57788 genes encoding two from the four enzymes in the CMP-Kdo biosynthesis pathway (3). However the correlation between your duplicated genes as well as the suggested terminal Kdo residue continues to be noted it generally does not represent a unifying feature for everyone bacteria formulated with these CPS set up systems because various other illustrations (e.g. K5 and K1 and group B to ask if they possess the same lipid terminus. The analysis uncovered a distinctive glycolipid terminus conserved in every three bacteria. Results Identification of a Conserved Lipid Terminus. Structural characterization of a lipid terminus and its linkage region is not feasible with heterogeneous preparations made up of high-molecular-mass CPS glycans. As a result prior studies have investigated material released from CPS preparations treated with acid. Although acid hydrolysates yield information on individual components they provide no insight into the linkage. Therefore we developed a strategy that generated highly purified CPS and then reduced the contribution of the CPS with specific endo-acting CPS depolymerases. These glycanases are tail-spike proteins from K1 and K5 CPS-specific bacteriophages (23 24 They rapidly depolymerize purified CPS (Fig. S1) but leave the terminal lipid (and any linker domain) intact and connected to the first few residues of the CPS repeat unit. The hydrophobic products from these enzyme digests were purified and analyzed by mass spectrometry (MS). The liquid chromatography (LC)-MS spectrum of the K1 terminus showed six major species and several minor ones (Fig. 1and Fig. S2). The spectrum for ion A revealed characteristic ions corresponding to Kdo and NeuAc in addition to a major ion at 483 corresponding to the mass of lyso-PG made up of palmitate as the acyl chain. MS/MS/MS of the 483 ion confirmed that it is indeed palmitoyl-phosphatidylglycerol based on the characteristic fragment ions: glycerol2-PO4 (227) and palmitate (255) (Fig. S3). Also detected in the MS/MS spectrum of ion A were ions corresponding to lyso-PG linked to multiple Kdo residues as well as multiple Kdo residues linked to NeuAc identifying a poly-Kdo linker between KU-57788 the PSA glycan and the lipid moiety. The difference between ions A and B lies in the identity of the acyl chain; ion A contains C16:0 whereas B contains C18:1 (Fig. 1and Fig. S2). The same is usually.