== Bacteriophage K1F-sensitivity assays and immunoblotting were performed as previously described (2,35). of the CPS via multiple residues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), referred to as a poly-Kdo linker. The Kdo residues are -linked, suggesting that they are synthesized by retaining glycosyltransferases. To date, the only characterized Kdo transferases are the inverting enzymes that catalyze the -linkages found in lipopolysaccharide. Here, we identify two conserved proteins from CPS assembly systems, KpsC and KpsS, as the -Kdo-transferases and demonstrate in vitro reconstitution of poly-Kdo linker assembly on BI-409306 a fluorescent phosphatidylglycerol acceptor. KpsS adds the first Kdo residue, and this reaction product is then extended by KpsC. Cross-complementation BI-409306 experiments demonstrate that theE. coliandN. meningitidisprotein homologs are functionally conserved. Many bacterial pathogens possess capsules, which are comprised of high-molecular-mass (>100,000 Da) polysaccharides, known as capsular polysaccharides (CPSs) (1,2). Capsules represent a highly hydrated surface layer, conferring protection against host defenses, primarily phagocytosis. In Gram-negative bacteria, most CPSs are assembled by one of two widely distributed systems: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways (1).Escherichia coliCPSs formed by the Wzy-dependent pathway have been called group 1, distinguishing them from the group 2 ABC transporter-dependent CPSs. The ABC transporter-dependent pathway is the focus of this study. TheE. coliK1 system provides an influential model for the ABC transporter-dependent pathway. K1 is one of more than 80 K (CPS) serotypes inE. coli(3), and its repeat-unit structure is composed of -2,8-linkedN-acetylneuraminic acid (polysialic acid; PSA) (4). The genetic locus for K1 biosynthesis and assembly was the first polysaccharide biosynthesis gene cluster cloned and expressed inE. coli, and it paved the way for understanding of this system and other systems (5). The K1 locus encodes serotype-specific proteins that synthesize the K1 repeat-unit glycan and several conserved (serotype-independent) proteins found in allE. coligroup 2 capsule loci. Most of the serotype-independent proteins are also found in other bacteria with CPSs synthesized by this pathway. Examples includeCampylobacter jejuni,Haemophilus influenzae,Neisseria meningitidisandPasteurella multocida(2). Four of the conserved proteins are involved in the transport of CPS from the cytoplasm, where it is synthesized, to the cell surface (reviewed in refs.2and68). These proteins include the system-defining ABC transporter (KpsMT inE. colinomenclature), an inner-membrane polysaccharide copolymerase (PCP-3) protein, designated KpsE, and KpsD, and an outer-membrane polysaccharide export (OPX) protein. Together, Rabbit polyclonal to KAP1 the KpsMTED proteins are thought to form a transenvelope complex analogous to the one proposed for tripartite efflux pumps (1,2,810). We recently reported that CPSs fromE. coliandN. meningitidiscontain the same reducing terminal BI-409306 (lyso)phosphatidylglycerol moiety, which is attached to the CPS via a poly-Kdo linker (11). The poly-Kdo linker is proposed to be a unifying feature of CPSs synthesized via BI-409306 the ABC transporter-dependent pathway (11). It has long been known that the ABC transporter proteins display no specificity for the CPS repeat unit (1,12,13) and that the conserved reducing terminal glycolipid provides an attractive candidate for an export signal. Although the enzymes and processes involved in biosynthesis of the poly-Kdo linker are unknown, the linker consists of BI-409306 -linked Kdo, and the donor sugar is likely CMP–Kdo (14), so the corresponding glycosyltransferase enzyme(s) are predicted to be retaining Kdo transferases (2). All Kdo transferases characterized to date are inverting enzymes that add -linked Kdo (or Kdo derivatives) to the inner core region of all lipopolysaccharides (LPSs) (15). The highly conserved WaaA -Kdo transferase provides the best-characterized example (16). In contrast, -Kdo is relatively rare. It has been found in nature as part of the repeat units of some LPS O antigens inProvidencia alcalifaciensand as the nonreducing chain terminating residue in the O12 antigen fromKlebsiella pneumoniae(17,18). It has also been identified in CPS repeat units fromE. coliserotype K12 (19),N. meningitidisgroup E (20), andActinobacillus pleuropneumoniae5a and 5b (21,22). However, the -Kdo transferases required for synthesis of these structures have not been identified. The genetic loci for model ABC transporter-dependent CPSs encode two additional conserved (serotype-independent) proteins, designated KpsC and KpsS inE. coli(23) andC. jejuni(24), HcsA and HcsB inH. influenzae(25), LipA and LipB inN. meningitidis(26), and PhyA and PhyB inP. multocida(27). The roles of these proteins in CPS assembly have been debated. InE. coliK1 and K5 andN. meningitidisgroup B, mutations in eitherkpsCorkpsS(or their homologs) result in intracellular accumulation of CPS (2830). This CPS is not.