Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved -Kdo oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two CMP-Kdodependent glycosyltransferase catalytic centres with different linkage specificities. The structure of the Nterminal -(2→4)-Kdo transferase from KpsC reveals two α/β domains, supplemented by several additional helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared to canonical GT-B folds in glycosyltransferases. The similar structure of the Cterminal -(2→7)-Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 -Kdo transferases. This enzyme provides the prototype for the GT107 family. repeat-unit glycan, before the completed molecule enters the export pathway via the assembly strategy-defining ATP-binding cassette (ABC) transporter. Activities of KpsC and its individual GT modules were established using synthetic Kdo mono-and disaccharide acceptors attached to aromatic tags 12 . However, the precise structure of the natural acceptor (KpsS product) has not been determined and its reactivity with KpsC GT modules remains unknown.KpsC is a potential target for small-molecule therapeutics development due to its pivotal role in assembling an essential virulence determinant in high-profile encapsulated pathogens, and the absence of β-Kdo in mammals and in many bacterial commensals. Understanding the structure and function of KpsC is an important step in this strategy. The objectives of this study were to establish a definitive structure for the natural glycolipid acceptor for CPS polymerization and elucidate the sequence of addition of Kdo residues in its biogenesis. Here, we also characterize single GT module KpsC homologs from the Gram-negative thermophile, Thermosulfurimonas dismutans. This provided an essential resource to solve enzyme structures from both E. coli and T. dismutans, identifying in a new GT family. ResultsBioinformatic and functional investigation of KpsC. Functional characterization of the E. coli KpsC enzyme is challenging, due to the instability of full-length protein and most truncated derivatives 11,12 . This led us to investigate KpsC homologs, with an emphasis on thermophiles anticipating that these would have improved stability. Using BLAST, we identified candidates in the Gram-negative thermophile, T. dismutans (Supplementary Fig. 1a,b). While most KpsC proteins have dual GT modules, T. dismutans produces two smaller proteins (318 and 348 amino acid residues). Sequence similarity indicates these are homologs of the individual GT modul...

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is an essential component of bacterial lipopolysaccharides, where it provides the linkage between lipid and carbohydrate moieties. In all known LPS structures, Kdo residues possess ␣-anomeric configurations, and the corresponding inverting ␣-Kdo transferases are well characterized. Recently, it has been shown that a large group of capsular polysaccharides from Gram-negative bacteria, produced by ATP-binding cassette transporter-dependent pathways, are also attached to a lipid anchor through a conserved Kdo oligosaccharide. In the study reported here, the structure of this Kdo linker was determined by NMR spectroscopy, revealing alternating ␤-(234)-and ␤-(237)-linked Kdo residues. KpsC contains two retaining ␤-Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the ␤-Kdo linker on its glycolipid acceptor. Full-length Escherichia coli KpsC was expressed and purified, together with the isolated N-terminal domain and a mutant protein (KpsC D160A) containing a catalytically inactivated N-terminal domain. The Kdo transferase activities of these proteins were determined in vitro using synthetic acceptors, and the reaction products were characterized using TLC, mass spectrometry, and NMR spectroscopy. The N-and C-terminal domains were found to catalyze formation of ␤-(234) and ␤-(237) linkages, respectively. Based on phylogenetic analyses, we propose the linkage specificities of the glycosyltransferase domains are conserved in KpsC homologs from other bacterial species.Pathogenic bacteria are often covered by a protective layer of polysaccharide known as the capsule. Capsules are important virulence determinants because, depending on the pathogen, they can protect bacteria against phagocytosis and complement-mediated killing, as well as helping in adherence to host cells (1). In Gram-negative bacteria, capsular polysaccharides (CPS) 4 are synthesized and exported onto the surface of the outer membrane via one of two widely disseminated and fundamentally different assembly systems (1, 2). The ATP-binding cassette (ABC) transporter-dependent system is the focus of this study. In the nomenclature of Escherichia coli, these systems generate "group 2" capsules (1, 2). In group 2 CPS assembly, biosynthesis is initiated by KpsS, which transfers a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue from its precursor (CMP-␤-Kdo) to (lyso)phosphatidylglycerol, in a reaction located at the cytoplasm-membrane interface (1). The KpsS product is then extended by KpsC to form the ␤-Kdo linker, which typically consists of five to nine Kdo residues (3). The serotype-specific glycosyltransferases (GTs) extend this linker to complete polymerization of CPS in the cytoplasm before it is exported by the pathway-defining ABC transporter and then translocated to the cell surface (1, 2).The native lipid-linked ␤-Kdo-oligosaccharides were isolated from E. coli K1 and K5 and Neisseria meningitidis group B by enzymatic depolymerization of high molecular ma...

BackgroundThe genus pestivirus within the family Flaviviridae includes bovine viral diarrhoea virus (BVDV) types 1 and 2, border disease virus (BDV) and classical swine fever virus. The two recognised genotypes of BVDV are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2.MethodsThree studies were conducted to investigate the phylogenetic diversity of pestiviruses present in Northern Ireland. Firstly, pestiviruses in 152 serum samples that had previously tested positive for BVDV between 1999 and 2008 were genotyped with a RT-PCR assay. Secondly, the genetic heterogeneity of pestiviruses from 91 serum samples collected between 2008 and 2011 was investigated by phylogenetic analysis of a 288 base pair portion of the 5’ untranslated region (UTR). Finally, blood samples from 839 bovine and 4,437 ovine animals imported in 2010 and 2011 were tested for pestiviral RNA. Analysis of animal movement data alongside the phylogenetic analysis of the strains was carried out to identify any links between isolates and animal movement.ResultsNo BVDV-2 strains were detected. All of the 152 samples in the first study were genotyped as BVDV-1. Phylogenetic analysis indicated that the predominant subtype circulating was BVDV-1a (86 samples out of 91). The remaining five samples clustered close to reference strains in subtype BVDV-1b. Out of the imported animals, 18 bovine samples tested positive and 8 inconclusive (Ct ≥36), while all ovine samples were negative. Eight sequences were obtained and were defined as BVDV-1b. Analysis of movement data between herds failed to find links between herds where BVDV-1b was detected.ConclusionGiven that only BVDV-1a was detected in samples collected between 1968 and 1999, this study suggests that at least one new subtype has been introduced to Northern Ireland between 1999 and 2011 and highlights the potential for importation of cattle to introduce new strains.

The 16S ribosomal RNA gene from the beer-spoilage organism, Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position of M. cerevisiae as a sister taxon of Megasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.

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