The O antigen (O polysaccharide), composed of many oligosaccharide repeats (O units), is a part of the lipopolysaccharide (LPS) of Gram-negative bacteria and the most structurally variable cell surface constituent. The O-antigen diversity is due to variations of O-antigen biosynthesis genes and is believed to offer various bacterial clones selective advantages in their specific ecological niches (1). The O antigen plays important and various roles in bacteriophage interactions with the host. Many bacteriophages employ the O antigen as a primary receptor that ensures reversible adsorption to the host cell followed by irreversible adsorption to a secondary receptor, most frequently an outer membrane protein (2-4). O-antigen modifications may prevent bacteriophage binding. For instance, phage SPC35 uses the Salmonella O12 antigen receptor, and phase-variable glucosylation of the O antigen confers transient SPC35 resistance to the bacteria (5). A temperate podovirus, Sf6, also uses O antigen of its host, Shigella flexneri, as a primary receptor (4). Interestingly, the Sf6 genome harbors the oac gene for O-antigen acetylase that causes O-serotype conversion of Sf6 lysogens, which precludes bacteriophage Sf6 adsorption to these cells (6). On the other hand, the O-antigen-carrying LPS of E. coli is able to prevent the access of phages and colicins to their outer membrane protein receptors, which are otherwise sufficient for a successful attack of the cell (7). O-antigen deficiency also enhances the sensitivity of E. coli to Shiga toxin 2-converting bacteriophages (3,8). A phage T5 mutant lacking L-shaped tail fibers that recognize polymannose O antigens showed a reduced rate of adsorption to the O-antigen-producing hosts but infected O-antigen-less strains as efficiently as the wild-type phage (9, 10).These data indicate that the O-antigen layer represents an effective shield that nonspecifically protects the bacteria from interactions of bacteriophages with their cell surface receptors. In order to penetrate this shield, the phages need to acquire the proteins that specifically recognize the O antigen, thus becoming dependent on a given O-polysaccharide type. Many bacteriophages that use the O antigen as a primary receptor possess enzymes that degrade or modify it (11, 12).N4-like bacteriophage G7C and its host E. coli 4s were isolated from horse feces in the course of an investigation of coliphage ecology in the equine gut ecosystem (13). In addition to G7C, E. coli 4s was used as a host for the isolation and propagation of several other G7C-related phages (14; A. K. Golomidova, unpublished data). Currently, E. coli 4s remains the only known host for bacteriophage G7C, but despite the extremely narrow host range, G7C-related phages persisted in the same horse population for several years (14). The mechanisms that help G7C avoid extinction despite the small fraction of the total E. coli population that is suitable for its growth are poorly understood (9). Elucidation of the molecular details of the initial steps of th...

is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with because genes outside the main (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Gal-(1→3)-β-d-Gal-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Gal side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-β-d-Gal-(1→3)-β-d-GlcNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in O-antigen carbohydrate structures based on the O2a backbone.

A poor understanding of the mechanisms by which antibiotics traverse the outer membrane remains a considerable obstacle to the development of novel Gram-negative antibiotics. Herein, we demonstrate that the Gram-negative bacterium Escherichia coli becomes susceptible to the narrow-spectrum antibiotic vancomycin during growth at low temperatures. Heterologous expression of an Enterococcus vanHBX vancomycin resistance cluster in E. coli confirmed that the mechanism of action was through inhibition of peptidoglycan biosynthesis. To understand the nature of vancomycin permeability, we screened for strains of E. coli that displayed resistance to vancomycin at low temperature. Surprisingly, we observed that mutations in outer membrane biosynthesis suppressed vancomycin activity. Subsequent chemical analysis of lipopolysaccharide from vancomycin-sensitive and -resistant strains confirmed that suppression was correlated with truncations in the core oligosaccharide of lipopolysaccharide. These unexpected observations challenge the current understanding of outer membrane permeability, and provide new chemical insights into the susceptibility of E. coli to glycopeptide antibiotics.

Polysaccharide capsules are surface structures that are critical for the virulence of many Gram-negative pathogenic bacteria. Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. It produces a capsular polysaccharide known as “Vi antigen,” which is composed of nonstoichiometrically O-acetylated α-1,4-linked N-acetylgalactosaminuronic acid residues. This glycan is a component of currently available vaccines. The genetic locus for Vi antigen production is also present in soil bacteria belonging to the genus Achromobacter. Vi antigen assembly follows a widespread general strategy with a characteristic glycan export step involving an ATP-binding cassette transporter. However, Vi antigen producers lack the enzymes that build the conserved terminal glycolipid characterizing other capsules using this method. Achromobacter species possess a Vi antigen-specific depolymerase enzyme missing in S. enterica Typhi, and we exploited this enzyme to isolate acylated Vi antigen termini. Mass spectrometry analysis revealed a reducing terminal N-acetylhexosamine residue modified with two β-hydroxyl acyl chains. This terminal structure resembles one half of lipid A, the hydrophobic portion of bacterial lipopolysaccharides. The VexE protein encoded in the Vi antigen biosynthesis locus shares similarity with LpxL, an acyltransferase from lipid A biosynthesis. In the absence of VexE, Vi antigen is produced, but its physical properties are altered, its export is impaired, and a Vi capsule structure is not assembled on the cell surface. The structure of the lipidated terminus dictates a unique assembly mechanism and has potential implications in pathogenesis and vaccine production.

Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of β-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. β-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the β-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype β-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena. The β-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/β domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities. microbial glycobiology | 3-deoxy-D-manno-oct-2-ulosonic acid | Kdo | glycosyltransferase | polysaccharide