Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10–13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and β-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.     

Relationships among the O-Antigen Gene Clusters of Salmonella enterica Groups B, D1, D2, and D3

The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzy_D3). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like wzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.     

O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis

Bacterial lipopolysaccharide (LPS) is an essential cell envelope component for gram-negative bacteria. As the most variable region of LPS, O antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in Escherichia coli with an N-terminal His₁₀-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemo-enzymatically synthesized the substrates of Wzy, the lipid-PP-linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that Wzy functions in a distributive manner.     

Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10⁶ and 10⁸ CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.     

Molecular Analysis of the O-Antigen Gene Cluster of Escherichia coli O86:B7 and Characterization of the Chain Length Determinant Gene (wzz)

Escherichia coli O86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster between galF and hisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions between galF and gnd are identical, including wzy genes. However, deletion of the two wzy genes revealed that wzy in O86:B7 is responsible for the polymerization of the O antigen, while the deletion of wzy in O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functional wzy gene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that the wzz genes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.     

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, Wzy_Sf. Oag chain length is regulated by chromosomally encoded Wzz_Sf and pHS-2 plasmid-encoded Wzz_pHS2. To identify functionally important amino acid residues in Wzy_Sf, random mutagenesis was performed on the wzy_Sf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzy_Sf proteins in the wzy_Sf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of Wzy_Sf and Wzz_Sf was investigated by transforming these mutated wzy_Sf plasmids into a wzy_Sf- and wzz_Sf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified Wzy_Sf mutants whose polymerization activities were Wzz_Sf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the Wzy_Sf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for Wzz_Sf in Wzy_Sf stability. Hence, in addition to its role in regulating Oag modal chain length, Wzz_Sf also affects Wzy_Sf activity and stability.     

Coordination of Polymerization, Chain Termination, and Export in Assembly of the Escherichia coli Lipopolysaccharide O9a Antigen in an ATP-binding Cassette Transporter-dependent Pathway

The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for O-PS synthesis and export by the ATP-binding cassette transporter-dependent pathway. Comparable systems are widespread in Gram-negative bacteria. The polymannose O9a O-PS is assembled on a polyisoprenoid lipid intermediate by mannosyltransferases located at the cytoplasmic membrane, and the final polysaccharide chain length is determined by the chain terminating dual kinase/methyltransferase, WbdD. The WbdD protein is tethered to the membrane via a C-terminal region containing amphipathic helices located between residues 601 and 669. Here, we establish that the C-terminal domain of WbdD plays an additional pivotal role in assembly of the O-PS by forming a complex with the chain-extending mannosyltransferase, WbdA. Membrane preparations from a ΔwbdD mutant had severely diminished mannosyltransferase activity in vitro, and no significant amounts of the WbdA protein are targeted to the membrane fraction. Expression of a polypeptide comprising the WbdD C-terminal region was sufficient to restore both proper localization of WbdA and mannosyltransferase activity. In contrast to WbdA, the other required mannosyltransferases (WbdBC) are targeted to the membrane independent of WbdD. A bacterial two-hybrid system confirmed the interaction of WbdD and WbdA and identified two regions in the C terminus of WbdD that contributed to the interaction. Therefore, in the O9a assembly export system, the WbdD protein orchestrates the critical localization and coordination of activities involved in O-PS chain extension and termination at the cytoplasmic membrane.Lipopolysaccharide (LPS)³ is a glycolipid unique to the outer membranes of Gram-negative bacteria. LPS has three structural domains in most bacteria (1). Hydrophobic lipid A is a major component of the outer leaflet of the outer membrane. A short core oligosaccharide (OS) serves as a linker between lipid A and a repeat unit polymer termed the O-polysaccharide (O-PS; O-antigen). The structure of lipid A is conserved among Gram-negative bacteria, whereas limited variability is observed among the core OSs of a given species. For example, five closely related core oligosaccharides have been described for Escherichia coli (2). In contrast, the O-PS structures vary extensively within species. O-PS structural variations include differences in the number and type of sugars in the repeat unit and the nature of the glycosidic linkages within and between repeat units. O-PS variations provide the basis for the O-antigen serotyping system, and there are over 180 O-antigen serogroups proposed for E. coli (3, 4).Lipid A-core OS and O-PS are synthesized independently at the cytoplasmic membrane and are subsequently linked together in the periplasm (reviewed in Ref. 1). O-PS assembly is initiated by transfer of a sugar-1-phosphate from a nucleotide sugar precursor to the 55-carbon lipid acceptor, undecaprenol phosphate. In the majority of E. coli serotypes, the initiating reaction is performed by the GlcNAc:Und-P GlcNAc-1-P transferase, WecA (5, 6). WecA is an integral membrane protein and is also essential for initiating synthesis of the enterobacterial common antigen (7). In E. coli, elongation and export of the undecaprenol-PP-linked intermediate proceeds through one of two fundamentally different O-PS assembly pathways. These pathways have been termed Wzy (polymerase)-dependent and ATP-binding cassette (ABC) transporter-dependent biosynthesis, respectively (reviewed in Ref. 1). In Wzy-dependent O-PS biosynthesis, single repeat units are assembled on the undecaprenol-PP-linked intermediate at the cytoplasmic face of the inner membrane. The lipid-linked repeat units are subsequently reoriented to the periplasm where they are assembled into polysaccharide by a process involving Wzy and a chain length regulator, Wzz. In contrast, in the ABC transporter-dependent pathway, the O-PS is elongated on the undecaprenol-PP-linked intermediate in the cytoplasm by sequential glycosyl transfer. Depending on the system, chain extension is terminated by the addition of a nonreducing terminal residue or by interaction with the ABC transporter (8). Full-length O-PS chains are then translocated across the inner membrane by the ABC transporter. The two O-PS assembly pathways converge at a ligation reaction, which transfers the O-PS from undecaprenol-PP to lipid A-core OS at the periplasmic face of the inner membrane. Once assembled, LPS molecules are shuttled to the outer membrane through a process involving the LptABCDE complex (reviewed in Ref. 9).The polymannose O-PS of E. coli O9a provides a model system for ABC transporter-dependent O-PS biosynthesis. The E. coli O9a PS biosynthesis gene cluster (see Fig. 1A) encodes three GDP-mannose-dependent mannosyltransferases (WbdA, WbdB, and WbdC) that assemble the O-PS on undecaprenol-PP-GlcNAc (10). Structural studies identify terminal capping residues in a number of O-PSs synthesized by the ABC transporter-dependent pathway (11). It has been proposed that the addition of a capping residue to the nonreducing end of the undecaprenol-PP-linked PS serves to regulate O-PS chain length by terminating elongation. In the case of the O9a PS, termination involves methylation and phosphorylation. The chain length of the O9a PS is strictly controlled by the activity of WbdD, and O-PS-substituted LPS molecules expressed on the cell surface exhibit a narrow size distribution. The E. coli O9a WbdD protein contains putative kinase and methyltransferase domains, and these activities have been confirmed in biochemical studies (12). In addition to the role in O-PS chain regulation, methyl and/or phosphoryl modification is required for binding of the O9a PS to the nucleotide-binding component (Wzt) of the ABC transporter (13, 14), a crucial initial step in O-PS export. Unmodified O9a PS does not bind to Wzt, and a wbdD mutant accumulates unmodified polysaccharide in the cytoplasm.Open in a separate windowFIGURE 1.Structure and biosynthesis of the E. coli O9a PS and schematic showing WbdD and mutant derivatives. A, the structure of the O9a PS shows the adaptor region, repeat unit, and terminating residues. The nonreducing end of the O-PS is capped by methylation and phosphorylation, but the nature of the linkage between capping residues and the repeat unit is unknown (11, 12). The O9a-PS biosynthesis and export genes are shown together with the functions of the encoded proteins. B, a linear representation of the wild-type WbdD protein from CWG634 is shown in context with the genomic wbdD mutations in CWG635 and CWG900. The methyltransferase (MTase) and kinase domains are shown within WbdD and have been described previously (12). In CWG635, the chromosomal wbdD ORF was disrupted by replacing a 500-bp SmaI restriction fragment with the aacC1 cassette. A potential ribosomal-binding site, initiation codon, and stop codon are shown and together define an ORF encoding amino acids 501–708 of WbdD. In CWG900, the entire wbdD ORF has been removed from the chromosome. C, a schematic of the truncated WbdD polypeptide derivatives encoded by plasmids used in this study. The numbers shown above the polypeptides refer to amino acid positions in the native WbdD protein. Each polypeptide contained either an N-terminal His₆ tag or the T25 fragment of B. pertussis adenylate cyclase (see plasmids in 15, 16). However, the variability of specific assembly proteins among different biosynthetic systems precludes development of a generalized model for a polysaccharide assembly complex. Here we present data revealing the mechanisms that target the O9a mannosyltransferases to the cytoplasmic membrane and identify essential protein-protein interactions within the biosynthesis complex.     

Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an “uncapped core,” which is devoid of O polysaccharide (O-PS), and a “capped core,” which contains the site of O-PS attachment. The inner core region lacks l(d)-glycero-d(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-d-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the “uncapped core” is attached to the glycerol and is composed of a linear α-(1→3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, α-d-allosamine, is attached to the glycerol at position 3. In the “capped core,” there is a three- to five-residue extension of α-(1→3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. β-d-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.Porphyromonas gingivalis is a gram-negative anaerobe which is strongly implicated in the etiology of periodontal disease. Several putative virulence factors are produced by this organism. These virulence factors include the cysteine proteases Arg-gingipains (Rgps) and Lys-gingipain (Kgp) specific for Arg-X and Lys-X peptide bonds, respectively, which are capable of degrading several host proteins (56), and lipopolysaccharide (LPS), which has the potential to cause an inflammatory response in the periodontal tissues of the host. These factors are important antigens in patients with periodontal disease and may account for a considerable proportion of the immune response directed against P. gingivalis (58).LPS is a major constituent of the outer membrane of gram-negative bacteria and facilitates interactions with the external environment. It consists of three regions: a hydrophobic lipid A embedded in the outer leaflet of the outer membrane, a core oligosaccharide (OS), and the O-polysaccharide (O-PS) side chain composed of several repeating units. The hydrophobic lipid A serves as an anchor for the LPS and consists of β-1,6-linked d-glucosamine disaccharide, which is usually phosphorylated at the 1 and/or 4′ positions and N and/or O acylated at positions 2, 3, 2′, and 3′ with various amounts of fatty acids. The rest of the LPS molecule projects from the surface. The core region is attached to lipid A and is composed of ∼10 sugars in most bacteria studied to date and can be further subdivided into an inner core and an outer core. The inner core usually contains l(d)-glycero-d-(l)-manno-heptose and 3-deoxy-d-manno-octulosonic acid (Kdo) residues, whereas the outer core is usually composed of hexoses. Attached to the outer core are the repeating units of O antigen (O-PS), which vary in composition, stereochemistry, and the sequence of O-glycosidic linkages between bacterial strains and thereby give rise to O-serotype specificity within bacterial species. Attachment of O antigen to core lipid A results in “smooth” LPS (S-type LPS), whereas LPS lacking O antigen is “rough” LPS (R-type LPS). Attachment of one repeating unit of O-PS to core lipid A results in SR-LPS (core-plus-one repeating unit) (41, 47, 48). In addition, the outer core OS region can be either “uncapped” or “capped.” The “uncapped” core OS is devoid of O-PS repeating units, whereas the “capped” core OS contains attached O-PS repeating units (47, 53) due to modifications in the outer core region.P. gingivalis W50 was originally thought to synthesize a single LPS composed of a tetrasaccharide repeating unit in the O-PS, [→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAc-(1→3)-α-d-Galp-(1→], which is modified by phosphoethanolamine (PEA) at position 2 of Rha in a nonstoichiometric manner (43). However, a second LPS in this organism, namely A-LPS (49), which has a phosphorylated mannan-containing anionic polysaccharide (A-PS), was identified in our laboratory. The A-PS repeating unit is built up of a phosphorylated branched d-Man-containing oligomer composed of an α1→6-linked d-mannose backbone to which α1→2-linked d-Man side chains of different lengths (one or two residues) are attached at position 2. One of the side chains contains Manα1→2-Manα-1-phosphate linked via phosphorus to a backbone Man residue at position O-2. Although A-LPS is predominantly composed of α-d-mannose residues, it cannot be referred to as a homopolymer due to the presence of Manα1→2Manα1-phosphate-containing OS side chains forming a nonglycosidic linkage between the backbone α-mannose and side chains. Hence, it is likely that the synthesis of A-PS (A-LPS) occurs via a “wzy-dependent” pathway in which repeating units formed on the cytoplasmic face of the inner membrane are polymerized at the periplasmic face following transport or flipping across the cytoplasmic membrane. A-LPS cross-reacts with monoclonal antibody (MAb) 1B5 raised against one of the isoforms of Arg-gingipains, a family of differentially glycosylated cysteine proteases (14, 19). Deglycosylation of the cross-reacting Rgps with anhydrous trifluoromethane sulfonic acid abolishes their immunoreactivity to MAb 1B5, indicating that this antibody recognizes a carbohydrate-containing epitope also present in A-LPS (14, 44). Hence, there appear to be common elements in the biosynthesis of A-LPS and the Arg-gingipains of this organism.Inactivation of P. gingivalis waaL (PG1051, O-antigen ligase) abolishes the synthesis of both O-LPS and A-LPS (49). Hence, the WaaL O-antigen ligase appears to have dual specificity and is capable of ligating both O-PS and A-PS chains to core lipid A. The dual specificity of WaaL in the final step of LPS biosynthesis has also been demonstrated in the synthesis of Escherichia coli O-LPS and M_LPS (38) and for Pseudomonas aeruginosa A-band and B-band LPSs (1).However, the linkage between O-PS and A-PS and core OS has not been identified in P. gingivalis. In this paper, we describe a structural investigation of the core OS of O-LPS in which we used R-LPS prepared from ΔPG1051 (49) and ΔPG1142 (putative O-antigen polymerase), which we hypothesized would synthesize an SR-LPS (core plus one repeating unit) (60). The putative O-antigen polymerase encoded at PG1142 (42) is a phenylalanine-rich membrane protein consisting of 347 amino acids which shows 46% similarity over 297 amino acids to EpsK of Lactobacillus delbrueckii subsp. bulgaricus. EpsK is proposed to be a polymerase on the basis of homology and topological similarity to the O-antigen polymerase (Wzy) of E. coli and is required for the synthesis of an exopolysaccharide composed of Gal, Glc, and Rha (5:1:1) containing repeating units in L. delbrueckii (32). Application of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and methylation and monosaccharide analyses using gas chromatography-mass spectrometry (GC-MS) to purified core-containing OSs isolated from LPS from ΔPG1051 and ΔPG1142 mutants enabled us to solve the LPS core structure of an oral gram-negative bacterium for the first time.     

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.     

Formation of a new O-polysaccharide in Escherichia coli O86 via disruption of a glycosyltransferase gene involved in O-unit assembly

The majority of hetero-polysaccharide biosynthesis in Gram-negative bacteria utilizes the wzy-dependent pathway, in which repeating O-units are first synthesized in the cytosol and then subsequently translocated to the periplasmic face of the inner membrane where polymerization is initiated by the Wzy polymerase. Wzy proteins share little primary sequence homology and are specific for their cognate O-unit structures. Our previous studies on O-polysaccharide biosynthesis in Escherichia coli O86 identified the wbnI gene, which encodes a galactosyltransferase responsible for the introduction of alpha-(1-->3)-Galp residues as side chains of the polysaccharide. In this work, we functionally inactivated the wbnI gene and showed that the mutant strain produced a different polysaccharide without the side chain Galp residue. The yield of the polysaccharide was substantially lower than the one produced by the wild-type strain. This study indicated that the complete O-unit structure is the preferred substrate for the polymerization, thus further confirming the specificity of Wzy. On the other hand, these studies also suggest that the Wzy polymerase might have moderate tolerance of side-chain truncated O-unit substrates.     

Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly⁻) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly⁻ P-gp (91, 94, and 99N→Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly⁻ P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly⁻ D (91, 94, 99N→D) and Gly⁻Δ (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly⁻ D and Gly⁻Δ Pgps were also expressed to the same level as the Gly⁻ mutant protein. ³⁵S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of ³⁵S-methionine/cysteine in full length Gly⁻ P-gp compared to wild-type protein, but the half-life (∼3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of ³⁵S-methionine/cysteine in Gly⁻ P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies. Received: 28 July 1999/Revised: 20 October 1999     

Discovery and Characterization of BlsE,a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]²⁺ cluster. Mutagenesis studies demonstrated that Cys₃₁, Cys_35, Cys₃₈ in the C×××C×MC motif and Gly₇₃, Gly₇₄, Glu₇₅, Pro₇₆ in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met₃₇ had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.     

Biochemical characterization of UDP-Gal:GlcNAc-pyrophosphate-lipid β-1,4-Galactosyltransferase WfeD, a new enzyme from Shigella boydii type 14 that catalyzes the second step in O-antigen repeating-unit synthesis

The O antigen is the outer part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria and contains many repeats of an oligosaccharide unit. It contributes to antigenic variability and is essential to the full function and virulence of bacteria. Shigella is a Gram-negative human pathogen that causes diarrhea in humans. The O antigen of Shigella boydii type 14 consists of repeating oligosaccharide units with the structure [→6-d-Galpα1→4-d-GlcpAβ1→6-d-Galpβ1→4-d-Galpβ1→4-d-GlcpNAcβ1→]n. The wfeD gene in the O-antigen gene cluster of Shigella boydii type 14 was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a β4-GalT that synthesizes the Galβ1-4GlcNAcα-R linkage. WfeD was expressed in Escherichia coli, and the activity was characterized by using UDP-[³H]Gal as the donor substrate as well as the synthetic acceptor substrate GlcNAcα-pyrophosphate-(CH₂)₁₁-O-phenyl. The enzyme product was analyzed by liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and galactosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn²⁺, Ni²⁺, and, surprisingly, also Pb²⁺ enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to be required for activity and may be involved in the binding of the negatively charged acceptor substrate. Our study revealed that the β4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian β4-GalT, although it catalyzes a similar reaction.Lipopolysaccharides (LPSs) consist of O-polysaccharide (O-antigenic) side chains covalently linked to a core polysaccharide and lipid A. LPSs are found in the outer membranes of Gram-negative bacteria, where they contribute to the structural integrity of the membrane and interact with the external environment (9, 10, 15). In the complex and dynamic microbial ecosystem of the human intestine, the communication between microorganisms and the gastrointestinal (GI) epithelium involves O-antigen and LPS binding molecules. Thus, the elimination of the O antigen may reduce virulence (2, 16, 21). Shigella is a genus of highly adapted bacterial pathogens that cause gastrointestinal disease, such as bacillary dysentery or shigellosis. A recent survey showed that shigellosis causes approximately 165 million cases of severe dysentery and more than 1 million deaths per year, mostly in children from developing countries (10). Shigella strains are categorized into four groups: S. boydii, S. dysenteriae, S. flexneri, and S. sonnei, each containing multiple subgroups of different serotypes, based on structural variations in their O antigens.O antigens consist of repeating units of oligosaccharides that are assembled individually, followed by the polymerization of units to form O antigens of different lengths. The glycosyltransferases involved in the biosynthesis of O antigens play a critical role in determining O-antigen structural diversity. The pentasaccharide repeating unit of S. boydii type 14 (B14) has the structure [→6-d-Galpα1→4-d-GlcpAβ1→6-d-Galpβ1→4-d-Galpβ1→4-d-GlcpNAcβ1→]n (12), suggesting the existence of five specific glycosyltransferases: a GlcNAc-phosphotransferase (WecA), three Gal-transferases, and a glucuronosyltransferase.Three distinct processes for the synthesis and translocation of O antigens have been described: the Wzx/Wzy-dependent pathway, the ATP binding cassette transporter-dependent process, and the synthase-dependent process (20, 25, 26). The biosynthesis of the S. boydii B14 O antigen that contains a variety of different sugar residues is expected to utilize the Wzy/Wzx-dependent pathway, where the synthesis of the repeating unit is initiated by WecA, catalyzing the transfer of sugar-phosphate (GlcNAcα-phosphate) from nucleotide sugar (UDP-GlcNAc) to a lipid carrier, undecaprenol-phosphate (Und-P), at the cytoplasmic side of the inner membrane. The wecA gene is present in the S. boydii B14 genome but outside the O-antigen gene cluster (1). The wecA gene is also involved in the synthesis of bacterial polysaccharides other than the O antigen. The extension of the chain is then mediated by specific glycosyltransferases that utilize nucleotide sugar donor substrates and are thought to be loosely associated with the inner membrane. In contrast, mammalian glycosyltransferases are usually membrane-bound proteins. Bacterial and mammalian glycosyltransferases, although they may have similar substrate specificities and form the same linkage, show significantly different amino acid sequences (4). Completed repeating units are then flipped across the membrane to the periplasmic side (by the flippase Wzx) and are polymerized (by Wzy) to form the O antigen under the control of a chain length regulator (Wzz). The repeating units are initially linked to the lipid carrier through GlcNAcα-phosphate. However, the S. boydii B14 O antigen has GlcNAc in the β linkage; thus, upon the polymerization of the completed repeating units, the linkage may be inverted, probably through the specific action of the polymerase Wzy. The entire polymer is then ligated to an outer core sugar based on lipid A. Upon completion, the LPS is extruded from the inner membrane and translocated to the outer membrane (19, 26). The latter-acting enzymes have multiple transmembrane regions that integrate them into the bacterial membranes.Genes involved in O-antigen biosynthesis are normally clustered between galF and gnd in Escherichia coli and Shigella and are classified into three different groups: (i) nucleotide sugar synthesis genes involved in the synthesis of donor substrates, (ii) glycosyltransferase genes, and (iii) O-antigen-processing genes, such as the flippase gene wzx and the polymerase gene wzy. The O-antigen gene cluster of B14 has been sequenced and analyzed (10). Four putative glycosyltransferase genes found in the B14 O-antigen synthesis gene cluster are wfeA, wfeB, wfeD, and wfeE. WfeD shares 38% identity and 57% similarity to the putative glycosyltransferase Orf9, which is involved in the synthesis of the E. coli O136 O antigen (our unpublished data). Since the O antigens of B14 and E. coli O136 share only one common linkage, d-Galpβ1→4-d-GlcpNAc (12, 23), wfeD was proposed to encode the galactosyltransferase (GalT) that transfers Gal to GlcNAcα-PP-Und in the β1-4 linkage, which is the second step in the biosynthetic pathway of the B14 O-antigen repeating unit.We have used biochemical approaches to assay the WfeD enzyme activity and to characterize this enzyme. The lipid carrier analog GlcNAcα-PO₃-PO₃-(CH₂)₁₁-O-phenyl [GlcNAc-PP-(CH₂)₁₁-OPh] has previously been used as a defined synthetic acceptor substrate for the characterization of glycosyltransferases from E. coli serotypes O7 (β1,3-GalT WbbD), O56 (β1,3-Glc-transferase WfaP), and O152 (β1,3-Glc-transferase WfgD) (6, 17). In this work, we showed that GlcNAc-PP-(CH₂)₁₁-OPh could also serve as an exogenous substrate for WfeD from B14. We were therefore able to prove that wfeD encodes a novel β1,4-GalT.     

Clinical and Veterinary Isolates of Salmonella enterica Serovar Enteritidis Defective in Lipopolysaccharide O-Chain Polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.     

Detection of Escherichia coli Serogroups O26 and O113 by PCR Amplification of the wzx and wzy Genes

PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as ≤10 CFU/ml. The O26- and O113-specific PCR assays can potentially be used for typing E. coli O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.     

Amino Acid Substitutions of MagA in Klebsiella pneumoniae Affect the Biosynthesis of the Capsular Polysaccharide

Mucoviscosity-associated gene A (magA) of Klebsiella pneumoniae contributes to K1 capsular polysaccharide (CPS) biosynthesis. Based on sequence homology and gene alignment, the magA gene has been predicted to encode a Wzy-type CPS polymerase. Sequence alignment with the Wzy_C and RfaL protein families (which catalyze CPS or lipopolysaccharide (LPS) biosynthesis) and topological analysis has suggested that eight highly conserved residues, including G308, G310, G334, G337, R290, P305, H323, and N324, were located in a hypothetical loop region. Therefore, we used site-directed mutagenesis to study the role of these residues in CPS production, and to observe the consequent phenotypes such as mucoviscosity, serum and phagocytosis resistance, and virulence (as assessed in mice) in pyogenic liver abscess strain NTUH-K2044. Alanine substitutions at R290 or H323 abolished all of these properties. The G308A mutant was severely impaired for these functions. The G334A mutant remained mucoid with decreased CPS production, but its virulence was significantly reduced in vivo. No phenotypic change was observed for strains harboring magA G310A, G337A, P305A, or N324A mutations. Therefore, R290, G308, H323, and G334 are functionally important residues of the MagA (Wzy) protein of K. pneumoniae NTUH-K2044, capsular type K1. These amino acids are also likely to be important for the function of Wzy in other capsular types in K. pneumoniae and other species bearing Wzy_C family proteins.     

Glycosylation of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans Is Dependent upon the Lipopolysaccharide Biosynthetic Pathway

The human oropharyngeal pathogen Aggregatibacter actinomycetemcomitans synthesizes multiple adhesins, including the nonfimbrial extracellular matrix protein adhesin A (EmaA). EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for collagen binding. Two forms of the protein have been identified, which are suggested to be linked with the type of O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) synthesized (G. Tang et al., Microbiology 153:2447-2457, 2007). This association was investigated by generating individual mutants for a rhamnose sugar biosynthetic enzyme (rmlC; TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase), the ATP binding cassette (ABC) sugar transport protein (wzt), and the O-antigen ligase (waaL). All three mutants produced reduced amounts of O-PS, and the EmaA monomers in these mutants displayed a change in their electrophoretic mobility and aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The modification of EmaA with O-PS sugars was suggested by lectin blots, using the fucose-specific Lens culinaris agglutinin (LCA). Fucose is one of the glycan components of serotype b O-PS. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion using an in vitro rabbit heart valve model, suggesting a role for the glycoconjugant in collagen binding. These data provide experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and for the dependence of the posttranslational modification of EmaA on the LPS biosynthetic machinery in A. actinomycetemcomitans.The Gram-negative, nonmotile, microaerophilic, and oropharyngeal bacterium Aggregatibacter actinomycetemcomitans preferentially colonizes the subgingival region of the human oral cavity. This microorganism is implicated as the etiological agent of localized aggressive periodontitis (9, 13) and causes extraoral infections, including pneumonia, osteitis (30), and infective endocarditis (6). Recent studies also link this periodontal pathogen to cardiovascular diseases, such as atherosclerosis (20).Typical of Gram-negative bacteria, the outer membrane of A. actinomycetemcomitans possesses an asymmetric lipid-protein bilayer. The inner leaflet of the outer membrane is mainly phospholipids, and the outer leaflet consists of lipopolysaccharide (LPS), phospholipids, and proteins (4). LPS molecules are ubiquitously distributed on the outer membrane and are essential for maintaining the membrane integrity (3). Intact LPS molecules are also required for the assembly of some large outer membrane proteins (3, 18, 41). A typical LPS molecule is composed of hydrophobic lipid A, a nonrepeat core oligosaccharide, and a repeating O-antigen or O-polysaccharide (O-PS). The distal O-PS is a major antigen, stimulating the host immune response, and the basis for serotyping Gram-negative bacteria (36), including A. actinomycetemcomitans (32, 50).Six different serotypes (a to f) and the corresponding genetic loci have been identified for A. actinomycetemcomitans (19, 22, 27, 44, 50, 54, 55). Serotype b remains one of the common serotypes found in the human oral cavity (9, 13, 51). The serotype b O-PS of A. actinomycetemcomitans is encoded by an operon composed of 21 genes, which are responsible for the biosynthesis of the repeating trisaccharide unit of this particular serotype (53, 55). Each O-PS unit of serotype b contains a disaccharide backbone composed of d-fucose (d-Fuc) and l-rhamnose (l-Rha), linked by a nonreducing d-N-acetylgalactosamine (d-GalNAc) at the O-3 position of l-Rha (33) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) O-PS structure of serotype b A. actinomycetemcomitans. (B) Silver-stained 5 to 15% polyacrylamide-SDS gel of serotype b LPS. A total of 1.0 ml of mid-logarithmic-phase cells were collected and lysed. Three lysates from each strain were combined and treated with proteinase K at 60°C for 60 min before electrophoresis, followed by silver staining. C, control: whole-cell lysate without proteinase K digestion; WT, wild type (VT1169); emaA, extracellular matrix protein adhesin A mutant; rmlC, rhamnose epimerase mutant; wzt, ATP-binding cassette sugar transport mutant. The dark brown staining of the high molecular weight (75,000 to 250,000) corresponds to polymerized O-PS.The assembly of LPS molecules in Gram-negative bacteria involve diverse enzymes and pathways due to the variation of the O-PS structures among different bacteria (36). RmlC (previously RfbD), Wzt (previously AbcA or RfbB), and WaaL are three enzymes involved in different stages of the LPS synthesis of some Gram-negative bacteria (7, 36, 37). A homologue of RmlC, TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase, which is required for l-Rha synthesis, has been identified in A. actinomycetemcomitans (53, 55). Wzt is an ATP binding cassette (ABC) transporter that exports saccharide polymers from the cytoplasm to the periplasmic space (7, 36). A homologue of wzt was originally identified from a serotype b strain of A. actinomycetemcomitans, based on protein sequence identity with Aeromonas salmonicida (55). Kaplan et al. (19) later showed that a serotype f wzt mutant strain of A. actinomycetemcomitans produces less O-PS. WaaL, an O-antigen ligase found in Escherichia coli and Pseudomonas aeruginosa, ligates an undecaprenol pyrophosphate-linked oligo- or polysaccharide onto the lipid A-core oligosaccharide in the periplasm (1, 36). A putative O-antigen ligase is located in the chromosome of a serotype b A. actinomycetemcomitans strain (HK1651), based on sequence homology (Oralgen, Los Alamos, NM).Our earlier work suggested a correlation between the type of LPS molecule and the form of EmaA synthesized by A. actinomycetemcomitans (46). The EmaA of serotype b A. actinomycetemcomitans is a 202-kDa protein that forms the antennae-like appendages found on the surface of A. actinomycetemcomitans and is required for collagen binding (40). The appendages are composed of three EmaA monomers that oligomerize to form an ellipsoidal structure required for the collagen binding activity (56, 57). The ellipsoidal structure corresponds to the amino termini of the proteins and is located at the distal end of a long stalk domain that is attached to the outer membrane by the carboxyl termini (57). The carboxyl termini of the proteins assume β-barrel structures required for pore formation and translocation of the molecules through the outer membrane, similar to those of other type V_c autotransporter proteins (14). Recently, we have demonstrated that EmaA is important in the initiation of infective endocarditis in a rabbit model of infectious endocarditis (45).Two transposon mutant strains (rmlC and wzt) and a waaL mutant strain generated by site-directed insertional mutagenesis have been developed and characterized in this study. The rmlC mutant did not synthesize l-Rha and did not produce detectable O-PS. The wzt and waaL mutant strains synthesized less O-PS than the wild-type strain. Complementation of the mutant strains restored the production of the serotype b O-PS to wild-type levels. An increase in the electrophoretic mobility of the EmaA monomer was observed in all three mutants, which suggests the presence of carbohydrate. The EmaA mobility reverted to wild-type mobility upon complementation. The presence of carbohydrate associated with EmaA was confirmed by lectin blotting, and in vitro collagen binding assessment demonstrated that the glycoconjugant is important for the full function of this adhesin. The experimental data suggest that EmaA contains carbohydrate similar to that present in O-PS and is a substrate for the O-antigen ligase of the LPS biosynthetic pathway of A. actinomycetemcomitans.     

The structural characterization of the O-polysaccharide antigen of the lipopolysaccharide of Escherichiacoli serotype O118 and its relation to the O-antigens of Escherichiacoli O151 and Salmonellaenterica O47

Mild acid hydrolysis of the lipopolysaccharide produced by Escherichiacoli O118:H16 standard strain (NRCC 6613) afforded an O-polysaccharide (O-PS) composed of d-galactose, 2-acetamidoylamino-2,6-dideoxy-l-galactose , 2-acetamido-2-deoxy-d-glucose, ribitol, and phosphate (1:1:1:1:1). From DOC-PAGE, sugar and methylation analyses, one- and two-dimensional NMR spectroscopy, capillary electrophoresis-mass spectrometry, hydrolysis, and sequential Smith-type periodate oxidation studies, the O-PS was determined to be an unbranched linear polymer having the structure:[6)-α-d-Galp-(1→3)-α-l-FucpNAm-(1→3)-β-d-GlcpNAc-(1→3)-Rib-ol-5-P-(O→]_nThe structure of the O-PS is consistent with the reported DNA data on the O-antigen gene-cluster of E. coli O118 and interestingly, the O-PS is similar to the structures of the O-antigens of Salmonellaenterica O47 and E. coli O151:H10 reference strain 880-67, as predicted from the results of DNA sequencing of their respective O-antigen gene-clusters.     

Structural and genetic characterization of the O-antigen of Salmonella enterica O56 containing a novel derivative of 4-amino-4,6-dideoxy-d-glucose

Based on the O-antigens (O-polysaccharides), one of the most variable cell constituents, 46 O-serogroups have been recognized in the Kauffmann-White serotyping scheme for Salmonella enterica. In this work, the structure of the O-polysaccharide and the genetic organization of the O-antigen gene cluster of S. enterica O56 were investigated. As judged by sugar and methylation analyses, along with NMR spectroscopic data, the O-polysaccharide has a linear tetrasaccharide O-unit, which consists of one residue each of d-ribofuranose, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, and a novel sugar derivative, 4-(N-acetyl-l-seryl)amino-4,6-dideoxy-d-glucose (d-Qui4NSerAc). The following structure of the O-polysaccharide was established:→3)-β-d-Quip4NSerAc-(1→3)-β-d-Ribf-(1→4)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen gene cluster of S. enterica O56 having 12 open reading frames was found between the housekeeping genes galF and gnd. A comparison with databases and using the O-antigen structure data enabled us to ascribe functions to genes for (i) synthesis of d-GalNAc and d-Qui4NSerAc, (ii) sugar transfer, and (iii) O-antigen processing, including genes for O-unit flippase (Wzx) and O-antigen polymerase (Wzy).