O-antigen Biosynthesis Research Articles

Variant O89 O-Antigen of E. coli Is Associated With Group 1 Capsule Loci and Multidrug Resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing strain – E. coli 26561 – represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) – 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

Development of a molecular serotyping scheme and a multiplexed luminex-based array for Providencia

Providencia is an opportunistic human pathogen that belongs to the Enterobacteriaceae family. The bacterial cell surface O-antigen is one of the most structurally variable cell constituents and serves as a basis for serotyping gram-negative bacteria. In this work, the genomes of 12 Providencia strains were sequenced, and genes driving O-antigen biosynthesis were analyzed. The O-antigen-synthesizing genes of Providencia are located in the O-antigen gene cluster (OGC) between the cpxA and yibK genes. The gene functions predicted in silico agreed with the known O-antigen structures. All clusters were found to contain both wzx and wzy and exhibit a high degree of heterogeneity. Based on the sero-specific genes, we developed a molecular serotyping system to detect 23 serotypes (from the present and previous studies) for the first time. Five Proteus strains, five Morganella strains, five uropathogenic Escherichia coli (UPEC) strains and 32 Providencia strains with other serotypes were used to assess the specificity of our multiplexed Luminex-based array. Five serogroups (O3, O8, O19, O38 and O52 strains) were used to determine the sensitivity of the suspension array. The detection sensitivity was 0.1 ng genomic DNA, 103 CFU/ml in pure culture, or 104 CFU/ml in mock urine specimens. Furthermore, 29 publicly available Providencia genomes (which have not been serotyped) were analyzed, and 23 novel putative OGC types were identified. In total, we identified 35 new OGCs and developed a molecular serotyping system based on the sero-specific genes. The established classification system can support promising applications in basic research, clinical diagnosis, and epidemiological surveillance.

Development of rapid and simple experimental and in silico serotyping systems for Citrobacter.

Aim:Members of the genus Citrobacter are important opportunistic pathogens responsible for high mortality rate. Therefore, in this study, we aimed to develop efficient and accurate Citrobacter typing schemes for clinical detection and epidemiological surveillance.Materials & methods:Using genomic and experimental analyses, we located the O-antigen biosynthesis gene clusters in Citrobacter genome for the first time, and used comparative genomic analyses to reveal the specific genes in different Citrobacter serotypes.Results:Based on the specific genes in O-antigen biosynthesis gene clusters of Citrobacter, we established experimental and in silico serotyping systems for this bacterium.Conclusion:Both serotyping tools are reliable, and our observations are biologically and clinically relevant for understanding and managing Citrobacter infection.

Wzx flippases exhibiting complex O-unit preferences require a new model for Wzx-substrate interactions.

The Wzx flippase is a critical component of the O‐antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram‐negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O‐unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O‐unit main‐chain, despite both being able to translocate several other structurally‐divergent O units. We also identified other Y. pseudotuberculosis Wzx that can translocate a structurally divergent foreign O unit with high efficiency, and thus exhibit an apparently relaxed substrate preference. It now appears that Wzx substrate preference is more complex than previously suggested, and that not all O‐unit residues are equally important determinants of translocation efficiency. We propose a new “Structure‐Specific Triggering” model in which Wzx translocation proceeds at a low level for a wide variety of substrates, with high‐frequency translocation only being triggered by Wzx interacting with one or more preferred O‐unit structural elements found on its cognate O unit(s).

Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.

A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.

Transposition Behavior Revealed by High-Resolution Description of Pseudomonas Aeruginosa Saltovirus Integration Sites.

Transposable phages, also called saltoviruses, of which the Escherichia coli phage Mu is the reference, are temperate phages that multiply their genome through replicative transposition at multiple sites in their host chromosome. The viral genome is packaged together with host DNA at both ends. In the present work, genome sequencing of three Pseudomonas aeruginosa transposable phages, HW12, 2P1, and Ab30, incidentally gave us access to the location of thousands of replicative integration sites and revealed the existence of a variable number of hotspots. Taking advantage of deep sequencing, we then designed an experiment to study 13,000,000 transposon integration sites of bacteriophage Ab30. The investigation revealed the presence of 42 transposition hotspots adjacent to bacterial interspersed mosaic elements (BIME) accounting for 5% of all transposition sites. The rest of the sites appeared widely distributed with the exception of coldspots associated with low G-C content segments, including the putative O-antigen biosynthesis cluster. Surprisingly, 0.4% of the transposition events occurred in a copy of the phage genome itself, indicating that the previously described immunity against such events is slightly leaky. This observation allowed drawing an image of the phage chromosome supercoiling into four loops.

O-Antigens of Escherichia coli Strains O81 and HS3-104 Are Structurally and Genetically Related, Except O-Antigen Glucosylation in E. coli HS3-104.

Glycerophosphate-containing O-specific polysaccharides (OPSs) were obtained by mild acidic degradation of lipopolysaccharides isolated from Escherichia coli type strain O81 and E. coli strain HS3-104 from horse feces. The structures of both OPSs and of the oligosaccharide derived from the strain O81 OPS by treatment with 48% HF were studied by monosaccharide analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. Both OPSs had similar structures and differed only in the presence of a side-chain glucose residue in the strain HS3-104 OPS. The genes and the organization of the O-antigen biosynthesis gene cluster in both strains are almost identical with the exception of the gtr gene cluster responsible for glucosylations in the strain HS3-104, which is located elsewhere in the genome.

Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella 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 Klebsiella 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 Klebsiella pneumoniae because genes outside the main rfb (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-Galp-(1→3)-β-d-Galf-(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-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-β-d-Galf-(1→3)-β-d-GlcpNAc-(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 Escherichia coli 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 Klebsiella O-antigen carbohydrate structures based on the O2a backbone.

A bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide provides protection against avian pathogenic Escherichia coli O1 and O2 infection

Avian pathogenic Escherichia coli (APEC), a leading cause of avian airsacculitis and colibacillosis, is responsible for significant economic loss in the poultry industry. APEC serogroups O1, O2, and O78 are predominantly associated with disease. Lipopolysaccharide (LPS) O-antigen has been shown to be a potent antigen for inducing specific protective immune responses. Therefore, we sought to develop a multivalent polysaccharide vaccine to prevent most APEC infections. We previously reported the stable expression of plasmid pSS27 encoding the APEC O1 O-antigen gene cluster (10.8 kb) in attenuated Salmonella enterica serovar Typhimurium S740 provided excellent protection against APEC O1 challenge. In this study, the plasmid pSS28 harboring the APEC O2 O-antigen polysaccharide gene cluster (15.5 kb) was constructed. Biosynthesis of pSS28-encoded APEC O2 O-antigen in Salmonella vaccine strain S740 was validated by Western blot. The recombinant Salmonella vaccine strain S740 (pSS28) elicited homologous protection against virulent wild-type APEC O2 challenge in a chicken model. Furthermore, through equal-volume mixing the two monovalent vaccine strains S740 (pSS27) and S740 (pSS28), a bivalent vaccine candidate against both APEC O1 and O2 was developed. Immunization of chickens with the bivalent vaccine elicited production of serum IgG and mucosal sIgA antibodies against the LPS of both APEC O1 and O2. Moreover, antibodies induced by the bivalent vaccine promoted opsonization, provoked complement-mediated bactericidal activity, and elicited protection against lethal challenge with both virulent APEC O1 and O2 strains. These results demonstrate that the bivalent vaccine comprised of S740 (pSS27) and S740 (pSS28) is a promising vaccine candidate against APEC O1 and O2 infection.

Architecture of a channel-forming O-antigen polysaccharide ABC transporter.

O-antigens are cell surface polysaccharides of many Gram-negative pathogens that aid in escaping innate immune responses.1 A widespread O-antigen biosynthesis mechanism involves the synthesis of the lipid-anchored polymer on the inner membrane’s (IM’s) cytosolic face, followed by transport to the periplasmic side where it is ligated to the lipid A core to complete a lipopolysaccharide (LPS) molecule2. The O antigen’s transport to the periplasm is mediated by an ATP-binding cassette (ABC) transporter, called Wzm/Wzt, Extended Data Fig. 1. We present the crystal structure of the Wzm/Wzt homolog from Aquifex aeolicus in an open conformation. The transporter forms a transmembrane (TM) channel sufficiently wide to accommodate a linear polysaccharide. It’s nucleotide binding domain (NBD) and a periplasmic loop form ‘gate helices’ at the cytosolic and periplasmic membrane interfaces, likely serving as substrate entry and exit points. Site-directed mutagenesis of the gates impairs in vivo O antigen secretion in the E. coli prototype. Combined with a closed structure of the isolated NBDs, our structural and functional analyses suggest a processive O antigen translocation mechanism, which stands in contrast to the classical alternating access mechanism of ABC transporters.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a.

We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

Defining the Genetic Features of O-Antigen Biosynthesis Gene Cluster and Performance of an O-Antigen Serotyping Scheme for Escherichia albertii.

Escherichia albertii is a newly described and emerging diarrheagenic pathogen responsible for outbreaks of gastroenteritis. Serotyping plays an important role in diagnosis and epidemiological studies for pathogens of public health importance. The diversity of O-antigen biosynthesis gene clusters (O-AGCs) provides the primary basis for serotyping. However, little is known about the distribution and diversity of O-AGCs of E. albertii strains. Here, we presented a complete sequence set for the O-AGCs from 52 E. albertii strains and identified seven distinct O-AGCs. Six of these were also found in 15 genomes of E. albertii strains deposited in the public database. Possession of wzy/wzx genes in each O-AGC strongly suggest that O-antigens of E. albertii were synthesized by the Wzx/Wzy-dependent pathway. Furthermore, we performed an O-antigen serotyping scheme for E. albertii based on specific antisera against seven O-antigens and a high throughput xTAG Luminex assay to simultaneously detect seven O-AGCs. Both methods accurately identified serotypes of 64 tested E. albertii strains. Our data revealed the high-level diversity of O-AGCs in E. albertii. We also provide valuable methods to reliably identify and serotype this bacterium.

An untypeable enterotoxigenic Escherichia coli represents one of the dominant types causing human disease.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children below 5 years of age in endemic areas, and is a primary cause of diarrhoea in travellers visiting developing countries. Epidemiological analysis of E. coli pathovars is traditionally carried out based on the results of serotyping. However, genomic analysis of a global ETEC collection of 362 isolates taken from patients revealed nine novel O-antigen biosynthesis gene clusters that were previously unrecognized, and have collectively been called unclassified. When put in the context of all isolates sequenced, one of the novel O-genotypes, OgN5, was found to be the second most common ETEC O-genotype causing disease, after O6, in a globally representative ETEC collection. It’s also clear that ETEC OgN5 isolates have spread globally. These novel O-genotypes have now been included in our comprehensive O-genotyping scheme, and can be detected using a PCR-based and an in silico typing method. This will assist in epidemiological studies, as well as in ETEC vaccine development.

Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate

Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs.

Structures and gene clusters of the O-antigens of Escherichia albertii O3, O4, O6, and O7

The O-specific polysaccharides (OPSs) called O-antigens were obtained by mild acid degradation of the lipopolysaccharides of Escherichia albertii serotypes O3, O4, O6, and O7 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure was established for the OPS of E. albertii O4, which, to our knowledge, is unique among known bacterial polysaccharide structures:→2)-α-l-Rhap-(1 → 2)-α-l-Fucp-(1 → 2)-β-d-Galp-(1 → 3)-α-d-GalpNAc-(1 → 3)-β-d-GlcpNAc-(1→The OPS structure of the strain of E. albertii O7 studied was identical to that of strain LMG 20973 (= Albert 10457), whose structure has been reported earlier (R. Eserstam et al. Eur. J. Biochem. 269 (2002) 3289–3295). E. albertii O3 and O6 shared the OPS structures with Escherichia coli O181 and O3, respectively, except for the lack of O-acetylation in E. albertii O3, which is present in E. coli O181. The gene clusters driving the O-antigen biosynthesis of the E. albertii strains were sequenced, the genes were annotated by comparison with sequences in the available databases, and the predicted functions of the encoded proteins were found to be consistent with the OPS structures established. In accordance with the relatedness of the OPS structures, the O-antigen gene clusters of E. albertii O3 and O6 contain the same genes and have the same organization as those of E. coli O181 and O3, the entire gene clusters being 83% and 98% identical, respectively.

Rapid customised operon assembly by yeast recombinational cloning.

We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

Structures and gene clusters of the O-specific polysaccharides of the lipopolysaccharides of Escherichia coli O69 and O146 containing glycolactilic acids: ether conjugates of D-GlcNAc and D-Glc with (R)- and (S)-lactic acid.

Based on the O-specific polysaccharides of the lipopolysaccharides (O-polysaccharides, O-antigens), strains of a clonal species Escherichia coli are classified into 184 O serogroups. In this work, structures of the O-polysaccharides of E. coli O69 and O146 were elucidated and gene clusters for their biosynthesis were characterized. The O-polysaccharides were released from the lipopolysaccharides by mild acid hydrolysis and studied by sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy before and after O-deacetylation. The O146 polysaccharide was also studied by Smith degradation. The O69 and O146 polysaccharides were found to contain ether conjugates of monosaccharides with lactic acid called glycolactilic acids: 2-acetamido-2-deoxy-4-O-[(R)-1-carboxyethyl]-D-glucose (D-GlcNAc4Rlac) and 3-O-[(S)-1-carboxyethyl]-D-glucose (D-Glc3Slac), respectively. Structures of the pentasaccharide repeats of the O-polysaccharides were established, and that of E. coli O69 was found to differ in the presence of D-GlcNAc4Rlac from the structure reported for this bacterium earlier (Erbing C, Kenne L, Lindberg B. 1977. Carbohydr Res. 56:371-376). The O-antigen gene clusters of E. coli O69 and O146 between conserved genes galF and gnd were analyzed taking into account the O-polysaccharide structures established, and functions of putative genes for synthesis of D-Glc3Slac and D-GlcNAc4Rlac and for glycosyltransferases were assigned based on homology with O-antigen biosynthesis genes of other enteric bacteria. It was found that in E. coli and Shigella spp. predicted enolpyruvate reductases of the biosynthesis pathway of glycolactilic acids, LarR and LarS, which catalyze formation of conjugates with (R)- or (S)-lactic acid, respectively, are distinguished by sequence homology and size.

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate.

Bacteria use long-chain isoprenoids like undecaprenyl phosphate (Und-P) as lipid carriers to assemble numerous glycan polymers that comprise the cell envelope. In any one bacterium, multiple oligosaccharide biosynthetic pathways compete for a common pool of Und-P, which means that disruptions in one pathway may produce secondary consequences that affect the others. Using the Gram-negative bacterium Escherichia coli as a model, we demonstrate that interruption of the biogenesis of O antigen, a major outer membrane component, indirectly impairs peptidoglycan synthesis by sequestering Und-P into dead-end intermediates. These results strongly argue that the functions of many Und-P-utilizing pathways must be reevaluated, because much of our current understanding is based on experiments that did not control for these unintended secondary effects.

Six Novel O Genotypes from Shiga Toxin-Producing Escherichia coli.

Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny.

Structure and Biosynthesis Gene Cluster of the O-Antigen of Escherichia coli O12.

Two polysaccharides were isolated from Escherichia coli O12, the major being identified as the O12-antigen and the minor as the K5-antigen. The polysaccharides were studied by sugar analysis, Smith degradation, and one- and two-dimensional (1)H and (13)C NMR spectroscopy. As a result, the following structure of the O12-polysaccharide was elucidated, which, to our knowledge, has not been hitherto found in bacterial carbohydrates: →2)-β-d-Glcp-(1→6)-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→. The →4)-β-d-GlcpA-(1→4)-α-d-GlcpNAc-(1→ structure established for the K5-polysaccharide (heparosan) is previously known. Functions of genes in the O-antigen biosynthesis gene cluster of E. coli O12 were assigned by comparison with sequences in the available databases and found to be consistent with the O12-polysaccharide structure.