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This review covers the structures and genetics of the 46 O antigens of Salmonella, a major pathogen of humans and domestic animals. The variation in structures underpins the serological specificity of the 46 recognized serogroups. The O antigen is important for the full function and virulence of many bacteria, and the considerable diversity of O antigens can confer selective advantage. Salmonella O antigens can be divided into two major groups: those which have N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) and those which have galactose (Gal) as the first sugar in the O unit. In recent years, we have determined 21 chemical structures and sequenced 28 gene clusters for GlcNAc-/GalNAc-initiated O antigens, thus completing the structure and DNA sequence data for the 46 Salmonella O antigens. The structures and gene clusters of the GlcNAc-/GalNAc-initiated O antigens were found to be highly diverse, and 24 of them were found to be identical or closely related to Escherichia coli O antigens. Sequence comparisons indicate that all or most of the shared gene clusters were probably present in the common ancestor, although alternative explanations are also possible. In contrast, the better-known eight Gal-initiated O antigens are closely related both in structures and gene cluster sequences., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Salmonella enterica serovar Typhimurium (or simply Typhimurium) is the most common serovar in both human infections and farm animals in Australia and many other countries. Typhimurium is a broad host range serovar but has also evolved into host-adapted variants (i.e. isolated from a particular host such as pigeons). Six Typhimurium strains of different phage types (defined by patterns of susceptibility to lysis by a set of bacteriophages) were analysed using Illumina high-throughput genome sequencing., Results: Variations between strains were mainly due to single nucleotide polymorphisms (SNPs) with an average of 611 SNPs per strain, ranging from 391 SNPs to 922 SNPs. There were seven insertions/deletions (indels) involving whole or partial gene deletions, four inactivation events due to IS200 insertion and 15 pseudogenes due to early termination. Four of these inactivated or deleted genes may be virulence related. Nine prophage or prophage remnants were identified in the six strains. Gifsy-1, Gifsy-2 and the sopE2 and sspH2 phage remnants were present in all six genomes while Fels-1, Fels-2, ST64B, ST104 and CP4-57 were variably present. Four strains carried the 90-kb plasmid pSLT which contains several known virulence genes. However, two strains were found to lack the plasmid. In addition, one strain had a novel plasmid similar to Typhi strain CT18 plasmid pHCM2., Conclusion: The genome data suggest that variations between strains were mainly due to accumulation of SNPs, some of which resulted in gene inactivation. Unique genetic elements that were common between host-adapted phage types were not found. This study advanced our understanding on the evolution and adaptation of Typhimurium at genomic level.

Bacterial Wzx flippases translocate (flip) short oligosaccharide repeat units (O units) across the inner membrane into the periplasm, which is a critical step in the assembly of many O antigens, capsules and other surface polysaccharides. There is enormous diversity in O antigens and capsules in particular, even within species. Wzx proteins are similarly diverse, but it has been widely accepted that they have significant specificity only for the first sugar of an O unit. In this study, we analysed the Wzx from the Salmonella enterica group C2 O antigen gene cluster, which is a unique and divergent member of a set of gene clusters that produce galactose-initiated O antigens. We demonstrate that this Wzx has a strong preference for the presence of an abequose side-branch, which manifests in a reduction of long-chain O antigen and a major growth defect. This contributes to a growing body of evidence that, contrary to earlier proposals, Wzx flippases commonly exhibit a strong preference for the structure of their native O unit. [ABSTRACT FROM AUTHOR]

The O-specific polysaccharide (OPS) is a variable constituent of the lipopolysaccharide of Gram-negative bacteria. The polymorphic nature of OPSs within a species is usually first defined serologically, and the current serotyping scheme for Yersinia pseudotuberculosis consists of 21 O serotypes of which 15 have been characterized genetically and structurally. Here, we present the structure and DNA sequence of Y. pseudotuberculosis O:10 OPS. The O unit consists of one residue each of d-galactopyranose, N-acetyl-d-galactosamine (2-amino-2-deoxy-d-galactopyranose) and d-glucopyranose in the backbone, with two colitose (3,6-dideoxy-l-xylo-hexopyranose) side-branch residues. This structure is very similar to that shared by Escherichia coli O111 and Salmonella enterica O35. The gene cluster sequences of these serotypes, however, have only low levels of similarity to that of Y. pseudotuberculosis O:10, although there is significant conservation of gene order. Within Y. pseudotuberculosis, the O10 structure is most closely related to the O:6 and O:7 structures. [ABSTRACT FROM PUBLISHER]

Abstract: The genus Salmonella consists of two species S. enterica and S. bongori. S. enterica has a well defined subspecies structure with seven subspecies consistently delineated by sequence variation. Frequency of recombination between subspecies and within a subspecies is markedly different. Subspecies I undergoes frequent recombination as demonstrated recently, demystifying the long-held belief that Salmonella is a highly clonal organism. The majority of disease causing serovars are from subspecies I with the most important serovars in human health being Typhimurium and Typhi. Typhimurium has developed considerable diversity and may be a very old serovar. The majority of the isolates belong to a single clonal complex by multilocus sequence typing. Typhimurium isolates are divided into phage types and some of the phage types do not have a single origin as determined using mutational changes. Phage type DT104 is heterogeneous and represented in multiple sequence types, with its multidrug-resistant variant most successful causing epidemics in many parts of the world. Typhi, a human restricted serovar, is relatively young compared to Typhimurium, and has a low level of sequence variation. Single nucleotide polymorphisms (SNPs) have been shown to be very useful for typing and resolving relationships within Typhi. Genome sequences of 19 isolates revealed more than 1700 SNPs. The fully resolved phylogenetic tree allows one to trace the mutational changes occurred during clonal diversification. Genome wide SNPs have greatly enhanced our understanding of the evolution of Salmonella clones. [Copyright &y& Elsevier]

The O-antigen translocase, Wzx, is involved in translocation of bacterial polysaccharide repeat units across the cytoplasmic membrane, and is an unusually diverse, highly hydrophobic protein, with high numbers of predicted α-helical transmembrane segments (TMS). The Salmonella enterica serovar Typhimurium Group B O-antigen Wzx was an ideal candidate for topological study as the O-antigen gene cluster is one of only a few that have been well characterized. The topology profile prediction for this protein was determined using five programs, with different recognition parameters, which consistently predict that 12 TMS are present. A membrane topology model was constructed by analysis of lacZ and phoA gene fusions at randomly selected and targeted fusion sites within wzx. Enzyme activity of these, and full-length C-terminal fusion proteins, confirmed the 12-TMS topology for this Wzx, and also indicated that the C-terminus was located within the cytoplasm, which is consistent with the predicted topology. [ABSTRACT FROM AUTHOR]

Focuses on the molecular evolution of the GDP-mannose pathway genes, manB and manC, in Salmonella enterica. Phylogenetic relationships of the derived manB and manC sequences; Origins of the colanic acid (CA)-like O antigen sequence; Distribution of polymorphic nucleotide sites within the CA and CA-like manB[sub OAg] genes.

Discusses the study which sequenced the O-antigen cluster and flanking genes from an O35 Salmonella enterica serovar Adelaide strain and compared this sequence with that of the Escherichia coli O111. Suggestion that the ancestor of E. coli and S. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species.

Examines the genetic variation of dtDP-L-rhamnose pathway genes in Salmonella enterica. Evidence of O antigen gene transfer; Role of rml genes in serogroup-specific gene subspecies transfer; Determination of gene cluster source.

Investigates the involvement of a galactosyl-1-phosphate transferase encoded by Salmonella enterica rfbP gene in O-antigen subunit processing. Mapping of rfbT mutation to rfbP; Non-accumulation of UndPP-linked long-chain O antigen ; Flippin gof UndPP-linked o unit from the cytopalsmic face to the periplasmic face of the cytoplasmic membrane.

We report the identification and sequence from Escherichia coli and Salmonelia enterica strains of the cid gene, encoding the chain-length determinant (CLO) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cid gene. [ABSTRACT FROM AUTHOR]

In vitro enzymic synthesis of CDP-D-abequose, CDP-D-[U-14C]abequose, CDP-6-deoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-o-x3,/o-4-hexulose was achieved using enzymes from cell extracts of cultures of Escherichia coli strains harbouring and expressing genes of the rfb gene cluster of Salmonella enterica LT2. From an initial synthesis step, CDP-6-deoxy-D-xylo-4-hexulose was isolated after 30 rain reaction, using CDP-D-glucose. NAD and CDP-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 90.6%). From that intermediate, CDP-3,6-dideoxy-D-xylo-4-hexulose was produced in a reaction using NADH and a crude extract containing the required enzymes. CDP-D-abequose synthesis was performed either in the presence of excess NADH and NADPH or using an enzymic system which regenerates low concentrations of the coenzymes. In a two-step reaction, CDP-D-glucose was first converted to CDP-6-deoxy-D- xylo-4-hexulose, then, following addition of the required coenzymes and enzymes. CDP-D-abequose was formed from this intermediary product in a I-h incubation. Starting from 250 mg CDP-D- glucose, the molar yield of CDP-D-abequose after protein precipitation and HPLC was 82%. corre- sponding to more than 200 mg. CDP-D-[U-14C]abequose was synthesised from α-D-[U14C]glucose 1-phosphate and CTP using purified glucose-l-phosphate cytidylyltransferase in a reaction preceding the later steps. GC-MS and NMR revealed that the hexose part of the end product was 3.6- dideoxy-D-galactose (abequose) and that the corresponding intermediates were 4-keto-6-deoxy-D- xylo-hexose and 4-keto-3,6-dideoxy-D-xylo-hexose, respectively. The synthesized CDP-6-deoxy-D-xylo-4-hexulose exhibited the characteristic ultraviolet light absorption at 318 nm but no corresponding absorption was found for CDP-3,6-dideoxy-D-xylo-4-hexulose. A HPLC technique, where the four CDP-sugars were baseline separated, was developed and used for enzyme assays and for the analysis of synthesized products. [ABSTRACT FROM AUTHOR]

We here report on the purification and characterization of glucose-1-phosphate thymidyly-transferase, the first of four enzymes commited to biosynthesis of dTDP-L-rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the rfbA gene encoding this enzyme. Pure enzyme was obtained by 109-fold enrichment in three chromatography steps. The glucose-1-phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a “ping-pong” mechanism. The K[sub m] values for dTTP and α-D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the K[sub m] values for dTDP-D-glucose and diphosphate are 0.083 mM and 0.15 mM. respectively. The enzyme also accepts UTP and UDP-D-glucose and α-D-glucosamine 1-phosphate is accepted equally as well as α-D-glucose 1-phosphate. The NH[sub 2]-terminal sequence of glucose-1-phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the orf6.1 gene of the rfb gene cluster. The SDS/PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the orf6.1 gene (32453 Da}. [ABSTRACT FROM AUTHOR]

Serovar Typhimurium of Salmonella enterica is a model organism for studies of pathogenesis that exhibits phage-type variation and variation in host range and virulence, but in a recent study showed no sequence variation in four genes, indicating the clonal nature of this serovar. We determined the relationships of 46 Typhimurium isolates of nine phage types using mutational changes detected either by matching AFLP (amplified fragment length polymorphism) fragments to computer-modeled LT2 AFLP fi-agments or by sequencing intergenic regions. Fifty-one polymorphic sites were detected, which gave a single phylogenetic tree. Comparison with genome sequences of five other serovars, Typhi, Paratyphi A, Gallinarum, Enteritidis, and Pullorum, enabled determination of the root of the tree. Only two parallel events were observed, giving high confidence in the tree branching order. The mutation-based tree provided a high level of consistency and a clear lineage for the Typhimurium isolates studied. This enabled us to show that for seven of the nine phage types used, the isolates studied have a single origin, but that two phage types clearly have more than one independent origin. We found that sequencing intergenic regions provides a good strategy for detection of mutational polymorphisms and study of phylogenetic relationships of closely related isolates and would be applicable to many other species. [ABSTRACT FROM AUTHOR]

Amplified fragment length polymorphism (AFLP) was applied to 35 and 34 isolates, respectively, of Salmonella enterica serovar Typhimurium phage types DT 9 and DT 135, using eight primer pair combinations. Eight and 17 AFLP types were observed in DT 9 and DT 135, respectively. DT 9 is rare in the UK and common in Australia, but one AFLP form dominated with 28 isolates, comprising 22 of 25 UK isolates, four of five Australian isolates, one Jamaican and one Spanish isolate. Of the others, two UK isolates are closely related to the major form, two from elsewhere are in the major cluster and three isolates from different countries are in a separate cluster. For DT 135, two closely related AFLP types of seven and 11 isolates form the major cluster, which also includes 11 isolates, mostly in single-isolate AFLP types, while five isolates from different countries form a well-separated minor cluster. For both DTs all isolates are grouped together if only the phage type specific bands identified earlier are used, confirming their value for molecular-based ‘phage typing’. Polymorphic markers identified in this study could also be used for subtyping within both phage types. The value of AFLP is in locating DNA fragments useful for typing, but implementation of a replacement typing scheme would probably involve multiplex PCR or microarray technologies. [Copyright &y& Elsevier]

The article discusses a group of Escherichia coli O serogroups and the Salmonella enterical serogroup O: 6, 14 (H), share a common four-sugar backbone O-subunit structure, and possess nearly identical O-antigen gene clusters. Results support the proposal that these serotypes have a common sugar backbone encoded by a conserved O-antigen gene cluster, with variation due to side branches encoded by genes located elsewhere in the genome.

Abstract: Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson''s index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing. [Copyright &y& Elsevier]

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes. [ABSTRACT FROM AUTHOR]

Characterizes the O-antigen gene cluster in Salmonella enterica serogroup. Classification of lipopolysaccharides; Variation of O-antigen structure in S. enterica group B; Inactivation of O-antigen derivatives by genetic deletion.

Provides information on a study sequencing the 16S-23S spacer regions of two ribosomal operons (rrnA and rrnE) in seven representatives of the Salmonella enterica subspecies. Methodology and materials used to conduct the study; Information on the variability among the ISR1 operons; Results of the study; Discussion on the results.