Serotype O:8 isolates in the Yersinia pseudotuberculosis complex have different O-antigen gene clusters and produce various forms of rough LPS

In Yersinia pseudotuberculosis complex, the O-antigen of LPS is used for the serological characterization of strains, and 21 serotypes have been identified to date. The O-antigen biosynthesis gene cluster and corresponding O-antigen structure have been described for 18, leaving O:8, O:13 and O:14 unresolved. In this study, two O:8 isolates were examined. The O-antigen gene cluster sequence of strain 151 was near identical to serotype O:4a, though a frame-shift mutation was found in ddhD, while No. 6 was different to 151 and carried the O:1b gene cluster. Structural analysis revealed that No. 6 produced a deeply truncated LPS, suggesting a mutation within the waaF gene. Both ddhD and waaF were cloned and expressed in 151 and No. 6 strains, respectively, and it appeared that expression of ddhD gene in strain 151 restored the O-antigen on LPS, while waaF in No. 6 resulted in an LPS truncated less severely but still without the O-antigen, suggesting that other mutations occurred in this strain. Thus, both O:8 isolates were found to be spontaneous O-antigen-negative mutants derived from other validated serotypes, and we propose to remove this serotype from the O-serotyping scheme, as the O:8 serological specificity is not based on the O-antigen.Peer reviewe

Two extremely divergent sequence forms of the genes that define Escherichia coli group 3 capsules suggest a very long history since their common ancestor

Capsules are a critical virulence factor in many pathogenic Escherichia coli, of which groups 2 and 3 capsules are synthesised by the ABC transporter pathway. The well-studied forms are in group 2 and much of our knowledge of group 3 is inferred from our understanding of group 2. We analyse six group 3 gene clusters including representatives of K10, K11 and K96, and find unexpected diversity. Groups 2 and 3 both have gene clusters with terminal regions 1 and 3 containing mostly genes shared by all members of both groups, plus a central region 2, that in group 2 has the genes for synthesising the serotype-specific repeat unit. We find that in all but one case group 3 gene clusters include, in addition to serotype-specific genes, a previously unrecognised set of shared genes in region 2 that probably codes for an additional structural element. Also, the six shared genes in regions 1 and 3 of group 3 exist in two very different sequence forms. It appears that the E. coli ABC transporter capsules have a very long history, with more fundamental diversity present in group 3, but greater diversity in the exposed strongly antigenic serotype-specific component encoded by region 2.</p

Genetics and evolution of the Salmonella galactose-initiated set of o antigens.

This paper covers eight Salmonella serogroups, that are defined by O antigens with related structures and gene clusters. They include the serovars that are now most frequently isolated. Serogroups A, B1, B2, C2-C3, D1, D2, D3 and E have O antigens that are distinguished by having galactose as first sugar, and not N-acetyl glucosamine or N-acetyl galactosamine as in the other 38 serogroups, and indeed in most Enterobacteriaceae. The gene clusters for these galactose-initiated appear to have entered S. enterica since its divergence from E. coli, but sequence comparisons show that much of the diversification occurred long before this. We conclude that the gene clusters must have entered S. enterica in a series of parallel events. The individual gene clusters are discussed, followed by analysis of the divergence for those genes shared by two or more gene clusters, and a putative phylogenic tree for the gene clusters is presented. This set of O antigens provides a rare case where it is possible to examine in detail the relationships of a significant number of O antigens. In contrast the more common pattern of O-antigen diversity within a species is for there to be only a few cases of strains having related gene clusters, suggesting that diversity arose through gain of individual O-antigen gene clusters by lateral gene transfer, and under these circumstances the evolution of the diversity is not accessible. This paper on the galactose-initiated set of gene clusters gives new insights into the origins of O-antigen diversity generally

The WbaK acetyltransferase of Salmonella enterica group E gives insights into O antigen evolution

O antigens are polysaccharides consisting of repeat units of three to eight sugars, generally assembled by genes in a discrete O antigen gene cluster. Salmonella enterica produces 46 forms of O antigen, and most of the variation is determined by genes in the gene cluster. However in some cases the structures are modified by enzymes encoded outside of the gene cluster, and several such modifications have been reported for Salmonella enterica group E, some with the genes on bacteriophages and one gene at a distant chromosomal site. We identified the enzyme, WbaK, that is responsible for O-acetylating the subgroup E1 O antigen, and found that the gene is located just downstream of the gene cluster as currently known. The wbaK gene appears to have been imported by a recombination event that also replaced the last 37 bp of the wbaP gene, indicating that homologous recombination was involved. Some of the group E strains we studied must have the original gene cluster, as they lack wbaK and the sequence downstream of wbaP is very similar to that in several other S. enterica O antigen gene clusters. In effect the gene cluster was extended by one gene in subgroup E1. It appears that a function that is usually encoded by a gene outside of the gene cluster has been added to the gene cluster, in this case giving an example of how such gene clusters can evolve.</p

O-antigen gene clusters of the Galactose-initiated <i>S. enterica</i> serogoups.

<p>Genes are colour coded by synthesis pathways. Height of the coloured blocks indicates level of sequence similarity to group B1 genes; if little or no sequence similarity, the gene is colour coded by a vertical strip according to the pathway. Transferase genes are indicated by bold horizontal boundaries. Major junctions between blocks of genes with different relationships or levels of similarity are indicated by dashed lines. Gene names are shown and gene remnants labelled. The O-unit polymerase is shown on the right, named after the group in which it was first found, together with the linkage that it forms. Drawn to scale.</p

Group A triplication.

<p>Alignment of group A sequences with and without the triplication of the <i>wbaV</i> region. Strain names are indicated on the left (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069306#tab1" target="_blank">Table 1</a> for details). Both strains have identical gene clusters with the exception of the triplication of the region indicated. Drawn to scale.</p

Relationships of group D1, D2 and E gene clusters.

<p>Gene clusters of groups D1, D2 and E are shown, with the D2 regions proposed to be derived from D1 and E indicated by red connecting lines. Genes are colour-coded as in other figures. Sequence alignments of D2 with D1 (above) and with E (below) are shown for the junctions between homologous and non-homologous sequence, with the <i>E. coli</i> K-12 H-repeat element, <i>yhhI</i>, used for the H-repeat comparison. Cartoons of 2 O-unit repeat structures of each group are shown to the right to highlight the structural differences that relate to the genetic differences between clusters: purple, polymerisation linkage; green, Man-Rha glycosidic linkage; orange, DDH sidebranch. Abbreviations: G, galactose; M, mannose; R, rhamnose; T, tyvelose.</p

Comparison of the segments between the <i>wbaV</i> and <i>wbaU</i> genes of Groups D1, D3, B2 and B1.

<p>Base positions are indicated and are taken from either published or deposited sequences, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069306#tab1" target="_blank">Table 1</a>. The junctions with the neighbouring <i>wbaV</i> and <i>wbaU</i> genes are indicated by solid lines. A box cartoon to the left shows the gene clusters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069306#pone-0069306-g004" target="_blank">Figure 4</a>. Red arrows indicate a proposed diversification pathway of segments.</p

Structures of the Galactose-initiated O units of <i>S. enterica</i>.

<p>A single O unit is shown as it is built on the lipid carrier, Und-PP. The transferase responsible for the addition of each sugar of the O unit is shown. The repeat unit is given in square brackets. The Wzy polymerisation linkage is also shown to the left of the repeat unit. Sugar abbreviations: Abe, abequose; Gal, galactose; Man, mannose; Par, paratose; Rha, rhamnose; Tyv, Tyvelose.</p

The 6 known group B1 and B2 forms of the region between <i>wbaV</i> and <i>wbaU.</i>

<p>This is an expansion of a small segment of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069306#pone-0069306-g004" target="_blank">Figure 4</a> to show detail of the differences. The box insert has a cartoon of the clusters as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069306#pone-0069306-g004" target="_blank">Figure 4</a>. Forms differ in the presence or absence of insertion sequences, and either a non-functional <i>wzy</i> remnant (marked with an asterisk; characteristic of B1), or the complete <i>wzy</i> gene that characterises B2. IS types are colour-coded and labelled. Ends of sequence deletion/insertions are indicated by dashed lines, and the junctions with the neighbouring <i>wbaV</i> and <i>wbaU</i> genes are indicated by solid lines. A representative of each form is named on the right, as is information regarding segment length, the number of isolates within each form and their subspecies distribution. The proposed diversification from an ancestral Schleissheim form to other forms is shown by red arrows. Dashed arrow lines indicate alternative pathways. Modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069306#B39" target="_blank">39</a>]..</p