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1 Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, 5200 Kiyotake, Miyazaki 889-1692, Japan
2 Division of Microbiology, Department of Infectious Disease, Faculty of Medicine, University of Miyazaki, 5200 Kiyotake, Miyazaki 889-1692, Japan
3 Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK
Correspondence
Tetsuya Hayashi
thayash{at}med.miyazaki-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1 intimin subclass and trigger actin polymerization by the Tir-TccP pathway. However, atypical O55 : H7 strains capable of triggering actin polymerization via the Tir-Nck pathway have recently been identified. In this study, we investigated the genotypic differences and phylogenetic relationships between typical and atypical O55 : H7 strains. We show that the atypical O55 : H7 strains, which express the 
intimin subclass and lack both tccP and tccP2, belong to an E. coli lineage distinct from the typical O55 : H7 and from the EPEC O55 : H6, which also uses the Tir-Nck actin polymerization pathway. We conducted genomic comparisons of the chromosomal regions covering the O-antigen gene cluster and its flanking regions between the three O55 lineages by RFLP analysis of PCR products and DNA sequencing analysis of about 65 kb chromosomal regions. This unexpectedly revealed that horizontal transfer of large fragments (
40 kb) encoding the O55-antigen gene cluster and part of the neighbouring colanic acid gene cluster was involved in the emergence of the three O55 E. coli lineages. The data provide new insights into the mechanisms involved in the generation of a wide variety of O-serotypes in Gram-negative bacteria.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB334576–AB334719 (housekeeping genes), AB334558–AB334567 (eae), AB355995–AB356000 (tccP), AB356001 (tccP2), AB334568–AB334575 (fliC) and AB353131–AB353133 (O55-antigen gene clusters and their flanking regions). The GEO accession number for the microarray data reported in this paper is GSE8889.
A supplementary figure and table are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In general, most genes required for O-antigen biosynthesis are clustered on the E. coli chromosome between the colanic acid biosynthesis gene cluster and the histidine biosynthesis (his) operon (Reeves et al., 1996). The organization of O-antigen biosynthesis gene clusters is O-antigen-specific. Sequence comparisons in many bacterial species and strains have shown that the variation between O-antigens has been generated by horizontal transfer of a part or the entire O-antigen gene clusters (Cunneen & Reeves, 2007; Curd et al., 1998; Fegan et al., 2006; Sugiyama et al., 1998; Wang et al., 2002; Xiang et al., 1994). Thus, the serotype of a strain correlates to some extent with its phylogeny, and often provides valuable information on its pathotype.
Enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important enteric pathogens for humans. EPEC is the most common bacterial cause of prolonged diarrhoea in infants in unindustrialized countries (Donnenberg et al., 1997; Nataro & Kaper, 1998), while EHEC, which causes diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome, is one of the most important intestinal pathogens in industrialized countries (Kaper, 1998; Nataro & Kaper, 1998). EPEC and EHEC colonize the intestinal epithelia via attaching and effacing (A/E) lesions (Nataro & Kaper, 1998), which are characterized by the degeneration of the epithelial cell brush border microvilli and the formation of actin-rich pedestals within the host enterocytes beneath the adherent bacteria (Nataro & Kaper, 1998). Most of the genes required to induce A/E lesions are located on the locus of enterocyte effacement (LEE) pathogenicity island (Elliott et al., 1998; Mellies et al., 1999), which encodes the outer-membrane adhesin intimin, a type III secretion system (T3SS), transcriptional regulators, and several T3SS effector proteins including the translocated intimin receptor (Tir).
LEE-positive strains can be classified into several subtypes based on sequence variation within LEE genes (Blanco et al., 2006; Lacher et al., 2006; McGraw et al., 1999; Ogura et al., 2007). Variation in the eae gene, which encodes intimin, has led to classification of more than 25 intimin subtypes (Lacher et al., 2006). Based on the sequence variation in Tir, the protein can be classified into two subtypes (DeVinney et al., 1999): Tir Y-P, which contains a tyrosine residue (Y474 in EPEC E2348/69) that is phosphorylated by host tyrosine kinases (Kenny, 1999) and uses Nck to trigger actin polymerization (Gruenheid et al., 2001), and Tir-S, which lacks a Y474 equivalent and typically triggers actin polymerization by the TccP pathway (Garmendia et al., 2004). The former is represented by the tir gene of EPEC O127 : H6 strain E2348/69 (thus referred to as tirE2348/68) and the latter by that of EHEC O157 : H7 strain Sakai (tirSakai) (Ogura et al., 2007). Previous studies have shown that tirE2348/68 is widely distributed in various EPEC and EHEC lineages (Ogura et al., 2007; Ooka et al., 2007), while tirSakai was found mainly in EHEC O157 : H7 and its close relatives O55 : H7 strains (Garmendia et al., 2005).
Most O55 : H7 strains are classified as EPEC (Pupo et al., 1997; Whittam et al., 1993), and the O157 : H7 lineage is known to have emerged from an O55 : H7-like EPEC ancestor by shifting the O-antigen from O55 to O157 and acquiring Shiga toxin (Stx1 and/or Stx2)-converting phages and a large virulence plasmid (pO157) (Wick et al., 2005). Therefore, O55 : H7 and O157 : H7 strains have a very similar or nearly identical genetic backbone. In fact, similarly to EHEC O157 : H7, most O55 : H7 strains possess an LEE island encoding 1-intimin and TirSakai (Garmendia et al., 2005). However, in a recent study (Garmendia et al., 2005), a few O55 : H7 strains were found to encode TirE2348/68. The aim of this study was to investigate the phylogenetic relationships and genotypic differences between typical O55 : H7 strains expressing TirSakai, atypical O55 : H7 strains expressing TirE2348/68 and EPEC O55 : H6 strains, which are also known to express TirE2348/68 (Ogura et al., 2007).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The sequenced EHEC O157 : H7 strain RIMD 0509952 (referred to as O157 Sakai) (Hayashi et al., 2001) was used as a reference strain in long PCR and microarray experiments. The bacterial strains were grown to stationary phase at 37 °C in Luria–Bertani broth. Genomic DNA was purified using the Genomic-tip 100/G and Genomic DNA buffer set (both from Qiagen) according to the manufacturer's instructions.
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). Detection of the nine pathotype-associated genes listed in Table 2 was also done by PCR. All PCRs were performed according to the protocols described previously (see references in Table 2).
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. PCR amplification was performed with an EX taq PCR kit (Takara Bio) by 30 amplification cycles of denaturation for 30 s at 96 °C, annealing for 30 s at 55 °C and primer extension for 60 s at 72 °C. Primers and PCR conditions for the analyses of 15 housekeeping genes were set up according to the protocol provided by the multi-locus sequence typing database for pathogenic E. coli (EcMLST: http://shigatox.net/mlst).
Phylogenetic analysis.
Multiple alignments of sequences were constructed by using the CLUSTAL W program (Thompson et al., 1994) in the MEGA3 software (Kumar et al., 2004), and then neighbour-joining (NJ) trees were generated by using Tamura–Nei model. A bootstrap test with 1000 replicates was used to estimate the confidence of the branching patterns of the trees.
Microarray analysis.
The protocol of comparative genomic hybridization (CGH) analysis using an O157 Sakai oligoDNA microarray has been described previously (Ogura et al., 2006). Briefly, probes were prepared for all protein-coding genes on the O157 Sakai genome (5447 genes in total). Probes were mostly 60-mer in length and two probes were prepared for each gene. Repeated genes with almost identical sequences (542 genes in total) were grouped into 151 repeated gene families, and each family was represented by a single probe. Three micrograms of genomic DNA from the reference strain (O157 Sakai) and each test strain were used to generate Cy5- and Cy3-labelled samples, respectively, and cohybridized on a single array. For each test strain, DNA labelling and hybridization were performed twice independently. Fluorescence intensities of the spots were collected using the ArrayVision 8.0 software (Imaging Research). After excluding low-quality signals with lower signal intensity and spotting abnormalities, the signal intensities of each spot were corrected by subtracting the local background, and subjected to the ‘presence or absence’ determination using the array-based genotyping software GACK (Kim et al., 2002). Finally, the presence or absence of each gene was determined according to the judgments of each spot in two independent hybridizations as described previously (Ogura et al., 2006). Hierarchical cluster analysis was performed using the Cluster 3.0 software (de Hoon et al., 2004), and the result was visualized using the Java TreeView software (Saldanha, 2004).
Long PCR and RFLP analyses.
The O55-anigen gene cluster and part of the his operon in the O55 : H7 strain TB182A have been sequenced previously (Wang et al., 2002). Based on the sequences of TB182A (accession no. AF461121) and O157 Sakai (BA000007), eight pairs of PCR primers were designed to amplify the 85.6 kb chromosomal region covering the O55-antigen gene cluster and its flanking regions (Table 2, Fig. 2). Each segment to be amplified (7.8–19.0 kb) overlapped its neighbours by 21–350 bp. Some primers used were previously designed for the whole-genome PCR scanning analysis of O157 strains (Ohnishi et al., 2002). The protocol for long PCR amplification was also described previously (Ohnishi et al., 2002). For the RFLP analysis of long PCR products, PCR products were purified with a QIAquick PCR purification kit (Qiagen), digested with HaeIII (Takara Bio), and analysed by agarose gel electrophoresis.
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). Each clone was sequenced with universal primers T7 and T3 using an ABI PRISM 3100 automated sequencer (Applied Biosystems). Sequence data were assembled by the Sequencher software, ver. 4.2.2 (Gene Codes). The open reading frame finder program in the in silico MolecularCloning Genomic Edition, version 1.4.6 (in silico biology, Kanagawa, Japan) was used for the initial gene finding and annotation. Final annotation was manually done according to the results of database searches using the BLASTP program (Altschul et al., 1997). Sequence alignments and comparisons were performed by the Sequencher and the CLUSTAL W programs. |
RESULTS |
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), six (TB182A, DEC5d, WC211, ICC57, 5905 and 12646) possessed tirSakai, while two (WC416 and ICC58) possessed tirE2348/68 (Garmendia et al., 2005). All the O55 : H7 strains with TirSakai were tccP positive (strain 5905 was also tccP2 positive) and possessed the eae gene (2805 bp) encoding 1 subclass intimin (Int-1), which was almost identical to the eae gene of O157 Sakai, with at most three nucleotide differences. In contrast, the two O55 : H7 strains with TirE2348/68 were tccP and tccP2 negative and possessed an eae gene (2808 bp) encoding the subclass intimin (Int-), which was almost identical to the eae gene of EHEC O111 : H8 strain DEC8b (accession no. AF449420), with a single nucleotide difference. Although the intimin of strain ICC58 had been classified as Int- in several previous studies where PCR-based intimin-typing systems were employed, the sequence analysis clearly indicated that strain ICC58 encodes Int-. On the basis of their virulence gene combination, the eight O55 : H7 strains examined here were divided into two subgroups referred to as O55 : H7/Int-1 and O55 : H7/Int-, respectively. The three O55 : H6 strains with TirE2348/68 were tccP and tccP2 negative, and possessed the eae gene (2820 bp) encoding the 
subclass intimin (Int-), which was almost identical to the eae gene of EPEC O127 : H6 strain E2348/69 (accession no. AF022236), with three nucleotide differences.
Other virulence gene profiles
The presence of other virulence genes known to be associated with EPEC, EHEC or enteroaggregative E. coli (EAEC) was examined in each of the O55 strains by PCR (Table 1). The O55 : H7/Int- and O55 : H7/Int-1 strains did not possess the EPEC adherence factor (EAF) plasmid or the bfpA gene encoding a subunit of bundle-forming pili. Therefore, they were categorized as atypical EPEC (Trabulsi et al., 2002), except for O55 : H7/Int-1 strains 5905 and 12646, which were stx2 or stx1 positive and thus categorized as EHEC. The O55 : H6 strains were typical EPEC as they harbour the EAF plasmid and carry the bfpA gene (Trabulsi et al., 2002). Of note is the fact that the O55 : H7/Int- strains were found to contain the irp2 gene, which encodes an iron-repressible high-molecular-weight protein HMWP2, one of the virulence gene markers of EAEC (Schubert et al., 1998). However, other EAEC markers, aggR (encoding a regulator for the expression of aggregative adherence fimbriae) and astA (encoding heat-stable enterotoxin EAST1), were both negative in the O55 : H7/Int- strains (data not shown).
Comparison of fliCH7 sequences
The fliC genes encoding H7 antigen are known to exhibit sequence variations, and 10 distinct subtypes (H7-1 to H7-10) have been described (Wang et al., 2000). All the fliCH7 sequences of the O55 : H7/Int-1 strains were identical to the H7-5 sequence of O157 : H7 strains. In contrast, those of the O55 : H7/Int- strains were almost identical to the H7-7 sequence of an O19ab : H7 strain, with one or two nucleotide differences. Eight nucleotide differences exist between the DNA sequences of H7-5 and H7-7 (Wang et al., 2000).
Phylogenetic relationship of O55 strains
We analysed the phylogenetic relationship of the 11 O55 strains (six O55 : H7/Int-1, two O55 : H7/Int- and three O55 : H6/Int-) based on the concatenated nucleotide sequences (7395 bp) of 15 housekeeping genes. As shown in Fig. 1, the O55 strains formed three distinct clusters. All the O55 : H7/Int-1 strains were tightly associated with the EHEC O157 : H7 lineage, while the two O55 : H7/Int- strains and the three O55 : H6/Int- strains formed distinct clusters. The nucleotide sequence identity of the housekeeping genes within each O55 lineage was >99.9 %, but those between the three lineages ranged from 97.2 to 97.6 %.
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1, two O55 : H7/Int- and three O55 : H6/Int- strains) by using the O157 Sakai oligoDNA microarray (Fig. 2). Most O157 Sakai genes are represented by two probes on this array (Ogura et al., 2006). The probes were divided into two groups by their homologies to the K-12 genome sequences: probes sharing 90 % identity and probes sharing <90 % identity. Genes were then classified into ‘conserved in K-12’ genes (both probes share 90 % identity), ‘partly conserved in K-12’ genes (one probe shares 90 % identity but the other <90 % identity), or ‘Sakai-specific’ genes (both probes share <90 % identity). Genes with more than one copy in the O157 Sakai genome (referred to as repeated genes) were analysed separately from singleton genes (see Supplementary Table S1, available with the online version of this paper).
Analysis of singleton genes and a hierarchical clustering tree are shown in Fig. 2 (for details, see Table S1). The O55 strains examined were divided into three clusters according to their gene contents. The clustering pattern was consistent with their phylogenies; the O55 : H7/Int-1 group was closely related to O157 Sakai.
Among the 4905 O157 Sakai singleton genes, ‘Conserved in K-12’ genes (3651 genes) were highly conserved in the O55 : H7/Int-1 strains (98.3–99.1 %) and also in other O55 strains, although to slightly lesser extents (88.3–95.9 % but most >90 %). ‘Partly conserved in K-12’ genes (101 genes) were also highly conserved in the O55 : H7/Int-1 strains (94.1–96.0 %), but intermediately in the other strains (51.5–75.2 %). ‘Sakai-specific’ genes (1153 genes) exhibited moderate conservation in the O55 : H7/Int-1 strains (55.4–61.5 %), but were very poorly conserved in the O55 : H7/Int- (22.1–25.0 %) and O55 : H6 (25.9–35.9 %) strains. ‘Sakai-specific’ genes that were found to be missing in the O55 : H7/Int-1 strains were mainly on prophage and prophage-like elements (see Supplementary Fig. S1, available with the online version of this paper). In the other O55 strains, the missing genes were dispersed on the genome. These results indicated that O55 : H7/Int- strains were very different from the O55 : H7/Int-1 group also with respect to their gene repertoire.
PCR-RFLP analysis of O55-antigen gene clusters and their flanking regions
The results described above clearly indicated that O55 : H7/Int-1, O55 : H7/Int- and O55 : H6 strains belonged to distinct E. coli lineages, but expressed the same O55 antigen. In order to understand the genetic mechanism underlying the emergence of O55 strains in the three different E. coli lineages, we compared the O-antigen gene clusters of the three O55 lineages. We first performed a PCR-RFLP analysis of eight chromosomal segments covering the 85.6 kb region where the O-antigen gene cluster is located (Fig. 3). Segment 3 encoding most of the O55 gene cluster and segment 4 encoding half of the colanic acid biosynthesis gene cluster exhibited an identical RFLP pattern in all the O55 strains. In contrast, segments 1, 2, 7 and 8 exhibited different patterns between the three lineages, but an almost identical pattern within a lineage, with only few minor variations. Segments 5 and 6 exhibited an identical pattern in all the O55 : H7/Int-1 and O55 : H6 strains with one exception (segment 6 of WC211), but those of O55 : H7/Int- strains were slightly different from the others. These results suggest that the three O55 lineages share an identical or nearly identical sequence of the O55 and part of the colanic acid biosynthesis gene clusters, and that the left and right junctions between the lineage-specific and shared sequences are located in segment 2 and in segments 5 or 7, respectively.
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1, WC416 for O55 : H7/Int- and ICC219 for O55 : H6). The sequence of the O55 gene cluster of TB182A was identical to that previously reported (Wang et al., 2002).
Overall gene organization.
The overall gene organization in the sequenced region was almost identical between the three strains, with a few exceptions in WC416 (Fig. 4), which lacked a 745 bp fragment encoding two small genes (corresponding to ECs2869 and 2870 of O157 Sakai), but contained a 684 bp fragment encoding the yegJ gene. The gene organization of the yegH–yegL region of WC416 was thus identical to that of E. coli K-12 strain WC3110 (data not shown). This may be linked to the relatively close phylogenetic relationship between H7/Int- strains and K-12 (Fig. 1).
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). Comparison of TB182A and WC416 defined the left and right junctions of the lineage-specific and shared sequences within hisI and wzc, respectively. The 42.3 kb region from hisI to wzc had only 29 single nucleotide polymorphisms (SNPs) and a 6 bp indel. The nucleotide sequence identities of lineage-specific sequences were 96.8 % on the left hand and 97.7 % on the right hand, showing a similar level of sequence diversity as that observed in the 15 housekeeping genes (97.5 %). Between WC416 and ICC219, the left and right junctions were localized within ugd and wzc, respectively. The 40.0 kb region from ugd to wzc had only 33 SNPs and a 1 bp and a 6 bp indel. The identities of lineage-specific sequences were 95.2 % (left) and 97.2 % (right). Similarly, between ICC219 and TB182A, the left and right junctions were within ugd and yegE, the 49.6 kb region from ugd to yegE had 27 SNPs and a 1 bp indel, and the identities of lineage-specific sequences were 94.9 % (left) and 96.0 % (right). It is noteworthy that the wzz gene and its upstream region of O55 : H6 strain ICC219 had a highly divergent sequence compared with the others.
Evolutionary relationship of flanking regions.
To evaluate the evolutionary relationships of the three O55 lineage specific sequences flanking the O55-antigen and colanic acid biosynthesis gene clusters, concatenated sequences of the hisGDCBHA genes (left hand), and of the alkA and yegDIKL genes (right hand) (5667 and 5583 bp, respectively) were compared. Both sequences generated phylogenetic trees similar to that of housekeeping genes (Fig. 5). This result indicated that both flanking regions were inherited in each lineage through their evolution. The phylogenetic relationship of the colonic acid biosynthesis genes and their homologues in other E. coli strains was also analysed by using the concatenated sequences of the wcaA–F genes (3897 bp) and the wcaK–M genes (5061 bp). In both cases, the sequences from three O55 lineages form a single cluster, but showed no close relationship to any other E. coli sequences currently available (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1 (O55 : H7/Int-1) and TccP, atypical O55 : H7 strains were also categorized as atypical EPEC but encoded Int- (O55 : H7/Int-) and lacked TccP or TccP2. Although Int- was originally identified in E. coli O111 strains (Tarr & Whittam, 2002), it has been found in other E. coli serotypes including O5 : H11, O35 : H25, O76 : H7, O84 : H25 and O103 : H2 (Ramachandran et al., 2003). Interestingly, an O55 : H7 strain with Int- (named LTO55-43) has been described before (Lacher et al., 2006) and the strain information is available at the STEC centre Isolate Database (http://www.shigatox.net/cgi-bin/stec/index). Extracting data from this resource has shown that the sequences of 15 housekeeping genes of this strain are identical to those of the two O55 : H7/Int- strains we analysed in this study. Strains ICC58 and LTO55-43 were isolated in Brazil but WC416 in Thailand (Table 1), suggesting the global distribution of the O55 : H7/Int--clonal group.
An intriguing genotype characteristic of the O55 : H7/Int- strains was the presence of the irp2 gene, which is located in an operon specifying the yersiniabactin-mediated iron-uptake system. This operon was originally identified on the high-pathogenicity island of Yersinia pestis (Fetherston et al., 1995). Schubert et al. (1998) reported that 93 % of 60 EAEC isolates were irp2 positive. The authors reported that the gene was detected in a few EPEC but not in EHEC strains. However, more recently, Girardeau et al. (2005) reported that 34 % of the strains belonging to the EHEC 2 clonal group also harboured this island. The irp2 gene could be used as an additional genetic marker to distinguish O55 : H7/Int- strains from O55 : H7/Int-1 strains.
Horizontal gene transfer (HGT) is widely regarded as a major genetic mechanism to generate variations in O-serotypes between and within bacterial species, although the mechanism is not well understood. Most evidence for the horizontal transfer of O-antigen gene clusters has been obtained by comparing their sequences between closely related strains with different serotypes or phylogenetically unrelated strains with the same serotype. An excellent example of the former approach is provided by analyses of Vibrio cholerae O1 and O139 Bengal. Several studies have shown that a V. cholerae O1 progenitor had acquired a new O-antigen gene cluster and emerged as O139 Bengal (Mooi & Bik, 1997; Stroeher & Manning, 1997; Stroeher et al., 1998). In this O-antigen shift, a 22 kb region for the O1-antigen gene cluster was replaced by a 35 kb fragment encoding the O139-antigen gene cluster. The O-antigen shift from EPEC O55 : H7 to EHEC O157 : H7 appears to be the result of recombination within the galF gene (Wang et al., 2002), but more details remained to be clarified.
The latter type of studies of O-antigen evolution mainly involved sequence comparisons of O-antigen gene clusters between strains with a common antigenicity but belonging to different species. The data from a comparison of E. coli O9a and Klebsiella O3 suggested that about two-thirds of the O-antigen gene cluster of E. coli O9a was derived from Klebsiella O3 (Sugiyama et al., 1998). It has also been shown that the gene organization of the O-antigen gene cluster of E. coli O98 and Yersinia kristensenii O11 is identical and the gene cluster of Y. kristensenii is flanked by remnants of insertion sequences (ISs), thus suggesting the IS-mediated transfer of the O-antigen gene cluster from E. coli O98 to Y. kristensenii O11 (Cunneen & Reeves, 2007). These and other studies have provided substantial evidence for the dissemination of O-antigen biosynthesis genes between species through HGT, but horizontally transferred sequences and/or their junctions have rarely been well defined.
The present sequence comparison of the O55-antigen gene clusters and their flanking regions between the three different E. coli lineages provided strong evidence that the O-antigen shifts have taken place via the horizontal transfer of large fragments (>40 kb) encoding not only the O-antigen gene cluster but also neighbouring colanic acid biosynthesis genes (Fig. 4). The possible recombination points on the chromosomes have also been identified (Fig. 4). Bacteriophage-mediated general transduction or plasmid-mediated conjugation appears to have been involved in the horizontal transfer of such large chromosomal fragments.
We cannot determine whether these fragments were transferred directly or indirectly to the three O55 lineages from a common ancestor. But, based on the sequence variations of 15 housekeeping genes, it can be estimated that three lineages were separated from a common ancestor 4.7–5.7x106 years ago if a uniform rate of synonymous substitution proposed for E. coli and Salmonella enterica (4.7x10–9 per site per year) is applied (Lawrence & Ochman, 1998). In contrast, the sequence variations of the 40 kb region shared by the different lineages imply that the three O55 gene clusters were separated 0.9–1.4x105 years ago. Thus, each lineage acquired the O55 gene cluster relatively recently.
Of interest is the fact that possible recombination points are not the same between the three lineages. No repetitive sequences were found around the recombination points, such as IS elements and Chi octamer sequences (GCTGGTGG), which may be involved in the transfer and/or recombination of incoming foreign DNA (Dixon & Kowalczykowski, 1993). Although an H-repeat is present in the O55 gene cluster, it is not located at the junctions (Fig. 4). Thus, in either lineage, the replacement of the incoming fragment seems to have been achieved by homologous recombination between the allelic regions. To better understand the genetic mechanism(s) generating a wide variety of O-serotypes, we need to know more about whether transfer of such large fragments is involved in O-antigen shifts in other E. coli lineages and also in other bacterial species.
Although the O55-antigen biosynthesis genes of the three lineages were almost identical, the DNA sequence of the wzz gene of ICC219 was significantly divergent from its counterparts in the other two lineages. The encoded protein showed only 89.3 % amino-acid sequence identity to those of strains TB182A and WC416. The Wzz protein is involved in the chain length determination of O-antigen. Guo et al. (2005), who analysed two E. coli strains of serotype O86 that contained Wzz proteins with divergent sequences (90 % identical in amino-acid sequence), reported that the difference in Wzz proteins strongly affects the length of O-antigen chains. The length of the O-antigen chain affects various properties of Gram-negative bacteria, including sensitivities to the serum (Guo et al., 2005), complement (Murray et al., 2006) and bacteriophages (Iguchi et al., 2007), and the function of T3SSs (West et al., 2005). As such, ICC219 and the two O55 strains representing the other lineages (TB182A and WC416) provide good experimental organisms to examine how difference in O-antigen chain lengths might affect virulence potential.
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ACKNOWLEDGEMENTS |
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Edited by: I. R. Henderson
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Received 15 September 2007;
revised 3 November 2007;
accepted 9 November 2007.
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