Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens

Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E. coli O55 and S. enterica O50. The O-antigen gene cluster sequences for E. coli O157 and E. coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica O30 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coli O157 cluster. We also determined the sequence of the S. enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E. coli O55 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.     

The Escherichia coli O111 and Salmonella enterica O35 gene clusters: gene clusters encoding the same colitose-containing O antigen are highly conserved

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. Escherichia coli and Salmonella enterica each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical S. enterica or E. coli genes (51%) is thought to indicate that the O-antigen DNA originated in species other than S. enterica or E. coli and was captured by lateral transfer. The O-antigen structure of Salmonella enterica O35 is identical to that of E. coli O111, commonly found in enteropathogenic E. coli strains. This O antigen, which has been shown to be a virulence factor in E. coli, contains colitose, a 3,6-dideoxyhexose found only rarely in the Enterobacteriaceae. Sequencing of the O35-antigen gene cluster of S. enterica serovar Adelaide revealed the same gene order and flanking genes as in E. coli O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between S. enterica and E. coli. We conclude that the ancestor of E. coli and S. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.     

Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain

The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterica serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O-antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopolysaccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.     

Structural and genetic characterization of enterohemorrhagic Escherichia coli O145 O antigen and development of an O145 serogroup-specific PCR assay

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.     

Structural and genetic relationships between the O-antigens of Escherichia coli O118 and O151

O-antigen (O-polysaccharide) is a highly variable part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which is used as the basis for bacterial serotyping and is essential for the full function and virulence of bacteria. In this work, the structure and genetics of the O-antigens of Escherichia coli O118 and O151 were investigated. Both O-polysaccharides were found to contain ribitol phosphate and have similar structures, the only difference between their backbones being one linkage mode (β1→3 in E. coli O118 vs. β1→2 in E. coli O151), which, most probably, is the linkage between the oligosaccharide repeats (O-units). The O-antigen gene clusters of the two bacteria are organized in the same manner and share high-level identity (>99%). Analysis of the wzy genes from E. coli O118 and O151 strains, which are responsible for the linkage between O-units, revealed only one nucleotide substitution, resulting in one amino acid residue substitution. The possible genetic events that may lead to the structural difference between two O-antigen structures are discussed. Salmonella O47 has the same O-unit backbone and a similar O-antigen gene cluster (OGC) (the DNA identity ranges from 74% to 83%) as E. coli O118 and O151. It was suggested that the OGCs of the three bacteria studied originated from a common ancestor.     

Identification of the two glycosyltransferase genes responsible for the difference between Escherichia coli O107 and O117 O-antigens

The O-antigen is one of the most variable Gram-negative cell constituents, and its specificity is important for bacterial niche adaptation. The observed diversity of O-antigen forms is mainly due to genetic variations in O-antigen gene clusters. Less common is a change of gene function due to nucleotide substitution; a new instance of which is reported here. The O-antigens of E. coli O107 and O117 have similar structures differing only in a single sugar residue (GlcNAc in O107 substituted for Glc in O117). These O-antigen gene clusters contain the same set of 11 genes and share 98.6% overall DNA identity. The function of the genes in the gene clusters have been proposed previously, and a glycosyltransferase gene (wclY) with nucleotide polymorphism in each strain was proposed to transfer different sugars in different strains. To identify the gene responsible for the transfer of different sugars, wclY mutants of E. coli O107 and O117 were constructed, and each mutant was complemented with the wclY genes cloned from both O107 and O117. Structural analysis of the O-antigens of the four recombinant strains identified wclY as a Glc-transferase in O117 and a GlcNAc-transferase in O107. The evolutionary relationship of E. coli O107 and O117 O-antigens is also discussed.     

Characterization of the Escherichia coli O59 and O155 O-antigen gene clusters: the atypical wzx genes are evolutionary related

O-antigens are highly polymorphic. The genes specifically involved in O-antigen synthesis are generally grouped together on the chromosome as a gene cluster. In Escherichia coli, the O-antigen gene clusters are characteristically located between the housekeeping genes galF and gnd. In this study, the O-antigen gene clusters of E. coli O59 and E. coli O155 were sequenced. The former was found to contain genes for GDP-mannose synthesis, glycosyltransferase genes and the O-antigen polymerase gene (wzy), while the latter contained only glycosyltransferase genes and wzy. O unit flippase genes (wzx) were found immediately downstream of the gnd gene, in the region between the gnd and hisI genes in these two strains. This atypical location of wzx has not been reported before, and furthermore these two genes complemented in trans despite the fact that different O-antigen structures are present in E. coli O59 and O155. A putative acetyltransferase gene was found downstream of wzx in both strains. Comparison of the region between gnd and hisI revealed that the wzx and acetyltransferase genes are closely related between E. coli O59 and O155, indicating that the two gene clusters arose recently from a common ancestor. This work provides further evidence for the O-antigen gene cluster having formed gradually, and selection pressure will eventually bring O-antigen genes into a single cluster. Genes specific for E. coli O59 and O155, respectively, were also identified.     

Molecular analysis of Shigella boydii O1 O-antigen gene cluster and its PCR typing

Shigella is an important human pathogen and is closely related to Escherichia coli. O-antigen is the most variable part of the lipopolysaccharide on the cell surface of Gram-negative bacteria and plays an important role in pathogenicity. The O-antigen gene cluster of S. boydii O1 was sequenced. The putative genes encoding enzymes for rhamnose synthesis, transferases, O-unit flippase, and O-unit polymerase were identified on the basis of homology. The O-antigen gene clusters of S. boydii O1 and E. coli O149, which share the same O-antigen form, were found to have the same genes and organization by adjacent gene PCR assay. Two genes specific for S. boydii O1 and E. coli O149 were identified by PCR screening against E. coli- and Shigella-type strains of the 186 known O-antigen forms and 39 E. coli clinical isolates. A PCR sensitivity of 103 to 104 CFU/mL overnight culture of S. boydii O1 and E. coli O149 was obtained. S. boydii O1 and E. coli O149 were differentiated by PCR using lacZ- and cadA-based primers.     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Structural elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 180/C3 and its immunochemical relationship with E. coli O5 and O65

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 180/C3 has been determined. Sugar and methylation analysis together with (1)H and (13)C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure: -->2)beta-D-Quip3NAc-(1-->3)beta-D-RIBf-(1-->4)beta-D-Galp-(1-->3)alpha-D-GalpNAc-(1-->. Analysis of NMR data indicates that the presented sequence of sugar residues also represents the biological repeating unit of the O-chain. The structure is closely related to that of O-antigen polysaccharide from E. coli O5 and partially to that of E. coli O65. The difference between the O-antigen from the 180/C3 strain and that of E. coli O5 is the linkage to the D-Quip3NAc residue, which in the latter strain is 4-O-substituted. The E. coli O65 O-antigen contains as part of its linear pentasaccharide repeating unit a similar structural element, namely -->4)-beta-d-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->2)-beta-D-Quip3NAc-(1-->, thereby indicating that a common epitope could be present for the two polysaccharides. Monospecific anti-E. coli O5 rabbit serum did not distinguish between the two positional isomeric structures neither in slide agglutination nor in an indirect enzyme immunoassay. The anti-O65 serum did react with both the 180/C3 and O5 LPS showing a partial cross-reactivity.     

Recent horizontal transmission of plasmids between natural populations of Escherichia coli and Salmonella enterica.

Seventy-one natural isolates obtained from a Salmonella reference collection were examined for the presence of plasmids closely related to the Escherichia coli F plasmid. The collection consists of several serovars of the S. enterica Typhimurium complex, subspecies I, to which 99% of pathogenic salmonellae belong. Molecular genetic techniques of DNA hybridization, along with PCR and DNA sequencing, were used to examine the occurrence, distribution, and genetic diversity of F-like plasmids among Salmonella strains. The F plasmid genes examined were finO, traD, traY, and repA, which map at dispersed positions on the F plasmid of E. coli. Comparative sequence analysis of each of the four genes in Salmonella plasmids showed them to be homologous (in some cases, virtually identical) to those found in F plasmids of E. coli natural isolates. Furthermore, the frequency of F-like plasmids in Salmonella strains was approximately the same as that observed in the E. coli Reference Collection. However, in Salmonella, the distribution was confined predominately to the serovars Typhimurium and Muenchen. The unexpected finding of a shared pool of F-like plasmids between S. enterica and E. coli demonstrates the significant role of conjugation in the histories of these important bacterial species.     

Overexpression of the waaZ gene leads to modification of the structure of the inner core region of Escherichia coli lipopolysaccharide,truncation of the outer core,and reduction of the amount of O polysaccharide on the cell surface

The waa gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in Escherichia coli and Salmonella: Homologs of the waaZ gene product are encoded by the waa gene clusters of Salmonella enterica and E. coli strains with the K-12 and R2 core types. Overexpression of WaaZ in E. coli and S. enterica led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an E. coli isolate with an R1 core (waaZ is lacking from the type R1 waa gene cluster) with a copy of the waaZ gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core.     

The Yersinia kristensenii O11 O-antigen gene cluster was acquired by lateral gene transfer and incorporated at a novel chromosomal locus

We have sequenced the O-antigen gene clusters for the Escherichia coli O98 and Yersinia kristensenii O11 O antigens. The basic structures of these O antigens are identical, and the sequence data indicate that Y. kristensenii O11 gained its O-antigen gene cluster by lateral gene transfer (LGT). Escherichia coli O98 has a typical O-antigen gene cluster between galF and gnd as is usual in E. coli. However, the O-antigen gene cluster of Y. kristensenii O11 is not located at the traditional Yersinia O-antigen gene cluster locus, between hemH and gsk, but at a novel chromosomal locus between aroA and cmk where it is flanked by remnant galF and gnd genes that indicate the probable source of the gene cluster. Phylogenetic analysis indicated that the source was not E. coli itself but a species in the Escherichia, Salmonella, and Klebsiella group of genera. Although other O-antigen studies imply LGT on the basis of the hypervariability of the loci and GC content, this report also identifies a potential donor and provides evidence for the mechanism involved. Remnant insertion sequence (IS) sequences flank the galF and gnd remnants and suggest that LGT of the gene cluster was IS mediated.     

Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement

A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzzST and wzzfepE) or three heterologous (wzzO139 of Vibrio cholerae and wzzSF and wzzpHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.     

Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts

Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been associated with contaminated sprouts. We examined how S. enterica serovars, E. coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts. Growth on and adherence to sprouts were not significantly different among different serovars of S. enterica, but all S. enterica serovars grew on and adhered to alfalfa sprouts significantly better than E. coli O157:H7. E. coli O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of S. enterica remained attached per sprout. S. enterica Newport adhered to 3-day-old sprouts as well as Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella aquatilis, whereas the growth rates of all four strains throughout seed sprouting were similar. S. enterica Newport and plant-associated bacteria adhered 10- to 1,000-fold more than E. coli O157:H7; however, three of four other E. coli serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than E. coli O157:H7 and as well as the Pseudomonas and Rahnella strains. Therefore, attachment to alfalfa sprouts among E. coli serotypes is variable, and nonpathogenic strains of E. coli to be used as surrogates for the study of pathogenic E. coli may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.     

Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test

AIMS: The aims of the study were to characterize the O91 O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O91 and to provide the basis for a specific PCR test for rapid detection of E. coli O91. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species were used to amplify the 10-kbp O91 O-antigen biosynthesis locus of STEC O91. A DNA library representative of this cluster allowed two O91 specific probes to be identified, and two specific PCR O91 serotyping tests to be successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.     

Structure and gene cluster of the O-antigen of Escherichia coli O109; chemical and genetic evidences of the presence of L-RhaN3N derivatives in the O-antigens of E. coli O109 and O119

O-antigen representing the O-polysaccharide chain of the lipopolysaccharide is the most variable constituent on the cell surface of Gram-negative bacteria and a player in their pathogenicity. The O-polysaccharide of Escherichia coli O109 was studied by sugar analysis and nuclear magnetic resonance spectroscopy and found to contain a rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy-l-mannose (l-RhaNAc3NAc). The following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established, which is closely related to that of Proteus penneri O66: Ac--4-β-L-RhapNAc3NAc -->4)-α-D-Glcp-(1-->3)-α-L-6dTalp-(1-->3)-β-D-GlcpNAc-(1-->. The O-antigen gene cluster of E. coli O109 was sequenced and all 14 genes found were assigned functions based on their similarity to genes from the available databases. Putative genes for synthesis of l-RhaN3N were found in E. coli O109 and their homologues in E. coli O119, whose O-antigen has been reported earlier to contain 2-acetamido-2,3,6-trideoxy-3-formamido-d-mannose (d-RhaNAc3NFo). Analysis by GLC of the (S)-2-octyl glycosides confirmed that the absolute configuration of RhaN3N in E. coli O119 should be revised from D TO L.